extfieldsofstudy: Biology Medicine

text:
Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system.

Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 106 clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.

The first step in the biosynthesis of steroid hormones is the conversion of cholesterol into pregnenolone. The rate-limiting process is the transport of cholesterol from the outer mitochondrial membrane to the inner membrane, where cytochrome P450 side-chain cleavage (P450scc) 1 enzyme resides. Steroidogenic acute regulatory (StAR) protein plays a key role in the intra-mitochondrial movement of cholesterol (1). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia, a condition in which cholesterol accumulates in cytoplasmic lipid droplets, and adrenal and gonadal steroidogenesis is severely impaired (2,3). StAR gene knockout mice have the same phenotype as that of humans with congenital lipoid adrenal hyperplasia, that is cholesterol accumulation predominantly in the adrenal gland and markedly reduced steroid hormone secretion (4,5).
The tropic hormones ACTH, luteinizing hormone, and follicle-stimulating hormone stimulate steroid hormone production in the adrenal glands and gonads through a cAMP-dependent pathway. StAR gene expression increased rapidly in response to cAMP stimulation (6,7). Although the human StAR gene promoter lacks cAMP-responsive elements, steroidogenic factor-1 (8), CCAAT/enhancer-binding proteins, and GATA-4 confer cAMP-dependent StAR gene expression (9 -11). The factors SREBPs, Sp1, insulin-like growth factors, transforming growth factor-␤, tumor necrosis factor-␣, DAX-1, and SIK have also been shown to regulate StAR gene expressions (12)(13)(14)(15)(16)(17)(18)(19). Increased progesterone synthesis prior to an increase in the level of StAR mRNA expression in response to cAMP induction has been revealed by analysis of hyperacetylation in the StAR gene promoter (20). Although this increased steroidogenesis is thought to be the result of phosphorylation of the StAR protein by protein kinase A (8), the mechanism is still not clear.
Following synthesis, the 37-kDa StAR pre-protein is imported into mitochondria with subsequent cleavage of the mitochondria targeting sequence, yielding a 30-kDa mature StAR protein (21,22). StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein), which contain the mitochondrial targeting sequence, has been reported to be as effective as wild-type StAR protein in stimulating steroidogenesis (23). This led to the conclusion that the C terminus of StAR protein encodes its biological function for steroidogenesis and that the StAR protein acts on the outer mitochondrial membrane to stimulate cholesterol translocation. The proposed site of action of StAR protein on the cytoplasmic face of the outer mitochondrial membrane raised questions about the mechanisms by which StAR protein trades with mitochondria and expresses its action in the cytoplasm. In the present study, we used a yeasttwo hybrid system to screen for proteins that interact with StAR protein and modulate its steroidogenic action.

MATERIALS AND METHODS
Plasmid Constructs-A plasmid expressing a GAL4-N-62-StAR fusion lacking 62 amino acid-terminal residues was constructed by inserting an EcoRI fragment, prepared by PCR using human StAR cDNA as a template, into a pACT2 vector, which has a GAL4-activating domain (GAD) (Clontech Laboratories, Inc., Palo Alto, CA). Plasmids expressing GAL4-StAR mutants (GAL4-R193X, GAL4-Q253X, and GAL4-frameshift) were also constructed by inserting an EcoRI fragment prepared by PCR from cDNA of human StAR mutants, which were previously constructed (2). A plasmid expressing a GAD-N-62-StAR fusion was * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Hokkaido University Graduate School of Medicine, Kita-ku, Kita 15, Nishi 7, Sapporo 060-8638, Japan. Tel.: 81-11-706-5047; Fax: 81-11-727-6006; E-mail: terusuga@med.hokudai.ac.jp. 1 The abbreviations used are: P450scc, P450 side-chain cleavage; StAR, steroidogenic acute regulatory; SBP, StAR-binding protein; DNA-BD, DNA-binding domain; GAD, GAL4-activating domain; AD, activation domain; RACE, rapid amplification of cDNA ends; siRNA, small interfering RNA; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; RFP, red fluorescent protein; PBR, peripheral-type benzodiazepine receptor. also constructed by inserting an EcoRI fragment prepared by PCR and cloned into a pACT2 vector, which has a GAD (Clontech). Plasmid pVP16-StAR was constructed by inserting the EcoRI fragment from human N-62 StAR cDNA into the pVP16 vector, which has an activation domain (AD) derived from the VP16 protein of herpes simplex virus. The clone 4 translated product was named StAR-binding protein (SBP). We produced plasmid pM-SBP by inserting the EcoRI/BamHI fragment from SBP cDNA into the pM vector, which has a GAL4 DNA-binding domain (DNA-BD). Reverse combinations were also prepared; pVP16-SBP was constructed by inserting the EcoRI/BamHI fragment of SBP into the pVP16 vector. To produce plasmid pM-StAR, the EcoRI fragment from N-62 StAR was cloned into the pM vector. pG5luc (Promega Corp., Madison, WI) contains the chloramphenicol acetyltransferase gene or the luciferase gene as a reporter. From RACE results, we prepared an expression plasmid (pSBP) by inserting the EcoRI fragment of the entire coding region amplified by PCR from testis cDNA into the pTarget vector (Promega). A plasmid expressing an SBP-GFP fusion protein was constructed with the N-terminal green fluorescent protein (GFP) (pSBP-GFP) by inserting an EcoRI fragment, prepared by PCR using SBP cDNA as a template, into pEGFP-N1 (Clontech). A StAR-RFP fusion protein was also constructed with the N terminus of red fluorescent protein (pStAR-REP) by inserting an EcoRI fragment prepared by PCR from the full-length cDNA of human StAR cDNA into pDsRed2-N1 (Clontech). The plasmids were prepared for transfection studies using a Qiagen Maxiprep system (Qiagen, Hilden, Germany).
Cell Culture-Mouse Y-1 adrenal tumor cells, COS-1 cells, and human Hep G2 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). Human adrenocortical carcinoma H295R cells and mouse MA-10 Leydig cells were a gift from Dr. Mitsuhiro Okamoto, Osaka University Medical School (Osaka, Japan). Human MCF-7 breast cancer cells were obtained from ATCC (Manassas, VA). Human granulosalike tumor KGN cells were a gift from Dr. Yoshihiro Nishi, Graduate School of Medical Sciences, Kyushu University (24). The Y-1 cells and COS-1 cells were grown in 35-mm plastic dishes. The Y-1 cells and COS-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 50 g/ml gentamycin. The KGN cells were grown in DMEM/F-12 containing 10% fetal calf serum and 50 g/ml gentamycin. H295R cells were grown in DMEM/F-12 containing 2% ULTROSER G (BioSepra, Cergy-Pontoise, France) and 1% ITS Premix (BD Biosciences).
Pull-down Experiment-A plasmid expression clone was constructed by inserting an EcoRI fragment prepared by PCR and cloned into a pCI vector (Promega). Translated protein was synthesized in vitro using a T7 RNA polymerase-based TNT-coupled reticulocyte lysate system (Promega). A plasmid expressing a His-tagged CBD-N-62-StAR fusion protein lacking 62 amino acid-terminal residues was constructed by inserting an EcoRI fragment prepared by PCR using a human StAR cDNA as a template into a pET38b vector, which has a C-terminal His tag (Novagen, San Diego). CBD-N-62-StAR protein was expressed in bac-teria according to the manual of the manufacturer and was used for pull-down assays. His-tagged N-62-StAR fusion protein bound to His-Bind Resin (Novagen) was incubated for 3 h with 50 l in vitro translated [ 35 S]methionine-labeled translated clone in a total volume of 250 l of incubation buffer (50 mM potassium phosphate, pH 7.4, 150 mM KCl, 1 mM MgCl 2 , 10% glycerol, 0.1% Triton X-100). Resin was collected by microcentrifugation and washed three times. Washed beads were resuspended in 20 l of 2ϫ SDS sample buffer, heated for 5 min, and pelleted in a microcentrifuge, and the supernatant was subjected to SDS-PAGE and autoradiography.
Mammalian Two-hybrid Assay-Culture cells at 40 -60% confluence were transfected with plasmids with 0.1 g of the pG5luc reporter plasmid, 0.5 g of a pM-N-62-StAR protein (GAL4-StAR protein) expression plasmid, 0.5 g of a pVP16-StAR-binding protein (VP16-SBP) expression plasmid, and 0.1 g of pCH110 using FuGENE 6 (3 l/g DNA) (Roche Applied Science). The cells were cultured for 48 h after transfection and then harvested. Luciferase assays were performed using a Luciferase Assay System (Promega). The assay results were normalized to ␤-galactosidase activity to compensate for variation in transfection efficiency. Each experiment was repeated at least three times.
Northern Blot Analysis-Northern blots each containing 2 g of poly(A) ϩ RNA from various human tissues were purchased from Clontech and probed with 2.3-kb of human clone 4 cDNA and ␤-actin cDNA according to the supplier's protocol. Northern blotting and hybridization using human clone 4 were carried out as described previously (25).
RACE Analysis of the 5Ј-Site of cDNA of SBP-Marathon-Ready cDNA, 5Ј-stretched human testis cDNA (Clontech), was amplified using primers. The sense primer was Adapter Primer 1 (AP1, 5Ј-CCATCCTA-ATACGACTCACTATAGGGC), and the antisense primer was 5Ј-CAAACTGGAAAGAGCCTCCTCGTGAG-3Ј, which was 198 bp downstream from the translation start site identified in clone 4. PCR was performed according to the supplier's protocol. The PCR products were electrophoresed, and DNA fragments were cut from the gels. The fragments were ligated to PCR 2.1 vectors using a manual protocol (Invitrogen). PCR products were sequenced, and the sequences were compared with the human genome sequence.
Transfection and Pregnenolone Immunoassay-COS-1 cells were transfected with F2, a vector for the cytochrome P450 cholesterol sidechain cleavage system (called the F2 system) kindly provided by Dr. Walter L. Miller of the University of California, San Francisco (26), pStAR (pSPORT StAR cDNA) (2), and pSBP using FuGENE 6. The cells were incubated for 48 h following transfection. Some dishes were treated with 22(R)-hydroxycholesterol (1 g/ml) during the final 24 h of culture. Forty eight hours after transfection, the medium was collected for radioimmunoassay of pregnenolone. The assay results were normalized by serum pregnenolone concentrations produced by cultures with 22(R)-hydroxycholesterol to compensate for variation in transfection efficiency. Each experiment included triplicate cultures for each treatment group and was repeated at least three times.
Inhibition of SBP Expression by Small Interfering RNA (siRNA)-Cultures of sub-confluent (40 -50% confluent) H295R cells and KGN cells were plated so that 35-mm plastic dishes received equal numbers of cells. Endogenous SBP mRNA was targeted in cells with transfection by the addition of 19-nucleotide duplex (siRNA-SBP-I) and 21-nucleotide duplex (siRNA-SBP-II) (Dharmacon, Inc., Lafayette, CO). These duplex RNAs target 106 and 393 nucleotides downstream of the start codon of SBP, respectively. siRNA were constructed using the ribooligonucleotide pairs SBP-I and SBP-II with the following sequences: 5Ј-CGGGAUGUUUCCAGUGACAdTdT-3Ј and 5Ј-UGUCACUGGAAA-CAUCCCGdTdT-3Ј (SBP-I) and 5Ј-GAACUUGGAAGAGGGGAGGC-AdTdT-3Ј, 5Ј-UGCCUCCCCUCUUCCAAGUUCdTdT-3Ј (SBP-II). As a control for the specificity of these duplexes, we used a scramble ribooligonucleotide pair (siRNA-Scramble) with the following sequences: and 5Ј-CGAAUCCUA-CAAAGCGCGCdTdT-3Ј. The oligonucleotides were annealed according to the Dharmacon protocol. Three hundred pmol of each duplex was introduced into cells using 15 l of metafectene (Biontex Laboratories GmbH, Munich, Germany) as recommended by the manufacturer. Dishes for H295R cells were treated with 20 M trilostane (provided by Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) 6 h after transfection to inhibit the enzyme activity of 3␤-hydroxysteroid dehydrogenase, which transforms pregnenolone into progesterone (27). 48 h after transfection, cells were collected for radioimmunoassay of steroid hormones. In some experiments, total RNA was extracted, and RT-PCR for SBP (27 cycles) and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (24 cycles) as a control was performed using the following primers for GAPDH: sense, 5Ј-TGCCGTCTAGAAAAACCTGC-3Ј; antisense, 5Ј-ACCCTGTTGCTGTAGCCAAA-3Ј.
Fluorescence Microscopy-COS-1 cells were used for microscopic analysis. Cells were maintained in DMEM containing 10% fetal bovine serum. Before transfection, cells were seeded on coverslips in culture dishes. Cells were transfected using FuGENE 6 according to the manufacturer's instructions. After transfection, cells were incubated for 24 h. Transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min. After washing twice in phosphatebuffered saline, each coverslip was mounted onto a glass microscope slide. Observations were made with a fluorescence microscope equipped with a mercury lamp with excitation wavelengths of 450 -490 and 546 nm for GFP and DsRed, respectively (Axiophot, Carl Zeiss Inc., Oberkochen, Germany). Emission was used with a 515-565-nm bandpass filter for GFP and a 590-nm longpass filter for DsRed RFP. A digital camera (DXM 1200, Nikon, Tokyo, Japan) was attached to the microscope. The soft view system (ACT-1) provided by Nikon was used for image capture. Adobe Photoshop 5.0 (Adobe System Inc., San Jose, CA) was used for image processing.

