Question: In mouse models, the loss of CSF1R facilitates MOZ-TIF2-induced leuekmogenesis.

Title: PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2 Content: Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony–stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1Rhigh cells), but not those expressing low amounts of CSF1R (CSF1Rlow cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the CSF1R promoter, we cured AML by ablation of CSF1Rhigh cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2–induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1Rhigh cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach.

Title: A tumor necrosis factor-α–mediated pathway promoting autosomal dominant polycystic kidney disease Content: Autosomal dominant polycystic kidney disease (ADPKD) is caused by heterozygous mutations in either PKD1 or PKD2, genes that encode polycystin-1 and polycystin-2, respectively. We show here that tumor necrosis factor-α (TNF-α), an inflammatory cytokine present in the cystic fluid of humans with ADPKD, disrupts the localization of polycystin-2 to the plasma membrane and primary cilia through a scaffold protein, FIP2, which is induced by TNF-α. Treatment of mouse embryonic kidney organ cultures with TNF-α resulted in formation of cysts, and this effect was exacerbated in the Pkd2+/− kidneys. TNF-α also stimulated cyst formation in vivo in Pkd2+/− mice. In contrast, treatment of Pkd2+/− mice with the TNF-α inhibitor etanercept prevented cyst formation. These data reveal a pathway connecting TNF-α signaling, polycystins and cystogenesis, the activation of which may reduce functional polycystin-2 below a critical threshold, precipitating the ADPKD cellular phenotype.

Title: Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice Content: Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.

Title: PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis Content: Osteogenesis during bone modeling and remodeling is coupled with angiogenesis. A recent study showed that a specific vessel subtype, strongly positive for CD31 and endomucin (CD31hiEmcnhi), couples angiogenesis and osteogenesis. Here, we found that platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces CD31hiEmcnhi vessel formation during bone modeling and remodeling. Mice with depletion of PDGF-BB in the tartrate-resistant acid phosphatase–positive cell lineage show significantly lower trabecular and cortical bone mass, serum and bone marrow PDGF-BB concentrations, and fewer CD31hiEmcnhi vessels compared to wild-type mice. In the ovariectomy (OVX)-induced osteoporotic mouse model, serum and bone marrow levels of PDGF-BB and numbers of CD31hiEmcnhi vessels are significantly lower compared to sham-operated controls. Treatment with exogenous PDGF-BB or inhibition of cathepsin K to increase the number of preosteoclasts, and thus the endogenous levels of PDGF-BB, increases CD31hiEmcnhi vessel number and stimulates bone formation in OVX mice. Thus, pharmacotherapies that increase PDGF-BB secretion from preosteoclasts offer a new therapeutic target for treating osteoporosis by promoting angiogenesis and thus bone formation.

Title: Disruption of the 5S RNP–Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia Content: Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2C305F knock-in mice that have a disrupted 5S RNP–Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2C305F reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP–Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP–Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

Title: Requirement of endogenous stem cell factor and granulocyte-colony-stimulating factor for IL-17-mediated granulopoiesis. Content: IL-17 is a novel, CD4+ T cell-restricted cytokine. In vivo, it stimulates hematopoiesis and causes neutrophilia consisting of mature granulocytes. In this study, we show that IL-17-mediated granulopoiesis requires G-CSF release and the presence or induction of the transmembrane form of stem cell factor (SCF) for optimal granulopoiesis. However, IL-17 also protects mice from G-CSF neutralization-induced neutropenia. G-CSF neutralization completely reversed IL-17-induced BM progenitor expansion, whereas splenic CFU-GM/CFU-granulocyte-erythrocyte-megakaryocyte-monocyte was only reduced by 50% in both Sl/Sld and littermate control mice. Thus, there remained a significant SCF/G-CSF-independent effect of IL-17 on splenic granulopoiesis, resulting in a preservation of mature circulating granulocytes. IL-17 is a cytokine that potentially interconnects lymphocytic and myeloid host defense and may have potential for therapeutic development.

Title: Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming Content: We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

Title: Foxp2 and Foxp1 cooperatively regulate lung and esophagus development. Content: The airways of the lung develop through a reiterative process of branching morphogenesis that gives rise to the intricate and extensive surface area required for postnatal respiration. The forkhead transcription factors Foxp2 and Foxp1 are expressed in multiple foregut-derived tissues including the lung and intestine. In this report, we show that loss of Foxp2 in mouse leads to defective postnatal lung alveolarization, contributing to postnatal lethality. Using in vitro and in vivo assays, we show that T1alpha, a lung alveolar epithelial type 1 cell-restricted gene crucial for lung development and function, is a direct target of Foxp2 and Foxp1. Remarkably, loss of a single Foxp1 allele in addition to complete loss of Foxp2 results in increased severity of morphological defects in mutant lungs and leads to perinatal loss of all Foxp2(-/-);Foxp1(+/-) mice. Expression of N-myc and Hop, crucial regulators of lung development, is compromised in Foxp2(-/-);Foxp1(+/-) mutants. In addition to the defects in lung development, esophageal muscle development is disrupted in Foxp2(-/-);Foxp1(+/-) embryos, a tissue where Foxp2 and Foxp1 are co-expressed. These data identify Foxp2 and Foxp1 as crucial regulators of lung and esophageal development, underscoring the necessity of these transcription factors in the development of anterior foregut-derived tissues and demonstrating functional cooperativity between members of the Foxp1/2/4 family in tissues where they are co-expressed.

Title: Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Content: Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.

Title: Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species. Content: Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

Title: Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. Content: Rho family proteins are known to regulate actin organization in fibroblasts, but their functions in cells of haematopoietic origin have not been studied in detail. Bac1.2F5 cells are a colony-stimulating factor-1 (CSF-1)-dependent murine macrophage cell line; CSF-1 stimulates their proliferation and motility, and acts as a chemoattractant. CSF-1 rapidly induced actin reorganization in Bac1 cells: it stimulated the formation of filopodia, lamellipodia and membrane ruffles at the plasma membrane, as well as the appearance of fine actin cables within the cell interior. Microinjection of constitutively activated (V12)Rac1 stimulated lamellipodium formation and membrane ruffling. The dominant inhibitory Rac mutant, N17Rac1, inhibited CSF-1-induced lamellipodium formation, and also induced cell rounding. V12Cdc42 induced the formation of long filopodia, while the dominant inhibitory mutant N17Cdc42 prevented CSF-1-induced formation of filopodia but not lamellipodia. V14RhoA stimulated actin cable assembly and cell contraction, while the Rho inhibitor, C3 transferase, induced the loss of actin cables. Bac1 cells had cell-to-substratum adhesion sites containing beta1 integrin, pp125FAK, paxillin, vinculin, and tyrosine phosphorylated proteins. These 'focal complexes' were present in growing and CSF-1-starved cells, but were disassembled in cells injected with N17Cdc42 or N17Rac1. Interestingly, beta1 integrin did not disperse until long after focal phosphotyrosine and vinculin staining had disappeared. We conclude that in Bac1 macrophages Cdc42, Rac and Rho regulate the formation of distinct actin filament-based structures, and that Cdc42 and Rac are also required for the assembly of adhesion sites to the extracellular matrix.

