chemokines ( cks ) are low molecular weight chemoattractant peptides , belonging to the cytokine family . differently from interleukins
, cks act via g protein - coupled receptors ( gpcrs ) , controlling cell migration and trafficking throughout the body , during immune response   and development [ 3 , 4 ] .
cks are also critical mediators of several physiological mechanisms such as wound - healing and tissue homeostasis [ 3 , 5 ] ; moreover , cks are expressed in the central nervous system ( cns ) [ 6 , 7 ] where they not only act as mediators of development , intercellular communication , and inflammatory processes but also function as neurotransmitters or neuromodulators , mainly involved in neuroendocrine regulations .
recently , it has been shown that cks play a relevant role in tumorigenesis , neoangiogenesis , tumor progression , and metastasization [ 9 , 10 ] .
evidence for autocrine / paracrine regulatory mechanisms in different normal and cancer cell types , driven by chemokine / receptors interaction on the same or a nearby cell , supports the potential role of cks in the control of physiological or tumoral endocrine functions .
in particular , the chemokine ( c - x - c motif ) ligand 12 ( cxcl12 ) and its receptors , cxcr4 and cxcr7 , have been involved in cancer cell proliferation , migration , and invasion [ 1113 ] .
they are classified by size ( microadenoma , < 10  mm or macroadenoma , > 10  mm ) and on the basis of their ability to produce hormones , as secreting or functioning tumors ( about 50% of adenomas ) or as clinically nonfunctioning pituitary adenomas ( nfpa ) that do not release hormones or , more often , secrete clinically nonrelevant ( i.e. , gonadotropins ) or nonbioactive hormones ( -subunit of glycoproteic hormones ) [ 14 , 15 ] .
almost all pituitary tumors display a benign clinical course being slow growing and show low incidence of metastasis ; however , they are frequently associated with high morbidity and mortality due to mass - related effects and paraneoplastic syndromes related to hormone hypersecretion . functioning
pituitary adenoma leads to hypersecretion of hormones that results in classic clinical syndromes , mainly acromegaly ( overproduction of gh ) , hyperprolactinemia ( excess of prl ) , and cushing 's disease ( overproduction of acth ) and , more rarely , secondary hyperthyroidism ( increased tsh secretion ) .
nfpas do not secrete sufficient hormones ( mainly fsh or lh ) to be detectable in the blood or to cause hormonal manifestations ; in other cases only biologically inactive -subunit is released and more rarely they are classified as true nonsecreting adenomas .
importantly , all pituitary adenomas may induce hypopituitarism and neurological symptoms ( for example compression of the optic chiasma ) due to mass effect [ 16 , 17 ] .
clinical relevance and recent advances in the comprehension of their molecular pathogenesis suggest that pituitary adenomas should be considered a more critical disease than a benign endocrine pathology .
thus , a deeper evaluation of the mechanisms at the basis of their tumorigenesis and better prognostic markers to identify tumors with a high risk of recurrence are most awaited to improve pituitary adenoma clinical outcome .
this review will focus on the diverse role of cxc chemokines and their receptors in normal pituitary cell functions and pituitary tumor development and progression , summarize recent progress in cxcr7 functions , and discuss the present issues and future perspectives .
cks are classified , according to the number and spacing of the first two cysteine residues of a conserved cysteine motif , into four groups : ( 1 ) cxc ( with a single nonconserved amino acid residue -x- between the first n - terminal c residues : cxcl1 - 17 ) ; ( 2 ) cc ( two adjacent cysteine residues : ccl1 - 28 ) ; ( 3 ) xc ( only one n - terminal cysteine : xcl1 - 2 ) ; ( 4 ) cx3c ( three nonconserved amino acid residues separating the n - terminal c residues : cx3cl1 ) [ 18 , 19 ] .
ck receptors are typical gpcrs , all of them signaling through heterotrimeric g proteins ( g , and g- subunits ) , with the remarkable exception of cxcr7 which is exclusively biased towards -arrestin - mediated signaling .