Yeast Two-hybrid Screening of Proteins Interacting with
StAR Protein-A Gal4-based yeast two-hybrid system was used to identify proteins interacting with StAR protein. A plasmid expressing a GAL-StAR fusion protein, containing human N-62 StAR protein lacking 62 amino acid-terminal residues, was used to screen a human testis cDNA library in yeast strain CG-1945. Nine clones were obtained from screening 1 ϫ 10 6 clones. DNA sequence and data base analysis revealed that these clones could be divided into three groups: ␣-helix coiledcoil rod homologue (HCR, GenBank TM accession number NM 019052), rabaptin-5 (RAB5EP, accession number NM 004703), and nucleobindin 2 (NUCB2, accession number NM 005013) ( Table I). Both HCR and NUCB2 expressed the LacZ phenotype. From the expected intracellular localization of proteins encoded in the clones, we chose clone 4, which contained a 2.3-kb insert encoding a putative cytoplasmic protein, for analysis. Clone 4 contained an open reading frame of 1971 nucleotides encoding a 657-amino acid protein and a 62-nucleotide 3Ј-untranslated sequence that ended in a poly(A) ϩ tail preceded 21 nucleotides upstream by an AATAAA sequence. Other clones were not examined in this study.
Interactions of StAR Protein and Clone 4 in the Yeast Twohybrid Assay-To determine the interaction between clone 4 and StAR protein in vivo, a plasmid expressing a GAD-clone 4 fusion and a plasmid expressing GAL4-N-62-StAR and GAL4-StAR mutant fusions were examined. Transfections were also performed with the reverse combinations of clone 4 fused to GAL4 and N-62 StAR fused to GAD. The yeast transfected with StAR, and clone 4 hybrid vector expressed the LacZ phenotype, but the yeast transfected with a StAR mutant and clone 4 did not express the LacZ phenotype (Table II), confirming that N-62 StAR interacts with clone 4 in the yeast.
Direct Interaction between StAR Protein and Clone 4 in Vitro-Pull-down assays were performed to examine the direct interaction between clone 4 and StAR protein. In vitro translated clone 4 (50 l) was mixed with CBD or CBD-N-62-StAR fusion protein (100 ng) and then subjected to SDS-PAGE and autoradiography. These experiments revealed that the clone 4 translated protein interacted with CBD-N-62-StAR protein but not with CBD (Fig. 1). We therefore named the clone 4 protein (HCR) StAR-binding protein (SBP).
Interaction between SBP and StAR Protein in Cells-To confirm further interaction between SBP and StAR protein, we used two-hybrid assays in Y-1 cells, COS-1 cells, and MA-10 cells. The empty vectors, GAL4 DNA-BD (pM) and AD (pVP16), did not activate the reporter genes. Cotransfection of GAL4 DNA-BD (pM) and AD-StAR (pVP16-StAR), pM and AD-SBP (pVP16-SBP), GAL4 DNA-BD-SBP (pM-SBP) and AD (pVP16), and GAL4 DNA-BD-StAR (pM-StAR) and AD (pVP16) did not increase reporter activity. However, the GAL4DNA-BD-StAR (pM-StAR) and AD-SBP (pVP16-SBP) fusion proteins induced 100-fold greater activation of the promoter compared with the activity observed when the pM and pVP16 vectors were cotransfected into Y-1 cells. The switched domain constructs, pM-SBP and pVP16-StAR, also activated the reporter in Y-1 cells (Fig. 2A). Cotransfection with pM-StAR and pVP16-SBP