Title: Derivation of novel human ground state naive pluripotent stem cells Content: Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Title: Cold Exposure Promotes Atherosclerotic Plaque Growth and Instability via UCP1-Dependent Lipolysis Content: Molecular mechanisms underlying the cold-associated high cardiovascular risk remain unknown. Here, we show that the cold-triggered food-intake-independent lipolysis significantly increased plasma levels of small low-density lipoprotein (LDL) remnants, leading to accelerated development of atherosclerotic lesions in mice. In two genetic mouse knockout models (apolipoprotein E(-/-) [ApoE(-/-)] and LDL receptor(-/-) [Ldlr(-/-)] mice), persistent cold exposure stimulated atherosclerotic plaque growth by increasing lipid deposition. Furthermore, marked increase of inflammatory cells and plaque-associated microvessels were detected in the cold-acclimated ApoE(-/-) and Ldlr(-/-) mice, leading to plaque instability. Deletion of uncoupling protein 1 (UCP1), a key mitochondrial protein involved in thermogenesis in brown adipose tissue (BAT), in the ApoE(-/-) strain completely protected mice from the cold-induced atherosclerotic lesions. Cold acclimation markedly reduced plasma levels of adiponectin, and systemic delivery of adiponectin protected ApoE(-/-) mice from plaque development. These findings provide mechanistic insights on low-temperature-associated cardiovascular risks.

Title: Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML Content: Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML). We have previously reported that CD34+CD38− human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rγnull mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.

Title: Induction of human neuronal cells by defined transcription factors Content: Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

Title: Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria Content: Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.

Title: An Antidepressant Decreases CSF Aβ Production in Healthy Individuals and in Transgenic AD Mice Content: Serotonin signaling suppresses generation of amyloid-β (Aβ) in vitro and in animal models of Alzheimer’s disease (AD). We show that in an aged transgenic AD mouse model (APP/PS1 plaque-bearing mice), the antidepressant citalopram, a selective serotonin reuptake inhibitor, decreased Aβ in brain interstitial fluid in a dose-dependent manner. Growth of individual amyloid plaques was assessed in plaque-bearing mice that were chronically administered citalopram. Citalopram arrested the growth of preexisting plaques and reduced the appearance of new plaques by 78%. In healthy human volunteers, citalopram’s effects on Aβ production and Aβ concentrations in cerebrospinal fluid (CSF) were measured prospectively using stable isotope labeling kinetics, with CSF sampling during acute dosing of citalopram. Aβ production in CSF was slowed by 37% in the citalopram group compared to placebo. This change was associated with a 38% decrease in total CSF Aβ concentrations in the drug-treated group. The ability to safely decrease Aβ concentrations is potentially important as a preventive strategy for AD. This study demonstrates key target engagement for future AD prevention trials.

Title: Inflammation-induced tumorigenesis in the colon is regulated by caspase-1 and NLRC4. Content: Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association is currently unknown. The inflammasome, a multiprotein complex formed by NOD-like receptor (NLR) family members, has recently been shown to orchestrate multiple innate and adaptive immune responses, yet its potential role in inflammation-induced cancer has been little studied. Using the azoxymethane and dextran sodium sulfate colitis-associated colorectal cancer model, we show that caspase-1-deficient (Casp1(-/-)) mice have enhanced tumor formation. Surprisingly, the role of caspase-1 in tumorigenesis was not through regulation of colonic inflammation, but rather through regulation of colonic epithelial cell proliferation and apoptosis. Consequently, caspase-1-deficient mice demonstrate increased colonic epithelial cell proliferation in early stages of injury-induced tumor formation and reduced apoptosis in advanced tumors. We suggest a model in which the NLRC4 inflammasome is central to colonic inflammation-induced tumor formation through regulation of epithelial cell response to injury.

Title: Activator protein-1 (AP-1) DNA binding activity is induced by hydroxyurea in organogenesis stage mouse embryos Content: Hydroxyurea is a potent teratogen; free radical scavengers or antioxidants reduce its teratogenicity. Activator Protein-1 (AP-1) and NF-kappaB are redox-sensitive transcription factors with important roles in normal development and the stress response. This study was designed to determine if exposure to teratogenic doses of hydroxyurea induces oxidative stress and alters gene expression by activating these transcription factors. Pregnant mice were treated with saline or hydroxyurea (400, 500, or 600 mg/kg) on gestation day 9 (GD 9) and killed either on GD 9, 0.5, 3, or 6 h after treatment, to assess oxidative stress and transcription factor activities, or on GD 18, to assess fetal development. Exposure to 400 mg/kg hydroxyurea did not affect the progeny, whereas exposure to 500 or 600 mg/kg resulted in dose-dependent increases in fetal resorptions and malformations, including curly tails, abnormal limbs (oligodactyly, hemimelia, and amelia), and short ribs. Hydroxyurea did not induce oxidative stress, as assessed by the ratio of oxidized to reduced glutathione, nor did it alter NF-kappaB DNA binding activity in the GD 9 conceptus. In contrast, exposure to hydroxyurea at any dose increased AP-1 DNA binding activity in embryos and yolk sacs 0.5 or 3 h after treatment. Hydroxyurea-induced c-Fos heterodimer activity in the embryo peaked 3-4-fold above control at 3 h and remained elevated by 6 h; in contrast, the activity of c-Jun dimers was not altered by drug exposure. A dramatic and region-specific increase in c-Fos immunoreactivity was found in hydroxyurea-treated embryos. The induction of AP-1 DNA binding activity by hydroxyurea represents an early, sensitive marker of the embryonic response to insult.

Title: Oct4-Induced Pluripotency in Adult Neural Stem Cells Content: The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

Title: Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function Content: Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.

Title: VEGF inhibits tumor cell invasion and mesenchymal transition through a MET/VEGFR2 complex. Content: Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor cell invasion through enhanced recruitment of the protein tyrosine phosphatase 1B (PTP1B) to a MET/VEGFR2 heterocomplex, thereby suppressing HGF-dependent MET phosphorylation and tumor cell migration. Consequently, VEGF blockade restores and increases MET activity in GBM cells in a hypoxia-independent manner, while inducing a program reminiscent of epithelial-to-mesenchymal transition highlighted by a T-cadherin to N-cadherin switch and enhanced mesenchymal features. Inhibition of MET in GBM mouse models blocks mesenchymal transition and invasion provoked by VEGF ablation, resulting in substantial survival benefit.