upon ligand binding , many ck receptors may form homo- and heterodimers that activate distinct intracellular signaling pathways from individual receptors .
the classification of ck receptors is based on the class of their ligands ( e.g. , cxc ligands bind cxc receptors ) .
cks may also bind a small group of so - called  atypical chemokine receptors  ( ackrs ) , which are unable to initiate downstream conventional g - protein - dependent signaling , resulting , from a functional point of view , in the inability to induce directional cell migration .
these receptors bind distinct and complementary range of cks and likely control ck networks during development and physiopathological processes by scavenging cks .
ackrs , including duffy antigen receptor for chemokines ( darc , ackr1 ) , c6 ( ackr2 ) , cxcr7 ( ackr3 ) , and ccx - ckr1 ( ackr4 ) ( for classification and nomenclature see ) , were proposed to serve as decoy receptors to scavenge inflammatory cks from the extracellular microenvironment , inhibiting their signaling .
indeed , recent evidence supports the ability of ackrs to transport , internalize , and degrade cks , leading to the formation of ck gradients in normal and cancer tissues , responsible for the functional modulation of their signaling .
cks belonging to the cxc family can be further grouped according to the presence or absence of a the tripeptide motif glu - leu - arg ( elr ) preceding the cxc domain ( elr or elr ) , which affects receptor binding specificity and biological effects .
notably , elr cxc chemokines ( i.e. , cxcl9 , -10 and -11 ) are interferon -inducible and act as potent angiostatic factors to impair angiogenic stimuli induced by growth factors .
conversely , elr cks ( cxcl1 , -2 , -3 , -5 , -6 , -7 , -8 ) are proangiogenic [ 24 , 25 ] . cxcl12 ( previously known as stromal cell - derived factor-1 , sdf-1 ) is an exception to this characterization , since it is elr but mediates tumor - promoting angiogenesis via its receptor cxcr4 [ 9 , 24 , 26 ] .
common features shared by all cks are pleiotropism , promiscuity , and redundancy , with a single ck able to bind several receptors , whereas multiple cks bind the same receptor resulting in the same functional outcome . upon ligand binding , ck receptors undergo conformational change that activates the g subunits sensitive to bordetella pertussis toxin ( ptx ) .
their activation dissociates the gtp - bound g subunit from the g- dimer , and both these active components trigger intracellular signals , such as activation of phospholipase c ( plc)/inositol triphosphate ( ip3)-ca / diacyl glycerol ( dag)/protein kinase c ( pkc ) and inhibition of adenylyl cyclase ( ac)-camp / protein kinase a ( pka ) .
moreover , these receptors control the activity of different kinases , including extracellular regulated kinases ( erk1/2 ) , c - jun n - terminal kinase ( jnk ) , p38 , phosphatidyl inositol 3 kinase ( pi3k)-akt , and the focal adhesion kinase ( fak ) .
the distinct transductional cascades regulated by ck receptors mainly depend on the g subfamily which they activate : gi inhibits ac but also activates tyrosine kinases of the src family , favoring signal integration ; gq increases plc activity , to cleave pip2 to form dag and ip3 . in turn
, dag activates pkc , whereas ip3 binds specific receptors on the endoplasmic reticulum inducing ca release from intracellular stores . finally , g12 controls the activity of the small g protein rhoa , via rho - gef . on the other hand , ck receptor activation of g subunits results in the activation of pi3k leading , through the phosphoinositide - dependent kinase 1 and 2 ( pdk1 - 2 ) , to akt phosphorylation and subsequent activation of its downstream signal proteins such as glycogen synthase kinase 3 ( gsk3 ) , mammalian target of rapamycin ( mtor ) , and fak , which control migration in different types of normal and tumor cells .
after stimulatory responses , the inactivation of ck receptor signaling occurs after the hydrolysis of gtp to gdp by the intrinsic gtpase activity of g subunit , followed by its reassociation with g/ in an inactive complex .
moreover , receptor desensitization , internalization , and lysosomal degradation are mediated by g protein - coupled receptor kinases ( grks ) and arrestins .