Analysis of interaction between clone 4 and a StAR
protein mutant by a yeast two-hybrid system Interaction between clone 4 and a StAR protein mutant was analyzed by using a yeast two-hybrid system. Yeast strain Y187 was transfected with GAL4-N-62-StAR hybrid expression vector and GAD-clone 4 hybrid expression vector. The yeast transfected with StAR protein and clone 4 hybrid vector expressed the LacZ phenotype, but the yeast transfected with the StAR protein mutant and clone 4 did not express the Lac phenotype. 2B) and MA-10 cells (Fig. 2C). These findings reflect the interaction between SBP and StAR protein in vivo. SBP mRNA Expression in Human Tissues-Northern blot analysis was performed to examine the expression of SBP. Northern blots that each contained 2 g of poly(A) ϩ RNA isolated from the indicated tissues were probed sequentially with SBP and ␤-actin cDNAs. Expression of the SBP gene was detected in all tissues examined. Its expression level was particularly high in the testis, where the sizes of transcripts were 2.4 and 3.8 kb (Fig. 3). To examine the expression of SBP in various cell lines, we performed RT-PCR using mRNA ex-tracted from Hep G2 cells, KGN cells, H295R cells, and MCF-7 cells. The expected amplification products (400 bp) were obtained in all of the cell lines examined (Fig. 4).
Identification of the Transcription Start Sites of SBP Gene Using Rapid Amplification of cDNA Ends (RACE)-For determination of the transcription start sites of the SBP gene, we employed a method based on anchored PCR using uncloned single-strand human testis cDNA with an anchor primer and a second primer that is specific to SBP. RACE products were electrophoresed, and DNA fragments were subcloned into PCR2 vectors for sequence analysis (Fig. 5A). Fifteen PCR

FIG. 3. Expressions of SBP mRNA in various human tissues.
Northern blots each containing 2 g of poly(A) ϩ RNA isolated from the indicated tissues were probed sequentially with SBP and ␤-actin cDNAs. The autoradiogram for the SBP probe was exposed for 48 h. The blots were exposed for 2 h for actin. products were sequenced, and the sequences were compared with the human SBP genome sequence (Fig. 5B). The SBP gene has two transcription start sites from alternative splicing and several minor transcription start sites. Although SBP has exon 1a and exon 1b, both 5Ј-sites interact with the same 3Ј-splice acceptors (Table III). The main transcription start sites are located 79 (exon 1a) and 430 bp (exon 1b) upstream of the translation start site (ATG) in exon 2. The first translation start site ATG in clone 4 is present in exon 4 in SBP in the testis. Clone 4 encodes an N-terminally truncated 656-amino acid region of the HCR protein (757 amino acids) (28) (Fig. 5C). From the RACE results, we prepared an expression plasmid (pSBP) by inserting the EcoRI fragment of the coding region of 757 amino acids amplified by PCR from testis cDNA into the pTarget vector (Promega).
Effect of SBP on Steroid Hormone Production-The SBP gene was found to be expressed in steroid hormone-producing cells, including H295R cells and KGN cells. To examine the effect of SBP on the action of StAR protein, COS-1 cells were cotransfected with F2, StAR, and SBP expression plasmids (pSBP). The amount of pregnenolone produced by COS-1 cells was increased by 138% compared with that produced by cells transfected by F2, pStAR, and an empty vector (Fig. 6).
siRNA Treatment Decreases the Production of Steroid Hormones-To examine further the role of SBP in the production of steroid hormones in cells, we performed an experiment to elim-inate SBP expression using siRNA (29) and assayed steroid hormones. Two target sequences of the SBP gene were selected for gene silencing. The effects of siRNAs were assayed by RT-PCR. SBP gene expression levels were reduced by both siRNA-SBP-I and siRNA-SBP-II. After treatment of cells transfected with the double-stranded RNAs, the amounts of pregnenolone produced by H295R cells (85 Ϯ 5.0 and 66 Ϯ 8.2 ng/dish when treated with siRNA-SBP-I and siRNA-SBP-II, respectively) were significantly less than (p Ͻ 0.05) that produced by H295R cells transfected with scramble siRNA (Fig. 7A). The amounts of pregnenolone produced by H295R cells treated with SBP-I and SBP-II siRNA were decreased by 56.5 and 37.5%, respectively, compared with the amount produced by H295R cells treated with scramble siRNA. The amounts of progesterone produced by KGN cells treated with SBP-I siRNA and SBP-II siRNA were also decreased by 71 and 55%, respectively, compared with the amount produced by KGN cells treated with scramble siRNA (Fig. 7B). Reducing the level of SBP gene expression by siRNA treatment of targeted SBP gene sequences resulted in a decrease in the production of steroid hormones.
Determination of SBP Localization in Cells-Fluorescent microscopic observations were performed to determine the subcellular localization of SBP in COS-1 cells that had been cotransfected with pSBP-GFP and pStAR-REP. Punctate cytoplasmic staining of SBP-GFR fusion protein was observed in cells. The size of punctate signals varied from one vesicle to another (Fig. 8A). StAR-REP fusion protein exhibited ovoidshaped signals in a reticulum pattern. These signal patterns are consistent with the characteristics of mitochondria (Fig.  8B). SBP-GFP fusion protein signals were partially overlapped with StAR-REP fusion protein signals (Fig. 8C). The findings indicate that SBP is localized in organelles, including endosomes and mitochondria, in the cytoplasm. DISCUSSION We screened a human testis cDNA library to identify proteins that interact with the StAR protein in the cytoplasm or outer mitochondrial membrane. Three clones, RAB5EP, NUCB2, and HCR, were identified by the screening. NUCB2, which functions as a DNA-binding protein, is a novel DNAbinding/EF-hand/leucine zipper (NEFA) (30). StAR homology domains are known as StAR-related lipid transfer (START) domains (31). START domains have been found in many kinds of proteins, including transcription factors (31). NUCB2 may be a transcription factor that interacts with the START domain that StAR-like transcription factors have. Rab5, which is a protein belonging to the family of Rab GTPases, regulates sequential transport steps along the endocytic and recycling pathway on early endosomes in cells (32). The secondary structure of SBP was predicted to consist of ␣-helical coils, which are features of the cytoskeleton, and it was therefore named ␣-helical coiled-coil rod homologue (HCR) (28). A BLAST search revealed that the amino acid sequence of SBP has little homology with those of known proteins. SBP did not interact with a StAR protein mutant that lacked the biologically active C terminus of the StAR protein (33). Thus, SBP protein interacts with the START domain of the C terminus of the StAR protein in cells. In mammalian two-hybrid assays, the interaction between StAR protein and SBP in cells transfected with plasmids of reverse orientation (pM-SBP and pVP16-N-62-StAR) was less than that in cells cotransfected with the expression vectors (pM-N-62-StAR and pVP16-SBP). These fusion protein structures may restrict interaction between SBP and StAR protein in cells.
SBP was found to be expressed in all tissues and in all cell lines examined, including steroid-producing cells. However, the expression level was not high except in the testis. After cotransfection with F2, pStAR, and pSBP, the activity of steroidogenesis was increased by 138%. The results of siRNA experiments also support the function of SBP, which have the effect of steroidogenesis in cells. Although the function of the larger transcription of SBP is not known, RACE results showed that SBP has several transcriptions start sites. Alternative transcription of the 5Ј-untranslated region can be regulated in a tissue by alternative usage of promoters. SBP may have a larger translated protein that acts as a pro-protein of SBP or may translate another protein that has a different function in the testis. Although many two-hybrid libraries contain partial rather than full-length cDNAs, small cDNA molecules may improve the sensitivity of the selection process (34). Clone 4 encodes an N-terminally truncated 656-amino acid region of SBP. Although clone 4 is supposed to be a partial cDNA, truncated SBP may function in the testis. Recently, StarD4, StarD5, and StarD6 proteins, which each contain a START domain, have been identified using cDNA microarrays. StarD4 and StarD5 are ubiquitously expressed, whereas StarD6 expression is limited to the testis. These proteins are thought to function in the intracellular shuttling of sterols or other lipids (35). It is possible that StAR protein and SBP not only function in steroidogenesis but also function together for the transport of cholesterol to regulate cellular metabolic processes, because StAR protein is present not only in Leydig cells but also in Sertoli cells in the testis (36).
Although steroidogenesis requires the continuous synthesis of new StAR proteins and is associated with the 37-kDa preprotein (37), an N-62-StAR recombinant protein stimulates the transfer of cholesterol from sterol-rich liposomes to mitochondria (38). Based on its physical characterization, StAR protein changes its conformation into a molten globule to associate  7. siRNA treatment decreased the production of steroid hormones in cells. Endogenous SBP gene expression was partially eliminated in H295R cells (A) and KGN cells (B) by transfection of a 19-nucleotide RNA duplex (siRNA-SBP-I) and 21-nucleotide RNA duplex (siRNA-SBP-II) directed to the SBP sequence. As a control, scramble sequences were introduced into the cells. The media were collected 48 h after treatment, and the amounts of pregnenolone (A) and progesterone were determined by radioimmunoassays. Values presented are means Ϯ S.E. of concentrations in the media in experiments in which each treatment group contained three replicate cultures. The effects of siRNAs were assayed by reversed RT-PCR for SBP (400 bp) and GAPDH gene (231 bp). * and **, significant difference in pregnenolone or progesterone production compared with that by cells transfected with scramble siRNA and that by cells treated with siRNA-SBP-I and siRNA-SBP-II; p Ͻ 0.05; p Ͻ 0.01. Each experiment was replicated on three separate occasions.
with the outer mitochondrial membrane and causes the transfer of cholesterol to the inner mitochondrial membrane (39 -41). These findings suggest that StAR protein functional sites of action are outside mitochondria and transfer cholesterol from the outer membrane to the inner membrane. The facility of N-62 StAR protein to transport cholesterol between membranes in a reconstitute system has been reported to be 1.8 molecules of cholesterol/molecule of the StAR protein (41). The facility in a reconstitute system is smaller than that in Y-1 cells, in which StAR protein is able to transfer up to 400 molecules of cholesterol (37). It is likely that another protein in the cytoplasm is required for effective cholesterol transport. The peripheral-type benzodiazepine receptor (PBR) has been shown to function in steroidogenesis, mitochondrial respiration, apoptosis, and cell proliferation (42,43). Because the PBR has been found in the outer mitochondrial membrane, where StAR protein is thought to act (42), StAR protein and SBP may work together in steroidogenesis with supporting functions of PBR. Alternatively, SBP may bind StAR protein and sustain StAR protein outside of the mitochondrial membrane to stimulate steroid hormone production, because the function of StAR protein on the outer mitochondrial membrane is regulated by its rate of import into mitochondria (44).
Lipid droplets in the cytoplasm, in which steroidogenic cholesterol is stored, are tightly attached to intermediate filaments. Mitochondria are also attached to intermediate filaments (45). The transport of cholesterol to mitochondria is associated with filaments and regulates steroidogenesis (46). MLN64, which resides in late endosomes and lysosomes, is proteolytically cleaved and includes START domains (47)(48)(49)(50). MLN64 has been reported to associate with the mobilization of low density lipoprotein-derived cholesterol through late endosomes to mitochondria. Observation under a confocal microscope revealed that MLN64 in late endosomal tubules and StAR protein in the microtubule-dependent mitochondrial matrix exist in close proximity but are not completely overlapped in cells (51). We have shown that signals of SBP, which is localized in organelles, including endosomes and mitochondria, in the cytoplasm are overlapped with StAR protein signals. SBP may interact with StAR protein in the cytoplasm. SBP has important functions with StAR protein in cells: one is steroidogenesis, as is well known, and the others are unknown. Further studies, including studies using mouse knockout models, are needed to determine the functions of SBP.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Creating genetically modified pigs by using nuclear transfer