Title: The Nuclear Effector of Wnt-Signaling, Tcf1, Functions as a T-Cell–Specific Tumor Suppressor for Development of Lymphomas Content: The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

Title: Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity Content: The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant Braf(V600E) mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in Braf(V600E) mouse melanoma cells, as well as in Nras(G12D) melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.

Title: Teratogen-Induced Oxidative Stress Targets Glyceraldehyde-3-Phosphate Dehydrogenase in the Organogenesis Stage Mouse Embryo Content: Exposure during the organogenesis stage of the mouse embryo to the model teratogen, hydroxyurea (HU), induces curly tail and limb malformations. Oxidative stress contributes to the developmental toxicity of HU. Reactive oxygen species (ROS) interact with polyunsaturated bilipid membranes to form α,β-unsaturated reactive aldehydes; 4-hydroxy-2-nonenal (4-HNE), one of the most cytotoxic of these aldehydes, covalently adducts with proteins, lipids, and nucleic acids. The goal of the current study is to determine if HU exposure of CD1 mice on gestation day 9 generates region-specific 4-HNE-protein adducts in the embryo and to identify the proteins targeted. The formation of 4-HNE-protein adducts was elevated in the caudal region of control embryos; HU exposure further increased 4-HNE-protein adduct formation in this area. Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism. The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%. Furthermore, HU exposure induced the nuclear translocation of GAPDH in the caudal region of exposed embryos; this nuclear translocation may be associated with the reactivation of oxidized proteins involved in DNA repair, such as apurinic/apyrimidinic endonuclease-1, and the stimulation of E1A-associated P300 protein/creb-binding protein (p300/CBP) activity, initiating cell death in a p53-dependent pathway. We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

Title: Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Content: Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

Title: Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice. Content: Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.

Title: A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer. Content: The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.

Title: Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells. Content: Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Title: ADAR1 Forms a Complex with Dicer to Promote MicroRNA Processing and RNA-Induced Gene Silencing Content: Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a complex with Dicer through direct protein-protein interaction. Most importantly, ADAR1 increases the maximum rate (Vmax) of pre-microRNA (miRNA) cleavage by Dicer and facilitates loading of miRNA onto RNA-induced silencing complexes, identifying a new role of ADAR1 in miRNA processing and RNAi mechanisms. ADAR1 differentiates its functions in RNA editing and RNAi by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively. As expected, the expression of miRNAs is globally inhibited in ADAR1(-/-) mouse embryos, which, in turn, alters the expression of their target genes and might contribute to their embryonic lethal phenotype.

Title: Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses. Content: We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Title: Loss of IGF-IEa or IGF-IEb impairs myogenic differentiation. Content: Actions of protein products resulting from alternative splicing of the Igf1 gene have received increasing attention in recent years. However, the significance and functional relevance of these observations remain poorly defined. To address functions of IGF-I splice variants, we examined the impact of loss of IGF-IEa and IGF-IEb on the proliferation and differentiation of cultured mouse myoblasts. RNA interference-mediated reductions in total IGF-I, IGF-IEa alone, or IGF-IEb alone had no effect on cell viability in growth medium. However, cells deficient in total IGF-I or IGF-IEa alone proliferated significantly slower than control cells or cells deficient in IGF-IEb in serum-free media. Simultaneous loss of both or specific loss of either splice variant significantly inhibited myosin heavy chain (MyHC) immunoreactivity by 70-80% (P < 0.01) under differentiation conditions (48 h in 2% horse serum) as determined by Western immunoblotting. This loss in protein was associated with reduced MyHC isoform mRNAs, because reductions in total IGF-I or IGF-IEa mRNA significantly reduced MyHC mRNAs by approximately 50-75% (P < 0.05). Loss of IGF-IEb also reduced MyHC isoform mRNA significantly, with the exception of Myh7, but to a lesser degree (∼20-40%, P < 0.05). Provision of mature IGF-I, but not synthetic E peptides, restored Myh3 expression to control levels in cells deficient in IGF-IEa or IGF-IEb. Collectively, these data suggest that IGF-I splice variants may regulate myoblast differentiation through the actions of mature IGF-I and not the E peptides.

Title: Bone progenitor dysfunction induces myelodysplasia and secondary leukemia Content: Mesenchymal cells contribute to the 'stroma' of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

Title: the Development of Autoimmune Myocarditis in Mice by an Content: BACKGROUND Interleukin (IL)-12 exerts a potent proinflammatory effect by stimulating T-helper (Th) 1 responses. This effect is believed to be mediated primarily through the activation of STAT4 and subsequent production of interferon (IFN)-gamma. Methods and Results- We examined the role of IL-12 receptor (IL-12R) signaling in the development of murine experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization. Both IL-12Rbeta1-deficient mice and STAT4-deficient mice were resistant to the induction of myocarditis. Treatment with exogenous IL-12 exacerbated disease. We questioned whether IFN-gamma is required for the disease-promoting activity of IL-12. On the contrary, we found that IFN-gamma suppresses EAM. Lack of IFN-gamma due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Spleens from IFN-gamma-deficient mice immunized with cardiac myosin showed increased cellularity; greater numbers of CD3+, CD4+, CD8+, and IL-2-producing cells; and heightened ability to produce cytokines on stimulation in vitro. Treatment of mice with recombinant IFN-gamma suppressed the development of myocarditis. CONCLUSIONS IL-12/IL-12R/STAT4 signaling promotes the development of EAM. In contrast, IFN-gamma plays a protective role. The disease-limiting effects of IFN-gamma might be explained by its ability to control the expansion of activated T lymphocytes.

Title: Cdk1 is sufficient to drive the mammalian cell cycle Content: Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division. In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis. This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks. Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks. Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins. Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation. In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors. Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins. However, they become immortal on continuous passage. We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1. These results indicate that Cdk1 is the only essential cell cycle Cdk. Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.

Title: E2F1-3 Switch from Activators in Progenitor Cells to Repressors in Differentiating Cells Content: In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.

Title: Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy. Content: Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABA(A)-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice.