cks are constitutively secreted by leukocytes , fibroblasts , endothelial , and epithelial cells to mediate cell activation , trafficking , and homing [ 5 , 30 ] . beside their basal expression , most cks are highly induced during inflammatory or infective processes driving different phases of immune response via a ck gradient which directs leukocyte recruitment to the site of inflammation .
furthermore , cks directly activate specialized effector lymphocytes during the different steps of immune response , for example , cxcl8 ( formerly named il8 ) recruits neutrophils , basophils , and eosinophils expressing its receptors , cxcr1 and cxcr2 .
adaptive immune responses are mediated by cks ( cxcl9-l10-l11 ) secreted by macrophages activated by inf- released by natural killer and t helper 1 ( th1 ) cells that express cxcr3 , the receptor for cxcl9-l11 , thus amplifying leukocyte recruitment and , finally , inflammation .
cks also play a key role in embryogenesis , organogenesis , angiogenesis , and germ cell migration , especially during neural development .
the constitutive expression of cks and their receptors in adult normal brain was initially identified in the immune - like competent cell populations such as microglia and astrocytes .
the subsequent detection of their expression in neurons [ 3234 ] broadened ck role as neuromodulators / neurotransmitters in neurological processes such as thermoregulation , pain perception , and stress conditions , as well as in pituitary functions . focusing on the cxcl12/cxcr4-r7 network ,
it exerts a variety of functions in cns development as well as in mature brain .
cxcl12 directs the migration of embryonic and adult stem cells in the developing central and peripheral nervous system [ 35 , 36 ] , controlling the formation of cerebellum , cerebral cortex , hippocampus , and dorsal root and sympathetic ganglia [ 25 , 37 , 38 ] .
postnatally , cxcr4 expression , while downregulated in many brain areas , persists in the hypothalamus where it modulates the hypothalamic - pituitary system and the hypothalamic - pituitary - gonadal axis , in particular , cooperating to the regulation of neuroendocrine and reproductive systems [ 3941 ] .
as far as cns development is concerned , the pivotal role of cxcl12/cxcr4 emerged from studies using knockout mice for either the ligand or the receptor .
both models exhibited a superimposable abnormal neuron migration in the cerebellum , dentate gyrus , and dorsal root ganglia [ 35 , 36 , 42 , 43 ] .
furthermore , cxcl12/cxcr4 axis controls migration and homing of cajal - retzius cells [ 44 , 45 ] , postmitotic neurons , cortical interneurons [ 40 , 4749 ] , and dopaminergic neurons .
cxcl12/cxcr4 regulation of stem cell positioning and migration persists in adults , in the neurogenic niches of brain and in the bone marrow , where hematopoietic progenitors cells are retained by the interaction between ligand and receptor   that also promotes their survival and proliferation .
interestingly , a similar homing mechanism has been demonstrated for adult neural progenitor cells ( npcs ) or neural stem cells ( nscs ) .
cxcr7 , the second cxcl12 receptor , has a 10-fold higher binding affinity than cxcr4 but also binds cxcl11 ( formerly known as ifn - inducible t cell  chemoattractant , i - tac , ) , which , in turn , interacts with cxcr3 . presently , the function of cxcr7 is still controversial .
cxcr7 does not mediate cxcl12-dependent cell migration [ 20 , 52 , 54 ] and displays atypical signaling pathways , failing to induce intracellular ca mobilization and inhibition of camp production , since this receptor does not seem to be coupled to gi. based on its ability to rapidly sequester and degrade cxcl12 and thus to suppress cxcr4 activity , cxcr7 was firstly proposed to be a decoy receptor [ 5457 ] ; currently , this activity is considered only a part of the possible mechanisms by which cxcr7 modulates cellular functions .