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture.

Introduction
Pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs. Despite the ease with which transgenic pigs can be generated this technique has limitations. The DNA integrates randomly and potentially in multiple copies. In addition, the random site of integration limits the ability to control expression in the desired tissues or at the appropriate level. Moreover, the animal's endogenous genes cannot be specifically altered by using this technique.
Successful nuclear transfer (NT) of cultured cells, which was first demonstrated in cattle [1], has provided an alternative for obtaining genetically modified pigs. McCreath et al. [2] reported the first success of obtaining gene-targeting sheep by using gene-targeted fibroblasts as a source of donor nuclei for NT. Although NT of pigs once lagged behind that in mice, cattle and sheep, since the first piglet from somatic cell NT was reported in 2000 [3], tremendous progress has been made. Successful production of pigs resulting from random genetic modification in vitro followed by NT [4][5][6], as well as those with a specific modification (knock out) have been reported [7][8][9][10] by several groups in a short period. The production of cloned transgenic pigs is now in the transition from investigation to practical application. In this paper, we discuss the present status of production efficiency of transgenic pigs by NT; as well as the problems and offer a few perspectives.

History of pig cloning
The first successful cloning experiment in pigs was reported as early as in 1989. Prather et al., [11] used blastomeres from 4-cell stage embryos as donor nuclei and in vivo-derived metaphase II oocytes as recipient cytoblasts. A total of 88 NT embryos were transferred to recipient gilts for continued development. A single piglet was born. Similar success was not reported with embryonic cells until more than 10 years later [12]. With a similar NT technique that produced Dolly -in which a cultured differentiated somatic cell is fused with a mature egg whose genetic material has been removed [13], successful cloning of pigs was not reported until 3 years later [3,14]. Pigs are physiologically similar to humans and so there has been intense interest in using genetically modified pigs as organ donors for transplantation to humans, as well as models of human disease.
In 2000, the first somatic cell cloning success was Polejaeva et al. [3] who announced the birth of five healthy cloned piglets. These animals were produced via a different technology from that generally used for NT. The authors first fused porcine granulosa-derived donor cells to enucleated mature oocytes. After 18 hours, the donor nucleus was removed from the first oocyte and transferred to the cytoplasm of a fertilized egg. The investigators adopted this double NT strategy because they surmised that in the original one-step method, the activation stimulus provided after NT was insufficient to support fullterm development of the embryo. This report lead the pig cloners to think that the procedures of pig cloning might be more complicated and difficult than other animals. However, almost at the same time, Onishi et al. [14] reported the birth of a live cloned piglet, by directly injecting porcine fetal fibroblast donor nuclei into enucleated oocytes with piezo-actuated microinjection. The significance of Onishi's success is that they proved that two-step NT is unnecessary to make somatic NT pig. Both groups used mature oocytes collected directly from female pigs rather than culturing immature oocytes in vitro. Matured oocytes are needed in large numbers and in vivo matured oocytes are very expensive to acquire. Thus many have chosen to use in vitro matured oocytes. Immature oocytes are derived from ovaries obtained from the slaughterhouse and subsequently matured in vitro. Betthauser et al. [15] had systematically optimized each step in the NT procedure, including the source of oocytes and their maturation in vitro, the culture of donor cells, the activation of oocytes following NT, and the in vitro culture of embryos and their transfer to recipient gilts. The result is a more reproducible methodology that enables strategies to genetically modify pigs.
Park et al. [4] reported the first pigs produced by genetically modifying the fibroblast cells prior to NT. This was followed by Lai et al. [7] showing the first pigs with a gene knocked out. Homologous recombination was used to disrupt the sequence of the α-1, 3-galactosyltransferase (GGTA1) gene in mini-pig fibroblasts. The targeted fibroblast nuclei were transferred into enucleated in vitro matured oocytes. Four live GGTA1 knockout pigs were successfully produced. An important practical significance of this experiment is to facilitate studies in xenotransplantation of pig organs to humans, as elimination of terminal α-1, 3-galactosyl epitopes from the pig is expected to be a solution to the problem of hyperacute and delayed vascular rejection.

Unique aspects of pig cloning technology
The efficiency of somatic cell NT, when measured as development to term as a proportion of oocytes used, has been very low (1-2%). A number of variables influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to NT, the method of artificial oocyte activation, embryo culture, possible loss of somatic imprinting in the nuclei of reconstructed embryos, failure of reprogramming the transplanted nucleus adequately, and the techniques employed for NT. In the pig, there is an additional difficulty that at least 4 good embryos are required to induce and maintain pregnancy. Therefore, to increase the chance of producing offspring efforts to minimize the inefficiencies at each step of NT procedure must be made. Some of the variables are discussed below.

Selection of cell types used to produce cloned pigs
In mice, at least eight types of fetal and adult origin from males and females and different genetic backgrounds have been tested. Live offspring were obtained with similar efficiency only with fibroblast, undefined fetal gonad and cumulus cells [16]. Many cell types (macrophages, spleen, brain and mature Sertoli cells) repeatedly failed to develop after implantation. In cattle, at least fifteen somatic cell donors of fetal, newborn and adult origin from males and female and different genetic backgrounds were tested [17] and all supported development in vitro, and live offspring were obtained from cumulus, oviduct, skin and liver cells. Ten different somatic cell types have proven complete cloning-competence so far. In pigs, fibroblast and cumulus cells have been clonable. The use of isolated precursor cells of adipocytes from the subcutaneous adipose tissue of adult pigs for NT [18] have also has been documented.