Title: Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis. Content: The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Title: Uncoupling Mitochondrial Respiration for Diabesity. Content: Until recently, the mechanism of adaptive thermogenesis was ascribed to the expression of uncoupling protein 1 (UCP1) in brown and beige adipocytes. UCP1 is known to catalyze a proton leak of the inner mitochondrial membrane, resulting in uncoupled oxidative metabolism with no production of adenosine triphosphate and increased energy expenditure. Thus increasing brown and beige adipose tissue with augmented UCP1 expression is a viable target for obesity-related disorders. Recent work demonstrates an UCP1-independent pathway to uncouple mitochondrial respiration. A secreted enzyme, PM20D1, enriched in UCP1+ adipocytes, exhibits catalytic and hydrolytic activity to reversibly form N-acyl amino acids. N-acyl amino acids act as endogenous uncouplers of mitochondrial respiration at physiological concentrations. Administration of PM20D1 or its products, N-acyl amino acids, to diet-induced obese mice improves glucose tolerance by increasing energy expenditure. In short-term studies, treated animals exhibit no toxicity while experiencing 10% weight loss primarily of adipose tissue. Further study of this metabolic pathway may identify novel therapies for diabesity, the disease state associated with diabetes and obesity.

Title: Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers Content: ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Title: SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance. Content: Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

Title: Plzf regulates limb and axial skeletal patterning Content: The promyelocytic leukaemia zinc finger (Plzf) protein (encoded by the gene Zfp145) belongs to the POZ/zinc-finger family of transcription factors. Here we generate Zfp145−/− mice and show that Plzf is essential for patterning of the limb and axial skeleton. Plzf inactivation results in patterning defects affecting all skeletal structures of the limb, including homeotic transformations of anterior skeletal elements into posterior structures. We demonstrate that Plzf acts as a growth-inhibitory and pro-apoptotic factor in the limb bud. The expression of members of the abdominal b (Abdb) Hox gene complex, as well as genes encoding bone morphogenetic proteins (Bmps), is altered in the developing limb of Zfp145−/− mice. Plzf regulates the expression of these genes in the absence of aberrant polarizing activity and independently of known patterning genes. Zfp145−/− mice also exhibit anterior-directed homeotic transformation throughout the axial skeleton with associated alterations in Hox gene expression. Plzf is therefore a mediator of anterior-to-posterior (AP) patterning in both the axial and appendicular skeleton and acts as a regulator of Hox gene expression.

Title: Cyclin A1 is required for meiosis in the male mouse Content: The mammalian A-type cyclin family consists of two members, cyclin A1 (encoded by Ccna1) and cyclin A2 (encoded by Ccna2). Cyclin A2 promotes both G1/S and G2/M transitions, and targeted deletion of Ccna2 in mouse is embryonic lethal. Cyclin A1 is expressed in mice exclusively in the germ cell lineage and is expressed in humans at highest levels in the testis and certain myeloid leukaemia cells. To investigate the role of cyclin A1 and possible redundancy among the cyclins in vivo, we generated mice bearing a null mutation of Ccna1. Ccna1-/- males were sterile due to a block of spermatogenesis before the first meiotic division, whereas females were normal. Meiosis arrest in Ccna1–/– males was associated with increased germ cell apoptosis, desynapsis abnormalities and reduction of Cdc2 kinase activation at the end of meiotic prophase. Cyclin A1 is therefore essential for spermatocyte passage into the first meiotic division in male mice, a function that cannot be complemented by the concurrently expressed B-type cyclins.

Title: Activation of AMP-activated protein kinase suppresses oxidized low-density lipoprotein-induced macrophage proliferation. Content: Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [(3)H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKalpha1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27(kip) suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21(cip) and p27(kip) expression via AMPK activation, and small interfering RNA (siRNA) of p21(cip) and p27(kip) restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.

Title: Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Content: Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Title: A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4 Content: S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

Title: Salt-inducible kinase 2 links transcriptional coactivator p300 phosphorylation to the prevention of ChREBP-dependent hepatic steatosis in mice. Content: Obesity and type 2 diabetes are associated with increased lipogenesis in the liver. This results in fat accumulation in hepatocytes, a condition known as hepatic steatosis, which is a form of nonalcoholic fatty liver disease (NAFLD), the most common cause of liver dysfunction in the United States. Carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, has emerged as a major player in the development of hepatic steatosis in mice. However, the molecular mechanisms enhancing its transcriptional activity remain largely unknown. In this study, we have identified the histone acetyltransferase (HAT) coactivator p300 and serine/threonine kinase salt-inducible kinase 2 (SIK2) as key upstream regulators of ChREBP activity. In cultured mouse hepatocytes, we showed that glucose-activated p300 acetylated ChREBP on Lys672 and increased its transcriptional activity by enhancing its recruitment to its target gene promoters. SIK2 inhibited p300 HAT activity by direct phosphorylation on Ser89, which in turn decreased ChREBP-mediated lipogenesis in hepatocytes and mice overexpressing SIK2. Moreover, both liver-specific SIK2 knockdown and p300 overexpression resulted in hepatic steatosis, insulin resistance, and inflammation, phenotypes reversed by SIK2/p300 co-overexpression. Finally, in mouse models of type 2 diabetes and obesity, low SIK2 activity was associated with increased p300 HAT activity, ChREBP hyperacetylation, and hepatic steatosis. Our findings suggest that inhibition of hepatic p300 activity may be beneficial for treating hepatic steatosis in obesity and type 2 diabetes and identify SIK2 activators and specific p300 inhibitors as potential targets for pharmaceutical intervention.

Title: CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Content: The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

Title: Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors. Content: Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.

Title: A20 is overexpressed in glioma cells and may serve as a potential therapeutic target. Content: OBJECTIVE A20 is a TNF-inducible primary response gene, which has been found to have antiapoptotic function in several cancer cells. This study investigates A20 expression in human glioma tissues and four glioma cell lines, and its effect on tumorigenesis of glioma cells and a mouse tumor model. METHODS Human glioma tissue samples and cells were subject to reverse transcription-PCR (RT-PCR), western blotting and immunohistochemistry. Glioma cells was tested by flow cytometry. A xenograft tumor model in mice was utilized to examine the knock-down effect of specific A20 siRNAs on tumorigenesis. RESULTS A20 was overexpressed in clinical glioma tissue samples (63.9%) and correlated with clinical staging. All four human glioma cell lines expressed A20, among which U87 displayed the strongest expression signals. Inhibiting A20 expression by siRNAs in vitro reduced the growth rates of glioma cells and resulted in G1/S arrest and increased apoptosis. In a mouse tumor model, local administration of siRNA significantly suppressed solid tumor growth. CONCLUSIONS A20 was overexpressed both in human glioma tissues and cell lines, and inhibiting A20 expression greatly slowed tumor cell growth in culture and in mice. These findings indicated that A20 is involved in tumorigenesis of human glioma, and may serve as a future therapeutic target.