indeed , emerging evidence suggests that cxcr7 can promote cell motility [ 5860 ] and trigger intracellular signals in different human normal and cancer cell types [ 6164 ] . in particular
, cxcr7 activates akt , map kinase ( mapk ) , and jak / stat3 cascades , either by direct modulation , through a -arrestin - dependent pathway [ 20 , 65 ] , or after heterodimerization with cxcr4 [ 59 , 6669 ] . preferential signaling through g - proteins or -arrestin
is influenced by both cxcr4-cxcr7 dimer formation and the oligomerization state of cxcl12 [ 70 , 71 ] .
cxcr7 was recently shown to activate mtor in human renal cancer cells through the modulation of erk1/2 and p38 activities , further suggesting that it is a fully signaling receptor although independent from g proteins .
however , cxcr7 knockout mice display a lethal phenotype due to a heart valve and vascular defects , a very similar scenario observed in mice with targeted disruption of the genes encoding cxcr4 and cxcl12 [ 67 , 74 ] . in the adult rat brain
, cxcr7 is expressed at high levels in vessels , pyramidal cells , and mature dentate gyrus granule cells , overlapping cxcl12 expression pattern [ 75 , 76 ] , and a functional role for cxcr7 in the control of neuronal migration to the subventricular and intermediate zone was suggested [ 69 , 77 ] . in rat mature neurons and blood vessels , cxcr7 appears to be the preponderant cxcl12 receptor , likely contributing to cxcl12-dependent neuronal development .
moreover , cxcr7 acts as scavenger on brain microvessel endothelium   and it is essential for inflammatory leukocytes to infiltrate the cns .
cxcr7 is also expressed in neural tube and brain of mice embryos . in rat cortex , cxcr7 is localized in gabaergic neuron precursors , and cajal - retzius cells and , unlike cxcr4 , it has been identified in neurons forming the cortical plate and in the developing dentate gyrus and cerebellar external germinal layer [ 75 , 80 ] .
migrating immature cortical interneurons co - express cxcr4 ( membrane surface expression ) and cxcr7 ( intracellular expression , mainly endosomes ) .
cxcr7 rapidly recycles from membrane to intracellular pools of interneurons , and its trafficking mediates cxcl12 endocytosis .
it is essential for the regulation of interneuron migration in the developing cerebral cortex since its removal causes an increase in extracellular cxcl12 content , which favors its binding to cxcr4 and consequently induces the endocytosis and degradation of cxcr4 .
thus , cxcr7 regulates cxcr4 expression and likely controls cxcl12 signaling to drive successful migration in the developing cerebral cortex .
in addition , since cxcr7 and cxcr4 mutant mice displayed opposite defects in interneuron motility and positioning , cxcr4 and cxcr7 were proposed to have distinct roles and signal transduction to regulate interneuron movement .
this fine tuning of cxcl12 response induced by cxcr7 occurs either directly modulating -arrestin - mediated signaling cascades or scavenging local cxcl12 availability .
deletion of one of the cxcl12 receptors is sufficient to generate a migration phenotype that corresponds to the cxcl12-deficient pathway and interfering with the cxcl12-scavenging activity of cxcr7 causes loss of cxcr4 function .
for example , during development , cxcl12 regulates the migration of gonadotropin - releasing hormone ( gnrh ) neurons , through cxcr4-mediated activation of the girk channel , but this effect is modulated by cxcr7 which controls cxcl12 content availability acting as a scavenger along the migratory path .
the relevance of cxcl12 and cxcr4-r7 system in cns ontogeny and functions is even more crucial in the view of their expression in both embryonic and adult brain stem cells , a subset of undifferentiated cells characterized by self - renewal through asymmetric division , differentiation into multiple lineages , and constant proliferation that in adults acts in tissue maintenance and repair .
the role of cxcl12 and cxcr4 in stemness maintenance has gained much attention also in the neuroendocrinology field due to the proposed role of stem cells in pituitary plasticity [ 84 , 85 ] .
both cxcl12 and cxcr4 are expressed in different anterior pituitary cell subtypes , as well as in nonhormonal cell types [ 8688 ] .