Treatment of donor cells
The use of cultured cell populations for production of animals by NT is now well documented in a number of species. Analysis of these cells has shown considerable variation in development between individual cell populations and at present has provided no definitive method for identification of cell populations that are best suited for NT. Factors that are thought to influence the suitability include the effects of oxidative damage associated with metabolism, genome instability and chromosomal pathologies. All of these factors may be influenced by the method of isolation and culture, and the number of population doublings in culture. Even different subclones of fibroblasts derived from same fetus and cultured in same conditions to same generation lead to different in vitro developmental potential of reconstructed embryos [19]. Another factor affecting the efficiency of NT is the cell cycle phase of donor cells, which is still a topic being debated in the NT field. Wilmut et al. [13] stated that the donor cells for NT must be in G0 of the cell cycle (quiescent phase), while Cibelli et al. [20] showed that cycling cells, which contain cells in different cycle stages, could be successfully used for NT in cattle. Cells in G2/M stage of the cell cycle may be another option as these have been used as nuclear donors to produce cloned mice [21], a pig [6], and sheep [22].

Possible donor cells for genetic modification
Cells to be used as a source of donor nuclei for the production of genetically modified pigs must meet two criteria: 1) they must be able to direct term development, and 2) they should possess a proliferative ability such that correct DNA modification can be selected. Fetal-derived fibroblasts are a popular choice of cells to begin studies as they are capable of extensive proliferation.
The problem with all somatic cells is that they tend to become senescent before sufficient rounds of gene transfer and/or targeting and selection can be performed. This problem may be overcome by isolation of readily transfectable and selectable cells with high proliferative potential and long-term karyotypical normalcy, similar to murine ES and EG cell lines. Primordial germ cell-derived lines have been isolated from pig fetuses, and transfected lines have been shown to contribute to chimera formation when injected into pig blastocysts [23,24], but in no case has germ line transmission been demonstrated. Thus further development is needed to create cells that are developmentally competent and able to proliferate indefinitely in vitro.

Construction of nuclear transfer embryos
Oocytes must be enucleated for use in NT. One of the common chemicals used to facilitate enucleation by permitting visualization of the chromosomes is bisbenzimide. Unfortunately, exposure to this compound has deleterious effects on the development of pig oocytes to the blastocyst stage. Since the condensed chromosomes are always located in cytoplasm underneath the first polar body, enucleation of in vitro matured metaphase II oocytes can be performed by aspirating the first polar body and adjacent cytoplasm without staining the chromatin. By using this "blind enucleation" method the enucleation rate varies between 85% and 90% [25].
There are two approaches to put the donor nuclei into the cytoplasts; one is direct injection of donor cells into enucleated oocytes; while the second approach is to inject the intact donor cell into the perivitelline space and subsequently fuse the donor cell with the recipient oocyte by electrical pulses. With direct microinjection, the plasma membrane and much of the cytoplasmic material of the donor cell is not transferred. In contrast, with cell fusion, all of the components of the donor cell (nuclear, cytoplasmic and plasma membrane) merge with the enucleated oocyte.
After the donor nuclei are transferred into the enucleated oocytes, the reconstructed embryos must be activated to initiate subsequent development. Activation of oocytes can be induced artificially by a variety of physical and chemical agents. Miyoshi et al. [26] found that a delay of 3 hours between fusion and activation improved the rate of blastocyst formation. The rate of development to the blastocyst stage after NT has been as low as 3%. When presumptive G0 fibroblast cells were transferred to oocytes and electrically activated, only 7% formed blastocysts. Betthauser et al. [15] reported 4-8% blastocysts resulting from NT that were activated by ionophore followed by 6dimethylaminourine. Recently, Kühholzer et al. [19] reported almost 20% blastocysts from electrically activated oocytes. Combined thimerosal/DTT treatment of the oocytes also could effectively activate porcine oocytes and 42.0% of the oocytes developed to the compact morula or blastocyst stage in vitro culture [27].

Embryo transfer
In the pig pregnancy recognition by the surrogate requires a signal from four or more embryos around day 12 of gestation [28]. To minimize any adverse effect on the in vitro conditions on the development of NT embryos transfer to the surrogate is generally at a very early stage. Since the NT embryos are generally of a low quality a large number are transferred into a single surrogate.
If not enough NT embryos are available for transfer, then two different strategies might be employed. The first is to co-transfer "helper embryos" as an aid to inducing and maintaining pregnancy. These "helper embryos" maybe parthenogenetic embryos that are capable of establishing a pregnancy but degenerate by day 30 of gestation [6,29] because of genomic imprinting. Alternatively the helper embryos might be derived from a normal mating [7]. Finally, administration of estradiol, the normal signal for maternal recognition of pregnancy, on day 12 can maintain the pregnancy of small litters [6,11]. Although it is difficult to document that any of these strategies were beneficial, they did not appear to hinder development.

Applications of pig cloning in genetic modification
Genetic modification in swine could have many agricultural and medical applications. In the agricultural field, modification of the genome could (1) alter the carcass composition such that it is a healthier product, (2) produce pork faster or more efficiently, (3) create animals that are resistant to specific diseases, (4) reduce the major losses normally observed during the first month of swine embryogenesis, and (5) create animals that are more environmentally friendly.
In the medical field, making specific genetic modifications in the pig provides the possibility of producing recombinant products in animals for biomedical or nutraceutical uses and the possibility of producing models of human genetic disease for research and drug development. Somatic NT could play important roles in genetic modification by the following three methods.

Improving generation of transgenic pigs
Transgenic technology developed to add genes has been widely applied to livestock species because it is technically simple, but inefficient. Not all injected eggs will develop into transgenic pigs, and then not all transgenic pigs will express the transgene in the desired manner. Somatic NT would allow more efficient generation of transgenic animals. Foreign DNA could be introduced into cell lines in culture, and cells containing the transgene in the right configuration could be grown up. The expression level can be detected in individual cells, which could be determined by the addition of a reporter gene with a target gene. The cells with high level foreign gene expression would be selected and used as a source of nuclei for transfer, ensuring that all offspring are transgenic and have high level expression. We added a gene for the enhanced green fluorescent protein (EGFP) to a fetal-derived cell line by using a replication-defective retrovirus [4]. These cells were then used as donors in a NT scheme that used oocytes that had been matured in a defined system. The genetically marked tissues (EGFP) from the pigs produced from these cells will likely be very useful for basic research where such marked cells are required. Our laboratory is looking forward to the possibility of EGFP expressing sperm, oocytes and embryos for our studies on fertilization and embryo development.

Improving propagation of transgenic pigs
Nuclear transfer would speed up the expansion of a successful transgenic line by using skin cells of the transgenic pigs to make more clones. Ear skin fibroblasts from a transgenic pig produced by oocyte transduction [30] and expressing eGFP were isolated and used as donor nuclei for NT. Four live cloned pigs were born. As in the nuclear donor pig, all of the offspring expressed the eGFP in sim-ilar tissues [31]. Bondioli et al. [32] (2001) produced cloned pigs from cultured skin fibroblasts derived from an H-transferase transgenic boar. The cells used in these studies were subjected to an extensive culture time, unsuccessful transformation, freezing and thawing, and clonal expansion from single cells prior to NT. One 90 day fetus and two healthy piglets resulted from NT by fusion of these fibroblasts with enucleated oocytes.

Generating Targeted Gene Alterations
The most powerful technology for genetic manipulation in mammals -gene targeting -was developed in mice, and depends on the ability of mammalian DNA, when added to cells in culture, to recombine homologously with nearly identical DNA sequences in the genome and replace it. In mice, specific mutations can be generated in cultured embryonic stem (ES) cells. For more than a decade, researchers have searched for ES cells from livestock, including pigs. But although some success has been reported, none has passed the crucial test of contributing to the germ cells. Nuclear transfer from non-pluripotent cell lines provides an alternative to the ES cell route for introduction of targeted gene alterations into the germ line. The first such example is that of knocking out a gene that is responsible for hyperacute rejection (HAR) when organs from swine are transferred to primates.
As described above, pig somatic NT has been established well enough to make live cloned pigs. To generate targeted gene alterations in pigs, creation of gene targeted somatic cell lines becomes another critical issue to be addressed.
Generally, fibroblasts cannot proliferate indefinitely in vitro. Senescence of primary fibroblasts in livestock is generally seen following approximately 30 populations doubling ex vivo in non-clonal cultures. In pig fetal fibroblasts, clonal isolates are generally lost at passage following 24-28 population doublings ex vivo. This presents a technical hurdle as compared to mouse ES cells. However, this problem can be overcome to some extent by using a gene trap strategy which may result in a higher targeting rate [7]. To target the GGTA1 locus a long region of homology to the GGTA1 locus was used in a vector constructed from the same inbred line of miniature pig from which the fetal fibroblasts were derived, thus providing for isogeneticity to the target locus. A stop codon and selection cassette was inserted into exon 9, upstream of the catalytic domain of the protein. The selection cassette contained no promoter and was preceded by an internal ribosome entry site. Gene trap designs of this nature have been shown to result in relatively high targeting rates, as the vast majority of non-targeted recombination events do not result in transcription. Additionally, transient expression of the selection cassette cannot occur.
In addition, transfecting the cells as early as possible after isolation (passage 2, or about 6 population doublings) proved to be another helpful measure to overcome the hurdle [7]. RT-PCR can be performed on crude cell lysates the day following transfection to quickly identify potentially targeted clones. In our experiments, an absolute targeting of 8 × 10 -7 was obtained, which is similar to that in mouse ES targeting (1 × 10 -6 ) [7], producing enough cells to be used as donor nuclei. However, it is unlikely we could achieve two targeting events in the one primary cell line and still maintain enough cells for NT. Rather, removal of the second allele would be accomplished by breeding or producing a cloned fetus, or piglet, thus providing cells which can be used for a second round of targeting and NT. To produce homozygous GGTA1 knockout piglets by natural breeding, assuming both male and female heterozygous knockout pigs are available at the same time and are fertile, is feasible but takes up to 12 months. However, by using a second-round knockout and cloning strategy, we could save up to 6 months and all cloned piglets would be GGTA1 double knockout (DKO). One group has chosen to use second-round knockout and cloning strategy to produce homozygous GGTA1 knockout piglets and successfully obtained five galactosyltransferase-deficient pigs [33]. Our group has also produced a GGTA1 DKO by selecting cells that do not express GGTA1 originally isolated from a heterozygous knockout pig (manuscript in preparation).
In conclusion, the techniques of NT in pigs, while developed, are not efficient. Nevertheless, the possibility of making specific genetic modifications to pigs offers great potential to both medicine and agriculture.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
2-Arachidonoylglycerol Induces the Migration of HL-60 Cells Differentiated into Macrophage-like Cells and Human Peripheral Blood Monocytes through the Cannabinoid CB2 Receptor-dependent Mechanism*