Title: Modification of kidney barrier function by the urokinase receptor Content: Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of αvβ3 integrin. Mice lacking uPAR (Plaur−/−) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active β3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate αvβ3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of αvβ3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Title: Functional inhibition of osteoblastic cells in an in vivo mouse model of myeloid leukemia. Content: Pancytopenia is a major cause of morbidity in acute myeloid leukemia (AML), yet its cause is unclear. Normal osteoblastic cells have been shown to support hematopoiesis. To define the effects of leukemia on osteoblastic cells, we used an immunocompetent murine model of AML. Leukemic mice had inhibition of osteoblastic cells, with decreased serum levels of the bone formation marker osteocalcin. Osteoprogenitor cells and endosteal-lining osteopontin(+) cells were reduced, and osteocalcin mRNA in CD45(-) marrow cells was diminished. This resulted in severe loss of mineralized bone. Osteoclasts were only transiently increased without significant increases in bone resorption, and their inhibition only partially rescued leukemia-induced bone loss. In vitro data suggested that a leukemia-derived secreted factor inhibited osteoblastic cells. Because the chemokine CCL-3 was recently reported to inhibit osteoblastic function in myeloma, we tested its expression in our model and in AML patients. Consistent with its potential novel role in leukemic-dependent bone loss, CCL-3 mRNA was significantly increased in malignant marrow cells from leukemic mice and from samples from AML patients. Based on these results, we propose that therapeutic mitigation of leukemia-induced uncoupling of osteoblastic and osteoclastic cells may represent a novel approach to promote normal hematopoiesis in patients with myeloid neoplasms.

Title: Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Content: TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.

Title: Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1 Content: In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.

Title: Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1. Content: Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Title: UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis Content: Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

Title: Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2 Content: Rett syndrome is caused by loss-of-function mutations in the gene encoding the methyl DNA-binding factor MeCP2. As brain mass and neuronal complexity tend to be diminished in Rett patients, we tested whether MeCP2 directly influences the morphological complexity of developing neurons. Our results show that cultured mouse neurons overexpressing MeCP2beta (MECP2A) develop more complex morphologies, having longer axonal and dendritic processes, and an increased number of axonal and dendritic terminal endings. We then tested whether overexpressing a mutant form of MeCP2beta lacking its carboxyl terminus would elicit the same effects. Interestingly, while neurons overexpressing this mutant failed to enhance axonal and dendritic process elongation, the complexity of their axonal and dendritic processes remained significantly elevated. Taken together, these data support the hypothesis that MeCP2 directly regulates neuronal maturation and/or synaptogenesis, and provides evidence that MeCP2 may influence neuritic elongation and process branching through different mechanisms.

Title: Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Content: Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1(G93A) mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.

Title: Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death. Content: Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.

Title: Brain-Specific Phosphorylation of MeCP2 Regulates Activity-Dependent Bdnf Transcription, Dendritic Growth, and Spine Maturation Content: Mutations or duplications in MECP2 cause Rett and Rett-like syndromes, neurodevelopmental disorders characterized by mental retardation, motor dysfunction, and autistic behaviors. MeCP2 is expressed in many mammalian tissues and functions as a global repressor of transcription; however, the molecular mechanisms by which MeCP2 dysfunction leads to the neural-specific phenotypes of RTT remain poorly understood. Here, we show that neuronal activity and subsequent calcium influx trigger the de novo phosphorylation of MeCP2 at serine 421 (S421) by a CaMKII-dependent mechanism. MeCP2 S421 phosphorylation is induced selectively in the brain in response to physiological stimuli. Significantly, we find that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription. These findings suggest that, by triggering MeCP2 phosphorylation, neuronal activity regulates a program of gene expression that mediates nervous system maturation and that disruption of this process in individuals with mutations in MeCP2 may underlie the neural-specific pathology of RTT.

Title: Redefining the Genetic Hierarchies Controlling Skeletal Myogenesis: Pax-3 and Myf-5 Act Upstream of MyoD Content: We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.

Title: Autocrine VEGF Signaling Synergizes with EGFR in Tumor Cells to Promote Epithelial Cancer Development Content: It is established that tumor cell-derived VEGF acts on endothelial cells to promote angiogenesis and tumor growth. Here, we demonstrate that in K5-SOS-dependent mouse skin tumors, autocrine VEGF is required for tumor cell proliferation in a cell-autonomous and angiogenesis-independent manner. VEGF is upregulated in SOS-expressing tumors, and its deletion in epidermal cells delays tumorigenesis by suppressing angiogenesis and tumor cell proliferation. Epidermis-specific Flt1 deletion also impairs tumorigenesis and proliferation. Surprisingly, complete tumor inhibition occurs in the absence of VEGF in EGFR mutant mice, demonstrating that VEGFR and EGFR synergize in neoplastic cells to promote tumor growth. Mechanistically, K5-SOS upregulates VEGF, Flt1, and Neuropilin-1 in an Erk-dependent manner, thereby activating an autocrine proliferation loop, whereas EGFR prevents tumor cells from apoptosis. Moreover, Flt1 is upregulated in human SCC, and its inhibition in SCC cells impairs proliferation. Thus, in addition to regulating angiogenesis, VEGF has to be considered as a potent growth factor for epidermal tumors.

Title: Chronic Helicobacter pylori infections induce gastric mutations in mice. Content: BACKGROUND & AIMS Helicobacter pylori is an important etiologic factor in the development of gastric cancer. The aim of this study was to analyze the role of H. pylori infections in the induction of mutagenic events in gastric epithelial cells. The effect of a high-salt diet as a genotoxic risk factor was also investigated. METHODS Big Blue transgenic male mice (C57Bl/6) were inoculated with H. pylori (strain SS1) or Helicobacter felis (strain CS1) for 6 and 12 months. The frequency and spectrum of mutations at the stomach level were assessed. Inflammatory host response and inducible nitric oxide synthase (iNOS) expression by reverse-transcription polymerase chain reaction and immunohistochemistry analysis were also performed. RESULTS After 6 months, the gastric mutant frequency was 4-fold and 1.7-fold higher in mice infected with H. pylori and H. felis, respectively, than in uninfected mice. It was associated with a high frequency of transversions (AT --> CG and GC --> TA) known to result from oxidative damages. The Helicobacter-infected mice exhibited severe gastritis and a high level of iNOS messenger RNA expression. Hyperplasia developed 12 months after inoculation, and both the mutagenic effects and iNOS expression decreased in H. pylori- and H. felis-infected mice. No synergistic effects of a high-salt diet and Helicobacter infection were observed regarding the frequency of gastric mutation. CONCLUSIONS A direct gastric mutagenic effect due to H. pylori infection in the Big Blue transgenic mouse model has been shown 6 months after inoculation. This genotoxicity can be attributable to oxidative DNA damage involving the inflammatory host response.