the chemotactic activity of this ck could be also relevant in folliculostellate ( fs ) cell , non - fs nestin cell , and stem cell migration [ 86 , 89 ] . therefore
, understanding the ck - dependent mechanisms associated with candidate stem cells within pituitary might help to clarify their activity in development or in normal mature hormone - producing and tumor pituitary cells [ 84 , 90 , 91 ] .
the stem cell concept applied to cancer has radically changed the research approach to tumorigenesis and treatment , since the subset of cancer cells , namely , cancer stem cells ( cscs ) , seems responsible for tumor initiation , metastasis , and resistance to therapy .
although , at present , all factors and signals that regulate cscs are not completely clarified , accumulating evidence suggests a key role of the cxcl12/cxcr4 axis in csc maintenance and growth [ 62 , 93 ] .
moreover , interactions between cscs and tumor microenvironment through secreted cks ( e.g. , cxcl12 ) , possibly occurring also in pituitary adenomas , may act as chemoattractant to recruit fibroblasts , endothelial , mesenchymal , and inflammatory cells to the tumor , via cxcr4 .
through the release of growth factors ( bfgf , egf , and vegf ) , cyto / chemokines , and neuroendocrine proteins ( steroid hormones , prolactin , growth hormone , ghrelin , erythropoietin , catecholamines , etc . )
neuronal and neuroendocrine pathways regulate fundamental functions within the cns and its interaction with the immune system .
complex autocrine / paracrine signals through neuropeptides ( e.g. , egf and vip ) , neurotransmitters , cytokines ( il-1 , il-6 ) , and cks occur also in pituitary regulation , differently from the classical hypothalamic input and feedback signals from the periphery [ 9597 ] . egf expression has been observed at all stages of pituitary development and in the adult pituitary , and the egfr pathway contributes to pituitary physiology and tumorigenesis .
il-1 receptors were detected in pituitary cells , and their activation inhibits prolactin ( prl ) secretion from dispersed rat pituitary cells through the regulation of ac and plc activities , and ca fluxes [ 100103 ] . il-6 and its receptors are also expressed in the pituitary gland [ 104 , 105 ] where their interaction regulates apoptosis and proliferation of endocrine cells in vitro .
il-6 is mainly produced by the fs cells and activates a paracrine loop on the hormone - secreting cells [ 107 , 108 ] regulating acth , prl , lh , and gh secretion [ 110112 ] via the modulation of ac and plc activities .
interestingly , il-6 exerts opposite effects on normal and adenomatous pituitary cells : it is inhibitory for normal anterior pituitary   and stimulatory for adenoma cells .
il-18 was also proposed to exert paracrine effects in pig anterior pituitary being the ligand and its receptor expressed by different subsets of gh secreting cells .
finally , interleukins ' regulation of the hypothalamic - pituitary - adrenal axis also involves the modulation of vasoactive intestinal peptide- ( vip- ) secreting pituitary cells to control , in a paracrine manner , prl release .
cks can affect pituitary hormone secretion via the hypothalamic - pituitary axis or autocrine / paracrine regulation .
cxcl1 is expressed in the posterior pituitary , in the paraventricular nucleus ( pvn ) of the hypothalamus and the median eminence . in response to stressful stimuli ,
this ck is released in the median eminence   to reach its receptor ( cxcr2 ) expressed in pituitary cells and induce the release of prl and gh and the inhibition of lh and fsh secretion .
the generation of transgenic rats ( s100-gfp rats ) that express green fluorescent protein in s100-positive pituitary fs cells in the anterior pituitary   led to the characterization of s100-positive cells [ 86 , 121 ] and transcripts of cxcl10 ( ifn- inducible protein 10  kda , ip-10 ) were identified in a subpopulation of these cells .
importantly , cxcr3 , the receptor for cxcl10 , was shown to be expressed in corticotrophs , suggesting a possible autocrine / paracrine effect of cxcl10 , released from fs cells , on acth - producing cells .
cxcl12/cxcr4 is the major regulatory axis not only connecting the immune and nervous systems , but also playing a role in neuroimmune regulation of the anterior pituitary physiological functions .