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2) and has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo . Recently, we proposed that 2-arachido-noylglycerol is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB2) are primarily 2-arachidonoylglycerol receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of 2-arachidonoylglycerol on the motility of HL-60 cells differentiated into macrophage-like cells. We found that 2-arachidonoylglycerol induces the migration of differentiated HL-60 cells. The migration induced by 2-arachido-noylglycerol was blocked by treatment of the cells with either SR144528, a CB2 receptor antagonist, or pertussis toxin, suggesting that the CB2 receptor and G i /G o are involved in the 2-arachidonoylglycerol-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to anandamide, another cannabinoid failed to the The 2-arachidonoylglycerol-induced was also for two other types of macrophage-like cells, the cells and THP-1 cells, as well as peripheral blood monocytes. These results strongly suggest that 2-arachidonoylglycerol induces the migration of several types of leukocytes such as macrophages/monocytes through a CB2 receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses.

From the Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan 2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2) and has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we proposed that 2-arachidonoylglycerol is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB2) are primarily 2-arachidonoylglycerol receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of 2-arachidonoylglycerol on the motility of HL-60 cells differentiated into macrophage-like cells. We found that 2-arachidonoylglycerol induces the migration of differentiated HL-60 cells. The migration induced by 2-arachidonoylglycerol was blocked by treatment of the cells with either SR144528, a CB2 receptor antagonist, or pertussis toxin, suggesting that the CB2 receptor and G i /G o are involved in the 2-arachidonoylglycerol-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, failed to induce the migration. The 2-arachidonoylglycerol-induced migration was also observed for two other types of macrophage-like cells, the U937 cells and THP-1 cells, as well as human peripheral blood monocytes. These results strongly suggest that 2-arachidonoylglycerol induces the migration of several types of leukocytes such as macrophages/monocytes through a CB2 receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses.
⌬ 9 -Tetrahydrocannabinol (⌬ 9 -THC) 1 is a major psychoactive constituent of marijuana and is known to exert a variety of biological effects in experimental animals and human such as altered perception, inhibition of memory, immobility, analgesia, and the inhibition of immune response, although the mechanism of these actions of ⌬ 9 -THC remained elusive until the late 1980's. In 1988, Devane et al. (1) demonstrated the presence of a specific binding site for cannabinoids in rat brain synaptosomes. Later, Matsuda et al. (2) and Munro et al. (3) cloned the cDNAs for the cannabinoid receptors (CB1 and CB2). It has been assumed that the diverse actions of the cannabinoids are mediated in a large part through these receptors. In 1992, Devane et al. (4) isolated N-arachidonoylethanolamine (anandamide) from pig brain as an endogenous cannabinoid receptor ligand. This compound has been shown to exhibit various cannabimimetic activities in vitro and in vivo (5)(6)(7)(8). However, the levels of anandamide in various living tissues were very low (9,10). Furthermore, anandamide was found to act as a partial agonist at the cannabinoid receptors (10). These observations strongly suggested the existence of another endogenous ligand in mammalian tissues.
In 1995, we (11) and Mechoulam et al. (12) reported that 2-arachidonoylglycerol (2-AG) is the second endogenous ligand for the cannabinoid receptors. 2-AG has been shown to exhibit a strong binding activity toward the cannabinoid receptors (11,12) and a variety of cannabimimetic activities (5-8, 10, 13, 14). Importantly, 2-AG was found to act as a full agonist at the cannabinoid receptors (15)(16)(17)(18)(19). Moreover, 2-AG can be rapidly formed from arachidonic acid-containing phospholipids, such as inositol phospholipids, through the combined actions of phospholipase C and diacylglycerol lipase or the combined actions of phospholipase A 1 and phospholipase C in various types of tissues and cells upon stimulation (20 -26). Noticeably, the levels of 2-AG in various mammalian tissues are markedly higher than that of anandamide. Based on these results, we proposed that 2-AG, and not anandamide, is the intrinsic natural ligand for the cannabinoid receptors (15,16,27).
Despite their potential physiological and pathophysiological importance, the exact functions of the CB1 and CB2 receptors and their endogenous ligand 2-AG have not yet been fully elucidated. As for the CB1 receptor, several lines of evidence strongly suggested that 2-AG suppresses the neurotransmission through acting at the CB1 receptor expressed predominantly in the presynapse (10,13,14). It is becoming evident that 2-AG is a novel type of neuromodulator of profound physiological significance. On the other hand, the functions of the CB2 receptor, which is abundantly expressed in the immune system, still remain an enigma. Little is known concerning the biological activities of 2-AG toward inflammatory cells and immune competent cells. It is essential to investigate in detail the functions of the CB2 receptor and 2-AG to better under-stand the precise regulatory mechanisms of inflammatory reactions and immune responses.
In this study, we investigated the possible biological activity of 2-AG toward HL-60 cells differentiated into macrophage-like cells. We found that 2-AG induces the migration of differentiated HL-60 cells through a cannabinoid CB2 receptor-dependent mechanism. A similar effect was also observed with human monocytes. The physiological and pathophysiological meanings of the 2-AG-induced migration of macrophages/monocytes are discussed.
Cells-Human promyelocytic leukemia HL-60 cells, human monocytic leukemia U937 cells, and THP-1 cells were grown at 37°C in RPMI 1640 medium (Asahi Technoglass Co., Chiba, Japan) supplemented with 10% fetal bovine serum in an atmosphere of 95% air and 5% CO 2 . HL-60 cells were differentiated into macrophage-like cells by treatment with 100 nM 1,25-(OH) 2 vitamin D 3 for 5 days. U937 cells and THP-1 cells were also differentiated by treatment with 100 nM 1,25-(OH) 2 vitamin D 3 for 5 days. Human monocytes were separated from the peripheral blood of young healthy donors as follows: 1 ⁄4 volume of 6% dextran T-500 (Amersham Biosciences) in saline was added to heparinized blood to sediment the erythrocytes, the supernatant (leukocyterich fraction) was aspirated and centrifuged at 400 ϫ g for 10 min, the cells were washed once with Ca 2ϩ , Mg 2ϩ -free Hanks' balanced salt solution containing 5 mM HEPES (pH 7.4), and then the cells were transferred onto Lymphoprep TM (Axis Shield, Oslo, Norway) and centrifuged at 800 ϫ g for 20 min. The mononuclear leukocyte fraction (the interface layer) was collected and washed with Hanks' balanced salt solution. Monocytes were separated from other mononuclear leukocytes by negative selection using a MACS monocyte isolation kit (Miltenyi Biotec Gmbh, Gladbach, Germany). The purity of the monocytes was 93% as assessed by a nonspecific esterase assay described below.
Migration Assay-The migration of the differentiated HL-60 cells, U937 cells, THP-1 cells, and human monocytes was assayed using Transwell TM inserts (pore size, 5 m) and 24-well culture plates (Corning Costar, Cambridge, MA). Briefly, the cells (10 6 for the differentiated HL-60 cells, U937 cells, and THP-1 cells and 2.5 ϫ 10 5 for human monocytes) suspended in 0.1 ml of RPMI 1640 medium containing 0.1% bovine serum albumin were transferred to the Transwell TM insert (the upper compartment). 2-AG was dissolved in Me 2 SO and added to 0.6 ml of the RPMI 1640 medium containing 0.1% bovine serum albumin in the well of the culture plate (the lower compartment) (the final concentration of Me 2 SO was 0.2%). After the incubation at 37°C (for 4 h for HL-60 cells, U937 cells, and THP-1 cells and 2 h for human monocytes) in an atmosphere of 95% air and 5% CO 2 , the number of cells that migrated from the upper compartment to the lower compartment was counted using a hemocytometer.
NBT Reduction Assay-The NBT reduction assay was performed as described previously (28) with some modifications. Cells were suspended in 25 mM HEPES-Tyrode's solution (pH 7.4) containing 0.05% NBT and incubated at 37°C for 7 min. Phorbol 12-myristate 13-acetate was added to the cell suspension at a concentration of 4 M, and the suspension was incubated for 30 min at 37°C. After the addition of 10 mM EDTA to stop the reaction, the percentage of positive cells (bluestained cells) was determined using a hemocytometer.
Estimation of the Amount of Remaining 2-AG Following the Incubation with the Cells-HL-60 cells differentiated into macrophage-like cells (4 ϫ 10 6 ) were suspended in 0.4 ml of RPMI 1640 medium. The cells were then incubated with 1 M 2-AG for 30 min, 1 h, 2 h, and 4 h. Following the incubation, the supernatant was aspirated, and the lipids were extracted by the method of Bligh and Dyer (29). Butylated hydroxytoluene (final, 0.05%) was added to avoid lipid peroxidation, and 2-heptadecanoylglycerol was added as an internal standard. The lipids were fractionated by TLC using development with petroleum ether: diethyl ether:acetic acid (20:80:1, v/v) in a tank sealed with N 2 gas. The area corresponding to standard monoacylglycerol was scraped off the TLC plate, followed by extraction from the silica gel by the method of Bligh and Dyer (29). The extraction was conducted in the presence of butylated hydroxytoluene (0.001%) in an N 2 gas-sealed tube. The purified monoacylglycerols were converted to their 1-anthroyl derivatives and then analyzed with a high pressure liquid chromatography system equipped with a reverse phase column (CAPCELL PAK C18 SG120, 4.6 mm ϫ 250 mm; Shiseido Co., Tokyo, Japan) and a fluorescence detector (excitation at 370 nm, emission at 470 nm). The mobile phase was acetonitrile:2-propanol:water (90:4:6, v/v), and the flow rate was 1.4 ml/min as described previously (30).
Statistical Analysis-Statistical analysis was performed using the Student's t test.