Title: CDE-1 Affects Chromosome Segregation through Uridylation of CSR-1-Bound siRNAs Content: We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

Title: A Null Mutation in Inositol Polyphosphate 4-Phosphatase Type I Causes Selective Neuronal Loss in Weeble Mutant Mice Content: Weeble mutant mice have severe locomotor instability and significant neuronal loss in the cerebellum and in the hippocampal CA1 field. Genetic mapping was used to localize the mutation to the gene encoding inositol polyphosphate 4-phosphatase type I (Inpp4a), where a single nucleotide deletion results in a likely null allele. The substrates of INPP4A are intermediates in a pathway affecting intracellular Ca(2+) release but are also involved in cell cycle regulation through binding the Akt protooncogene; dysfunction in either may account for the neuronal loss of weeble mice. Although other mutations in phosphoinositide enzymes are associated with synaptic defects without neuronal loss, weeble shows that Inpp4a is critical for the survival of a subset of neurons during postnatal development in mice.

Title: Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100. Content: The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RARalpha knockin mouse were genetically modified with firefly luciferase (APL(luc)) to allow tracking by bioluminescence imaging. Coculture of APL(luc) cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL(luc) cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AML-niche interactions.

Title: Harnessing transposons for cancer gene discovery Content: Recently, it has become possible to mobilize the Tc1/mariner transposon, Sleeping Beauty (SB), in mouse somatic cells at frequencies high enough to induce cancer. Tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of the genes and signalling pathways that drive tumour formation. A conditional SB transposition system has also been developed that makes it possible to limit where SB mutagenesis occurs, providing a means to selectively model many types of human cancer. SB mutagenesis has already identified a large collection of known cancer genes in addition to a plethora of new candidate cancer genes and potential drug targets.

Title: Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension Content: Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH.

Title: Telomeric 3′ Overhangs Derive from Resection by Exo1 and Apollo and Fill-In by POT1b-Associated CST Content: A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.

Title: Helicobacter hepaticus does not induce or potentiate colitis in interleukin-10-deficient mice. Content: Helicobacter hepaticus has been reported to induce colitis, hepatitis, and hepatocellular carcinoma in several different murine models. The aim of this study was to determine if H. hepaticus will cause colitis in monoassociated mice lacking the interleukin-10 gene (IL-10(-/-) mice) and potentiate colitis in specific-pathogen-free (SPF) IL-10(-/-) mice. Germfree IL-10(-/-) mice on either a mixed (C57BL/6 x 129/Ola) or inbred (129/SvEv) genetic background were monoassociated with H. hepaticus ATCC 51448 by oral feeding and rectal enemas. In a second experiment, germfree IL-10(-/-) mice were colonized with stool from SPF mice that harbored or did not harbor endogenous H. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was isolated for histology and IL-12 secretion, and mesenteric lymph node cells were assessed for T-cell activation markers. It was found that IL-10(-/-) mice monoassociated with H. hepaticus for up to 16 weeks showed almost no histologic colitis or increased IL-12 production. SPF IL-10-knockout mice had no significant difference in weight loss, mortality rate, histologic scores, colonic IL-12 secretion, or T-cell activation with or without H. hepaticus. We conclude that H. hepaticus does not induce or potentiate disease in our IL-10(-/-) mice and therefore is not required to induce colitis in genetically susceptible hosts.

Title: Differential requirement for CARMA1 in agonist-selected T-cell development. Content: Caspase recruitment domain-containing membrane-associated guanylate kinase protein-1 (CARMA1) is a critical component of the NF-kappaB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist-selected T-cell subsets such as Treg, NKT cells, and CD8-alpha alpha T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1-deficient mice (CARMA1(-/-)) have altered populations of specific subsets of agonist-selected T cells. Specifically, CARMA1(-/-) mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild-type mice. Interestingly, CD8-alpha alpha T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1(-/-) mice, whereas memory-phenotype CD4(+) T cells (CD62L(low)/CD44(high)) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist-selected T-cell subsets. Overall, these data reveal an important but differential role for CARMA1-mediated TCR signaling in T-cell development.

Title: Wip1 phosphatase regulates p53-dependent apoptosis of stem cells and tumorigenesis in the mouse intestine. Content: Colorectal cancer is one of the major causes of cancer-related deaths. To gain further insights into the mechanisms underlying its development, we investigated the role of Wip1 phosphatase, which is highly expressed in intestinal stem cells, in the mouse model of APC(Min)-driven polyposis. We found that Wip1 removal increased the life span of APC(Min) mice through a significant suppression of polyp formation. This protection was dependent on the p53 tumor suppressor, which plays a putative role in the regulation of apoptosis of intestinal stem cells. Activation of apoptosis in stem cells of Wip1-deficient mice, but not wild-type APC(Min) mice, increased when the Wnt pathway was constitutively activated. We propose, therefore, that the Wip1 phosphatase regulates homeostasis of intestinal stem cells. In turn, Wip1 loss suppresses APC(Min)-driven polyposis by lowering the threshold for p53-dependent apoptosis of stem cells, thus preventing their conversion into tumor-initiating stem cells.

Title: Nuclear Factor-�B Affects Tumor Progression in a Mouse Model of Malignant Pleural Effusion Content: We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-κB activity in vitro and in vivo, which we used to drive expression of a NF-κB–dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-κB–dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-κB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-κB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-α levels. These studies indicate that tumor NF-κB a...

Title: Development of a syngeneic mouse model for events related to ovarian cancer. Content: Mouse ovarian surface epithelial cells (MOSEC) were obtained from virgin, mature mice by mild trypsinization and were repeatedly passaged in vitro. Early passage cells (<20 passages) exhibited a cobblestone morphology and contact inhibition of growth. After approximately 20 passages in vitro, cobblestone morphology and contact inhibition of growth was lost. Tumor forming potential was determined by s.c. and i.p. injection of early and late passage cells into athymic and syngeneic C57BL6 mice. Subcutaneous tumors formed in approximately 4 months and were present only at the injection site. Intraperitoneal injection of late passage MOSEC into athymic and syngeneic mice resulted in growth of tumor implants throughout the abdominal cavity, and production of hemorrhagic ascitic fluid. Early passage MOSEC did not form tumors in vivo. Histopathologic analysis of tumors revealed a highly malignant neoplasm containing both carcinomatous and sarcomatous components. Late passage MOSEC expressed cytokeratin and did not produce ovarian steroids in response to gonadotropin stimulation in vitro. Ten clonal lines were established from late passage MOSEC. Each clone formed multiple peritoneal tumors and ascitic fluid after i.p. injection into C57BL6 mice. Three cell lines examined cytogenetically were polyploid with near-tetraploid modal chromosome numbers. Common clonal chromosome gains and losses included +5, +15, +19 and -X, -3, -4. One cell line had a clonal translocation between chromosomes 15 and 18 and another had a small marker chromosome; common structural abnormalities were not observed. These data describe the development of a mouse model for the study of events related to ovarian cancer in humans. The ability of the MOSEC to form extensive tumors within the peritoneal cavity, similar to those seen in women with Stage III and IV cancer, and the ability of the MOSEC to produce tumors in mice with intact immune systems, makes this model unique for investigations of molecular and immune interactions in ovarian cancer development.