cxcl12 was detected in both rat pituitary   and hypothalamus   and its expression in hypothalamic neurons , concomitant with cxcr4 positivity at pituitary level , corroborated the hypothesis that this ck could represent a hypothalamic regulatory factor of anterior pituitary function . as a consequence
, the chemokinergic regulation of anterior pituitary cells might derive from coordinate activity of cxcl12 originating from both hypothalamic neurons and systemic circulation .
a regionalized constitutive expression of cxcl12 was reported in adult rat brain , particularly in arginine vasopressin- ( avp- ) expressing neurons   where its interaction with cxcr4 leads to modulates induced plasma avp release in vivo . the expression pattern of this chemokine and its receptor in the rat hypothalamo - neurohypophyseal system was further investigated : they colocalize within avp - expressing neurons in both supraoptic ( son ) and paraventricular ( pvn ) nucleus as well as in dense core vesicles of avp - positive nerve terminals in the posterior pituitary , showing a similar distribution . since avp controls body fluid homeostasis ,
the interaction between cxcl12 and avp was studied in avp - deficient brattleboro rats that show low expression of both cxcl12 and cxcr4 , correlated with avp protein expression level in son , pvn , and posterior pituitary . however , since cxcl12 mrna is increased , it was hypothesized that cxcl12 synthesis is present in these cells but ,
being costored with avp , a concomitant massive release of both peptides is responsible for their low content at both hypothalamic and posterior pituitary levels .
avp and cxcl12 expression is dependent on water balance and is centrally regulated , further strengthening the role of cxcl12 in neuroendocrine functions .
however , cxcl12 and cxcr4 are also coexpressed in rat pituitary cells   and a further autocrine / paracrine regulation of pituitary functioning was hypothesized .
complete colocalization between cxcr4 and gh was reported in normal rat pituitary , suggesting that cxcr4 is a rather specific regulator of somatotroph activity , in rats .
indeed , cxcl12 stimulates gh transcription and secretion in both primary rat anterior pituitary cells and the gh - producing pituitary adenoma cell line , gh3 .
interestingly , rat fs cells also express cxcr4 and secrete cxcl12 , which acts as a potent chemoattractant for these cells .
the activation of this autocrine loop facilitates the formation of f - actin in fs cells and the subsequent directional extension of their cytoplasmic processes toward other fs cells .
cxcl12/cxcr4 interaction induces invasion and interconnection of fs cells to near lobular structures likely forming a circuit that causes or maintains local cellular arrangement in the anterior pituitary . in humans ,
scattered expression of both cxcr4 and cxcl12 within the anterior lobe was detected by immunohistochemistry , revealing a nonhomogeneous positivity for both proteins throughout the tissue , including large negative areas , others showing few positive cells and rare zones with higher expression   ( figure 1 ) .
interestingly , in all these areas , cxcr4 expression resulted largely higher than its ligand , although all the cxcl12-positive cells express cxcr4 , as well .
cxcr4-expressing cells do not belong to specific secreting cell type , being present in gh , prl , or acth - secreting cells , while no expression was observed in human fs cells .
however , some cxcr4-expressing cells do not coexpress any hormones and no colocalization of either cxcr4 or cxcl12 was observed in fs cells ; thus it was proposed that cxcl12/cxcr4 system may also label undifferentiated / progenitor cells .
notably , this ck - receptor pair was undetectable in human posterior pituitary lobe , contrarily to what was observed in rats .
thus , from these data it is evident that , in normal pituitary , cxcl12 is secreted by cell subpopulations that , cooperating with hypothalamic factors ( including cxcl12 itself ) , may contribute to paracrine modulation of pituitary functioning ( figure 2 ) .
consequently , alterations of the endocrine regulatory pathways due to upregulation of hypothalamic / pituitary cxcl12/cxcr4 axis might lead to the development of pituitary adenomas [ 127 , 129 ] .
the activity of cxcr7 in normal pituitary deserves further investigation ; however its expression in pituitary adenoma tissues [ 130 , 131 ] suggests possible involvement in pituitary function regulation .