RESULTS
1,25-(OH) 2 vitamin D 3 is known to induce the differentiation of HL-60 cells into macrophage-like cells (31). We first examined the effect of 1,25-(OH) 2 vitamin D 3 -treatment on several cellular markers of differentiation and the CB2 receptor mRNA level in HL-60 cells. As shown in Fig. 1A, the percentage of NBT reduction assay-positive cells in undifferentiated HL-60 cells was low (10.0%). On the other hand, the proportion of NBT reduction assay-positive cells was elevated to 65.7% following the differentiation with 1,25-(OH) 2 vitamin D 3 . A similar result was observed for nonspecific esterase-positive cells (data not shown). We next examined whether differentiated HL-60 cells actually possess the CB2 receptor mRNA. As shown in Fig. 1B, differentiated HL-60 cells were found to contain a substantial amount of the CB2 receptor mRNA. The ratio of the CB2 receptor mRNA/GAPDH mRNA in the differentiated HL-60 cells was almost the same as that in the undifferentiated HL-60 cells (the ratio of the CB2 receptor mRNA/GAPDH mRNA in the differentiated cells was 0.9 of that in the undifferentiated cells).
We then investigated the effect of 2-AG on the motility of HL-60 cells. The addition of 1 M 2-AG enhanced the migration of undifferentiated HL-60 cells to some extent, although the number of migrated cells was low; the percentages of the migrated cells were 0.05 and 0.30% for the control and 2-AGstimulated cells, respectively. On the other hand, 1 M 2-AG exerted dramatic effects on the migration of the HL-60 cells differentiated into macrophage-like cells by treatment with 1,25-(OH) 2 vitamin D 3 for 5 days. The proportion of migrated cells was 3.4% for the control and 26.7% for the 2-AG-stimulated cells.
The number of migrated HL-60 cells differentiated into macrophage-like cells was augmented with time (FIG. 2A). The addition of 1 M 2-AG markedly accelerated the migration (Fig.  2A). The number of migrated cells also increased dose-dependently (Fig. 2B). The effect was observed from 10 nM 2-AG and reached a peak at 10 M. The EC 50 was around 430 nM.
We next examined whether the cannabinoid receptors are involved in the 2-AG-induced migration of differentiated HL-60 cells. We found that the addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells markedly reduced the migration induced by 2-AG (Fig. 3A). On the other hand, the treatment of the cells with SR141716A, a cannabinoid CB1 receptor-specific antagonist, exerted only a slight effect on the 2-AG-evoked migration. These results indicate that the 2-AGinduced migration is mediated mainly via the CB2 receptor. The effect of PTX treatment on the 2-AG-induced migration of the differentiated HL-60 cells was next examined. As shown in Fig. 3B We then compared the activities of the various cannabinoid receptor ligands to induce migration. As shown in Fig. 5, the activity of 2-AG (1 M) was the highest among those of the various cannabinoid receptor ligands examined in the present study. 2-AG ether (1 M), a metabolically stable ether-linked analog of 2-AG, exhibited appreciable migration-inducing activity, although its activity was much lower than that of 2-AG. CP55940 (1 M) and WIN55212-2 (1 M) were also found to possess weak activities. However, anandamide (1 M) was almost totally inactive.
We then examined whether the 2-AG-induced migration is because of chemotaxis (the directional movement along a concentration gradient) or chemokinesis (stimulated movement in no specific direction). The migration of the cells from the upper compartment to the lower compartment in the absence of 2-AG was 4.4% (Fig. 7A). The proportion of migrated cells was elevated to 21.5% when 2-AG (1 M) was added to the lower compartment. The migration was slightly reduced when 2-AG (1 M) was added to both the upper compartment (with cells) and the lower compartment (18.5%). On the other hand, the

2-Arachidonoylglycerol-induced Cell Migration
presence of 2-AG (1 M) in the upper compartment alone (with cells) did not evoke cell migration (5.2%). Because 2-AG is known to be rapidly metabolized by a variety of cells (10, 13), we examined whether exogenously added 2-AG exists as an intact molecule following the co-incubation with the cells. We found that more than 99% of the exogenously added 2-AG (1 M) was metabolized within 30 min of incubation. Thus, it is rather difficult to determine whether the migration is because of chemotaxis or chemokinesis under the present experimental conditions using 2-AG as a stimulant. To settle this problem, we added 2-AG ether, a metabolically stable analog of 2-AG, instead of 2-AG (Fig. 7B). We found that the percentage of migrated cells when 2-AG ether (1 M) was added to both the upper compartment (with cells) and the lower compartment (5.8%) was significantly lower than that observed when 2-AG ether (1 M) was added only to the lower compartment (8.3%), the proportion of migrated cells in the former case being close to the level of the control (4.6%). We also found that the addi-tion of 2-AG ether (1 M) to the upper compartment alone (with cells) did not enhance the migration of cells from the upper compartment to the lower compartment (4.9%). These results strongly suggest that 2-AG ether elicited mainly chemotaxis rather than chemokinesis.
We next investigated whether 2-AG induces the migration of other types of macrophage-like cells. In this study, we employed two types of human monocytic leukemic cells, U937 cells and THP-1 cells, which were differentiated by treatment with 1,25-(OH) 2 vitamin D 3 before use as in the case of the HL-60 cells. We found that 2-AG (1 M) significantly enhanced the migration of the differentiated U937 cells and THP-1 cells (Fig.   FIG. 4. 8), although the magnitude of augmentation was rather small compared with the case of the differentiated HL-60 cells.

2-Arachidonoylglycerol-induced Cell Migration
Finally, we examined whether human peripheral blood monocytes respond to 2-AG. As shown in Fig. 9, 2-AG (1 M) markedly accelerated the migration of human monocytes. The effect of 2-AG was abolished by treatment of the cells with SR144528 (1 M), a CB2 receptor-specific antagonist, as in the case of differentiated HL-60 cells.