Title: OPA1 gene therapy prevents retinal ganglion cell loss in a Dominant Optic Atrophy mouse model Content: Dominant optic atrophy (DOA) is a rare progressive and irreversible blinding disease which is one of the most frequent forms of hereditary optic neuropathy. DOA is mainly caused by dominant mutation in the OPA1 gene encoding a large mitochondrial GTPase with crucial roles in membrane dynamics and cell survival. Hereditary optic neuropathies are commonly characterized by the degeneration of retinal ganglion cells, leading to the optic nerve atrophy and the progressive loss of visual acuity. Up to now, despite increasing advances in the understanding of the pathological mechanisms, DOA remains intractable. Here, we tested the efficiency of gene therapy on a genetically-modified mouse model reproducing DOA vision loss. We performed intravitreal injections of an Adeno-Associated Virus carrying the human OPA1 cDNA under the control of the cytomegalovirus promotor. Our results provide the first evidence that gene therapy is efficient on a mouse model of DOA as the wild-type OPA1 expression is able to alleviate the OPA1-induced retinal ganglion cell degeneration, the hallmark of the disease. These results displayed encouraging effects of gene therapy for Dominant Optic Atrophy, fostering future investigations aiming at clinical trials in patients.

Title: IAP antagonists induce anti-tumor immunity in multiple myeloma Content: The cellular inhibitors of apoptosis (cIAP) 1 and 2 are amplified in about 3% of cancers and have been identified in multiple malignancies as being potential therapeutic targets as a result of their role in the evasion of apoptosis. Consequently, small-molecule IAP antagonists, such as LCL161, have entered clinical trials for their ability to induce tumor necrosis factor (TNF)-mediated apoptosis of cancer cells. However, cIAP1 and cIAP2 are recurrently homozygously deleted in multiple myeloma (MM), resulting in constitutive activation of the noncanonical nuclear factor (NF)-κB pathway. To our surprise, we observed robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and in patients with relapsed-refractory MM, where the addition of cyclophosphamide resulted in a median progression-free-survival of 10 months. This effect was not a result of direct induction of tumor cell death, but rather of upregulation of tumor-cell-autonomous type I interferon (IFN) signaling and a strong inflammatory response that resulted in the activation of macrophages and dendritic cells, leading to phagocytosis of tumor cells. Treatment of a MM mouse model with LCL161 established long-term anti-tumor protection and induced regression in a fraction of the mice. Notably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all of the treated mice.

Title: The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Content: IL-4 promotes the differentiation of naive CD4+ T cells into IL-4-producing T helper 2 (Th2) cells. Previous work provided suggestive but not conclusive evidence that the transcription factor c-Maf directed the tissue-specific expression of IL-4. It was not known whether c-Maf controlled the transcription of other Th2 cytokine genes. To elucidate the role of c-Maf in vivo, we examined cytokine production in mice lacking c-Maf (c-maf(-/-)). CD4+ T cells and NK T cells from c-maf(-/-) mice were markedly deficient in IL-4 production. However, the mice produced normal levels of IL-13 and IgE, and, when differentiated in the presence of exogenous IL-4, c-maf(-/-) T cells produced approximately normal levels of other Th2 cytokines. We conclude that c-Maf has a critical and selective function in IL-4 gene transcription in vivo.

Title: LIN28B enhanced tumorigenesis in an autochthonous KRASG12V-driven lung carcinoma mouse model Content: LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

Title: Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors. Content: Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.

Title: Enhanced pathological angiogenesis in mice lacking β3 integrin or β3 and β5 integrins Content: Inhibition of αvβ3 or αvβ5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking β3 integrins or both β3 and β5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither β3 nor β5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of β3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the β3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in β3-null endothelial cells. These data indicate that αvβ3 and αvβ5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of αv-integrin antagonists in anti-angiogenic therapeutics.

Title: Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance Content: Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Title: Control of Nutrient Stress-Induced Metabolic Reprogramming by PKCζ in Tumorigenesis Content: Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.

Title: GEP100-Arf6-AMAP1-Cortactin Pathway Frequently Used in Cancer Invasion Is Activated by VEGFR2 to Promote Angiogenesis Content: Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy. Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.

Title: Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice Content: Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element-binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Title: BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells into Embryonic and Extraembryonic Lineages Content: BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.

Title: The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor+ Innate Lymphoid Cells Content: Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-α produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

Title: A complex secretory program orchestrated by the inflammasome controls paracrine senescence Content: Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

Title: PHD3-dependent hydroxylation of HCLK2 promotes the DNA damage response. Content: The DNA damage response (DDR) is a complex regulatory network that is critical for maintaining genome integrity. Posttranslational modifications are widely used to ensure strict spatiotemporal control of signal flow, but how the DDR responds to environmental cues, such as changes in ambient oxygen tension, remains poorly understood. We found that an essential component of the ATR/CHK1 signaling pathway, the human homolog of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2), associated with and was hydroxylated by prolyl hydroxylase domain protein 3 (PHD3). HCLK2 hydroxylation was necessary for its interaction with ATR and the subsequent activation of ATR/CHK1/p53. Inhibiting PHD3, either with the pan-hydroxylase inhibitor dimethyloxaloylglycine (DMOG) or through hypoxia, prevented activation of the ATR/CHK1/p53 pathway and decreased apoptosis induced by DNA damage. Consistent with these observations, we found that mice lacking PHD3 were resistant to the effects of ionizing radiation and had decreased thymic apoptosis, a biomarker of genomic integrity. Our identification of HCLK2 as a substrate of PHD3 reveals the mechanism through which hypoxia inhibits the DDR, suggesting hydroxylation of HCLK2 is a potential therapeutic target for regulating the ATR/CHK1/p53 pathway.