DISCUSSION
The cannabinoid CB2 receptor is a seven transmembrane, G protein-coupled receptor and is expressed abundantly in various types of inflammatory cells and immune competent cells such as macrophages, natural killer cells, and B lymphocytes (32)(33)(34). Previously (16), we examined in detail the structureactivity relationship of a number of CB2 receptor ligands using HL-60 cells, which express the CB2 receptor and exhibit a Ca 2ϩ response when challenged with the CB2 receptor ligands. We found that the structure of 2-AG is strictly recognized by the CB2 receptor (16). The agonistic activity of 2-AG was most potent among various structural analogs. Noticeably, 2-AG acted as a full agonist at the CB2 receptor whereas anandamide acted as a weak partial agonist. Gonsiorek et al. (18) also demonstrated that 2-AG is a full agonist, and anandamide is a partial agonist using the membrane fraction of Sf9 cells transfected with the human CB2 receptor cDNA. We proposed that 2-AG, but not anandamide, is the intrinsic natural ligand for the cannabinoid CB2 receptor, and the CB2 receptor is primarily a 2-AG receptor (16).
Not much information is currently available concerning the biological activities of 2-AG toward inflammatory cells and immune competent cells. Previously, Kaminski and co-workers (35) reported that 2-AG affects lymphocyte proliferation. They also demonstrated that 2-AG suppresses the interleukin 2 gene expression in murine T lymphocytes through down-regulation of the nuclear factor (36). In addition, Chang et al. (37) demonstrated recently that 2-AG inhibited the production of interleukin 6 in J774 macrophage-like cells. It remains unclear, however, whether these effects of 2-AG are mediated through the cannabinoid receptor. Recently, we found that 2-AG induces rapid phosphorylation and activation of the p42/44 MAP kinase in HL-60 cells (38). 2-AG-induced activation of the p42/44 MAP kinase was abolished when the cells were pretreated with either SR144528 or PTX, indicating that the response was mediated through the CB2 receptor and G i /G o . We also found that rapid phosphorylation of the p38 MAP kinase and c-Jun N-terminal kinase takes place in 2-AG-stimulated HL-60 cells. 2 The 2-AG-induced activation of the p38 MAP kinase and c-Jun N-terminal kinase has also been reported by several investigators (39,40). These results strongly suggest that 2-AG plays some essential role in the inflammation and immune responses, although the exact physiological functions of 2-AG in inflammatory cells and immune competent cells still remain unclear.
In this study, we explored the effect of 2-AG on the motility of HL-60 cells. We found that 2-AG induces the migration of HL-60 cells differentiated into macrophage-like cells (see Figs. 2-7). Similar effects were observed with other macrophage-like cells of human origin such as U937 cells and THP-1 cells and human peripheral blood monocytes (see Figs. 8 and 9), suggesting that 2-AG-induced migration is a common event in human macrophages/monocytes. The 2-AG-induced migration of differentiated HL-60 cells was markedly reduced when the cells were pretreated with either SR144528 or PTX (Fig. 3), suggesting that the migration was mediated through the CB2 receptor and G i /G o . Arachidonic acid and its metabolites did not participate in the 2-AG-induced migration, because free arachidonic acid was not capable of inducing the migration (data not shown). This was also confirmed by the fact that 2-AG ether was able to induce the migration (Fig. 5), although its activity was rather weak compared with that of 2-AG. On the other hand, the Rho kinase, MEK and p38 MAP kinase, were suggested to be involved in the 2-AG-induced migration of differentiated HL-60 cells, because Y-27632 (a Rho kinase inhibitor), PD98059 (a MEK inhibitor), and SB203580 (a p38 MAP kinase inhibitor) suppressed the migration (Fig. 4). The inhibition of cell migration by Y-27632 (1-100 M) (41,42), PD98059 (10 -20 M) (43)(44)(45), and SB203580 (10 -50 M) (42,46) has already been reported for several types of cells stimulated with various chemoattractants, although there are conflicting results as to the inhibition by PD98059 (42,46). The relationship between Rho kinase and p38 MAP kinase, as well as MEK, is known to be complicated. Ashida et al. (42) reported that Rho kinase is upstream of p38 MAP kinase in monocyte chemoattractant protein-1-stimulated THP-1 cells. Details of the intracellular signaling pathways for 2-AG-induced migration of HL-60 cells will be clarified in the future.
The activity of 2-AG was highest among those of the vari-2 T. Sugiura and Y. Kobayashi, unpublished results.

2-Arachidonoylglycerol-induced Cell Migration
ous cannabinoid receptor ligands (Fig. 5). This is reasonable in view of the fact that 2-AG is the true endogenous ligand of the cannabinoid CB2 receptor. It has already been shown that 2-AG is present in appreciable amounts in various mammalian tissues (10, 13). 2-Monoacylglycerols containing saturated, monoenoic, dienoic, and hexaenoic fatty acids did not exhibit any appreciable activity, whereas 2-eicosa-5Ј,8Ј,11Јtrienoylglycerol and 2-eicosa-5Ј,8Ј,11Ј,14Ј,17Ј-pentaenoylglycerol induced the migration to some extent (Fig. 6). These results are in general agreement with the results of the Ca 2ϩ transient experiments reported previously (16). We have found that the presence of the double bond at the ⌬5-position is important for some characteristic conformation of the agonistic molecules (15,16).
The migration of HL-60 cells induced by 2-AG was assumed to mainly involve chemotaxis rather than chemokinesis, because 2-AG ether elicited mainly chemotaxis (Fig. 7). This was also confirmed by the fact that the migration of HL-60 cells observed in the presence of 2-AG in both the upper and lower compartments was markedly reduced compared with the case of the presence of 2-AG only in the lower compartment, when 0.5 mM diisopropylfluorophosphate, a monoacylglycerol lipase inhibitor, was added to block the hydrolysis of 2-AG: 9.3 ϩ 0.4, 26.7 ϩ 2.1, and 19.5 ϩ 3.3% for vehicle alone, 2-AG present only in the lower compartment, and 2-AG present in both compartments, respectively (the means Ϯ S.D. of four determinations). 3 We confirmed that 2-AG was rapidly metabolized during the co-incubation with the cells when the monoacylglycerol lipase inhibitor was not included in the incubation mixture as mentioned before. It cannot be ruled out, however, that some part of the migration induced by 2-AG was because of chemokinesis. Whatever the mode and the mechanism of action, the fact that 2-AG induces the migration of macrophage-like cells and monocytes is quite noticeable, because various types of proinflammatory molecules are known to induce the migration and recruitment of inflammatory cells. Very recently, Jorda et al. (47) also demonstrated that 2-AG induces the migration of mouse splenocytes and myeloid cells, yet the elucidation of the detailed mechanism of 2-AG-induced migration of these cells awaits further investigations.
Previously, Gallily et al. (48) reported that 2-AG suppresses the production of tumor necrosis factor ␣ in lipopolysaccharidestimulated mouse macrophages in vitro and in lipopolysaccharide-administered mice in vivo, although whether these effects of 2-AG are mediated through the CB2 receptor is uncertain. On the other hand, we found that the addition of 2-AG to HL-60 cells enhanced the production of chemokines such as interleukin 8 and MCP-1 through a CB2 receptor-and G i /G o -dependent mechanism. 4 Based on the results of a previous investigation on chemokine production and the present study on cell migration, we assume that 2-AG acts as a stimulator or accelerator, rather than as a suppressor, of inflammation reactions and immune responses.
As for ⌬ 9 -THC, it has been reported that ⌬ 9 -THC suppresses inflammation and immune responses in vivo (32)(33)(34). The mechanism by which ⌬ 9 -THC suppresses the inflammatory reactions and immune response has long remained obscure. Previously, we demonstrated that ⌬ 9 -THC is a weak partial agonist of the cannabinoid CB2 receptor (16). Bayewitch et al. (49) also reported that ⌬ 9 -THC acted as an antagonist toward the CB2 receptor. Noticeably, SR144528 and JTE-907, CB2 receptor antagonists/inverse agonists, inhibited inflammation in vivo (50). It is possible, therefore, that ⌬ 9 -THC blocks the action of the endogenous natural ligand of the CB2 receptor, that is, 2-AG, thereby inducing the suppression of inflammatory reactions and immune responses.
In conclusion, we found that 2-AG induces the migration of HL-60 cells differentiated into macrophage-like cells through the CB2 receptor-, G i /G o -, and several other signaling moleculedependent mechanisms. Similar effects were observed with other macrophage-like cells and human monocytes. The migration induced by 2-AG was mainly attributed to chemotaxis rather than chemokinesis. 2-AG is known to be generated from stimulated inflammatory cells and immune competent cells such as macrophages upon stimulation (20,(23)(24)(25) through an increased phospholipid metabolism such as inositol phospholipid turnover. It is possible that 2-AG, derived from a variety of stimulated tissues and cells, plays physiologically and pathophysiologically essential roles during the course of inflammatory reactions and immune responses.