Title: The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy. Content: Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started approximately 1 h after seizure onset, peaked at 3-6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5-10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Title: Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis. Content: Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

Title: The developmental potential of iPSCs is greatly influenced by reprogramming factor selection. Content: Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

Title: Bcl2 Regulation by the Melanocyte Master Regulator Mitf Modulates Lineage Survival and Melanoma Cell Viability Content: Kit/SCF signaling and Mitf-dependent transcription are both essential for melanocyte development and pigmentation. To identify Mitf-dependent Kit transcriptional targets in primary melanocytes, microarray studies were undertaken. Among identified targets was BCL2, whose germline deletion produces melanocyte loss and which exhibited phenotypic synergy with Mitf in mice. BCL2's regulation by Mitf was verified in melanocytes and melanoma cells and by chromatin immunoprecipitation of the BCL2 promoter. Mitf also regulates BCL2 in osteoclasts, and both Mitf(mi/mi) and Bcl2(-/-) mice exhibit severe osteopetrosis. Disruption of Mitf in melanocytes or melanoma triggered profound apoptosis susceptible to rescue by BCL2 overexpression. Clinically, primary human melanoma expression microarrays revealed tight nearest neighbor linkage for MITF and BCL2. This linkage helps explain the vital roles of both Mitf and Bcl2 in the melanocyte lineage and the well-known treatment resistance of melanoma.

Title: A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes Content: West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

Title: FGF-2 enhances intestinal stem cell survival and its expression is induced after radiation injury. Content: Fibroblast growth factors (FGFs) have mitogenic activity toward a wide variety of cells of mesenchymal, neuronal, and epithelial origin and regulate events in normal embryonic development, angiogenesis, wound repair, and neoplasia. FGF-2 is expressed in many normal adult tissues and can regulate migration and replication of intestinal epithelial cells in culture. However, little is known about the effects of FGF-2 on intestinal epithelial stem cells during either normal epithelial renewal or regeneration of a functional epithelium after injury. In this study, we investigated the expression of FGF-2 in the mouse small intestine after irradiation and determined the effect of exogenous FGF-2 on crypt stem cell survival after radiation injury. Expression of FGF-2 mRNA and protein began to increase at 12 h after gamma-irradiation, and peak levels were observed from 48 to 120 h after irradiation. At all times after irradiation, the higher molecular mass isoform ( approximately 24 kDa) of FGF-2 was the predominant form expressed in the small intestine. Immunohistochemical analysis of FGF-2 expression after radiation injury demonstrated that FGF-2 was predominantly found in the mesenchyme surrounding regenerating crypts. Exogenous recombinant human FGF-2 (rhFGF-2) markedly enhanced crypt stem cell survival when given before irradiation. We conclude that expression of FGF-2 is induced by radiation injury and that rhFGF-2 can enhance crypt stem cell survival after subsequent injury.

Title: Obesity induces a phenotypic switch in adipose tissue macrophage polarization. Content: Adipose tissue macrophages (ATMs) infiltrate adipose tissue during obesity and contribute to insulin resistance. We hypothesized that macrophages migrating to adipose tissue upon high-fat feeding may differ from those that reside there under normal diet conditions. To this end, we found a novel F4/80(+)CD11c(+) population of ATMs in adipose tissue of obese mice that was not seen in lean mice. ATMs from lean mice expressed many genes characteristic of M2 or "alternatively activated" macrophages, including Ym1, arginase 1, and Il10. Diet-induced obesity decreased expression of these genes in ATMs while increasing expression of genes such as those encoding TNF-alpha and iNOS that are characteristic of M1 or "classically activated" macrophages. Interestingly, ATMs from obese C-C motif chemokine receptor 2-KO (Ccr2-KO) mice express M2 markers at levels similar to those from lean mice. The antiinflammatory cytokine IL-10, which was overexpressed in ATMs from lean mice, protected adipocytes from TNF-alpha-induced insulin resistance. Thus, diet-induced obesity leads to a shift in the activation state of ATMs from an M2-polarized state in lean animals that may protect adipocytes from inflammation to an M1 proinflammatory state that contributes to insulin resistance.

Title: TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis. Content: The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

Title: Hypoxia‐Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis Content: Summary Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell‐intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF‐&kgr;B signaling and a transcription factor E2F1‐dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL‐induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine‐driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F‐metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction. Graphical Abstract Figure. No Caption available. HighlightsCOMMD1 is a negative regulator of osteoclast differentiationCOMMD1 suppresses bone loss in RA and inflammatory arthritis and osteolysis modelsCOMMD1 negatively regulates E2F1‐dependent metabolic pathways in macrophagesHypoxia suppresses COMMD1 expression to augment osteoclastogenesis &NA; Pathways that promote osteoclastogenesis are well characterized but less is known about negative regulators that suppress pathological bone loss. Murata et al. identify COMMD1 as an inhibitor of osteoclastogenesis that restrains NF‐&kgr;B‐ and E2F1‐CKB‐mediated metabolic pathways in macrophages.

Title: Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Content: Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2), encoding a transcriptional repressor, cause Rett syndrome and a variety of related neurodevelopmental disorders. The vast majority of mutations associated with human disease are loss-of-function mutations, but precisely what aspect of MeCP2 function is responsible for these phenotypes remains unknown. We overexpressed wild-type human protein in transgenic mice using a large genomic clone containing the entire human MECP2 locus. Detailed neurobehavioral and electrophysiological studies in transgenic line MeCP2(Tg1), which expresses MeCP2 at approximately 2-fold wild-type levels, demonstrated onset of phenotypes around 10 weeks of age. Surprisingly, these mice displayed enhanced motor and contextual learning and enhanced synaptic plasticity in the hippocampus. After 20 weeks of age, however, these mice developed seizures, became hypoactive and approximately 30% of them died by 1 year of age. These data demonstrate that MeCP2 levels must be tightly regulated in vivo, and that even mild overexpression of this protein is detrimental. Furthermore, these results support the possibility that duplications or gain-of-function mutations in MECP2 might underlie some cases of X-linked delayed-onset neurobehavioral disorders.

Title: p38MAPK controls expression of multiple cell cycle inhibitors and islet proliferation with advancing age. Content: Aging is a complex organismal process that is controlled by genetic, environmental, and behavioral factors. Accumulating evidence supports a role for different cell cycle inhibitors in mammalian aging. Little is known, however, about the upstream signals that induce their expression. Here, we explore the role of p38MAPK by generating a dominant-negative allele (p38(AF)) in which activating phosphorylation sites Thr180 and Tyr182 are mutated. Heterozygous p38(AF) mice show a marked attenuation of p38-dependent signaling and age-induced expression of multiple cell cycle inhibitors in different organs, including pancreatic islets. As a result, aged p38(AF/+) mice show enhanced proliferation and regeneration of islets when compared to wild-type littermates. We further find an age-related reduction in expression of the p38-specific phosphatase Wip1. Wip1-deficient mice demonstrate decreased islet proliferation, while Wip1 overexpression rescues aging-related decline in proliferation and regenerative capacity. We propose that modulation of p38MAPK activity may provide new avenues for treating certain age-related degenerative diseases.

Title: Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection Content: Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.