beside direct cxcr4-dependent activation of erk1/2 , transactivation of tyrosine kinase receptors is currently a relevant mechanism in tumor cell responses .
mainly , the transactivation of epidermal growth factor receptor ( egfr ) family members mediates the mitogenic activity of different cks in human cancer .
a cross - talk between cxcl12 and egfr and/or her2/neu phosphorylation was demonstrated in breast and ovarian cancer cells through g protein - dependent activation of kinases of the src family [ 132134 ] .
moreover , in breast cancer , cxcr4 interacts with the egfr variant , egfr viii , a constitutively active mutant highly expressed in cancer stem cells , to regulate invasion via p38 mapk .
cxcr4 signaling is negatively regulated by protein - tyrosine phosphatases ( ptps ) , such as the src homology - containing protein - tyrosine phosphatase 1 ( shp1 ) and the sh2 domain - containing inositol 5-phosphatases ( ship ) , while shp2 , constitutively associated with cxcr4 , potentiates ck signaling [ 137 , 138 ] .
these observations are particularly relevant since they highlight possible direct antagonisms between cxcr4 and somatostatin receptors ( sstr ) that are powerful activators of ptps [ 139141 ] .
this antagonistic activity could acquire clinical relevance in light of the fact that sstr agonists are the main pharmacological tool available for the treatment of pituitary adenomas [ 142 , 143 ] .
the concomitant expression of ligand - receptor pair in the same tumor cells , responsible of autocrine / paracrine activation , is one of the leading causes of clinical aggressive behavior in various cancer types [ 144 , 145 ] . exploiting the coexpression of cks and their receptors , cancer cells and cells of the tumor microenvironment
as previously described , this autocrine mechanism is maximally effective for cxcr4 , the most widely expressed ck receptor in human solid and hemopoietic malignancies .
the autocrine / paracrine loop of the cxcl12/cxcr4 pair has been deeply investigated in brain tumors in both in vitro and in vivo models : cxcl12 stimulates proliferation and migration of glioblastoma cells and xenografted tumors inducing erk1/2 and akt phosphorylation [ 147149 ] .
the mitogenic activity mediated by cxcl12-cxcr4 was also reported in meningioma , in which cxcr4 activation increased dna synthesis through activation of erk1/2 in primary cultures and its expression level significantly correlated with ki-67 proliferation index of the original tumor tissue [ 150 , 151 ] , while cxcr7 was mainly localized in tumor endothelia .
similar cxcr4 tumor - promoting effects were observed in breast carcinoma , suggesting cxcl12 as possible autocrine / paracrine growth factor .
interestingly , in breast cancer cells the synthesis and release of cxcl12 is under the control of 17-estradiol contributing to its proliferative effects and mediating , via a src - dependent mechanism , egfr transactivation [ 133 , 154 ] .
furthermore , cxcl12 is also indirectly implicated in tumor pathogenesis , acting as chemoattractant for cxcr4-positive cells , directing tumor cell migration [ 155157 ] and controlling invasive and metastatic properties of cxcr4-expressing cancer cells to distant organs [ 158 , 159 ] .
the invasive behavior of cancer cells might indirectly depend on locally released cxcl2 that , via an autocrine mechanism , binds to cxcr4 impairing chemotaxis towards cxcl12-producing target organs and metastatic spread .
in addition , normal cells forming tumor stroma ( i.e. , macrophages , lymphocytes , fibroblasts , and endothelial cells ) concur to cancer development and progression through cxcl12 secretion .
concentration gradient directs cancer cell motility in several tumors [ 10 , 161 ] , including aggressive solid neoplasms ( breast , colon , brain   ovarian , prostate , renal cell   and oral squamous cell   carcinomas , and melanoma ) .
importantly , as observed in a rat mammary adenocarcinoma cell line overexpressing both receptors , cxcr4 and cxcr7 play opposing roles in breast cancer metastasis : cxcr4 mediates cancer cell invasion allowing cells to follow the cxcl12 gradient generated by metastatic targets whereas cxcr7 favors tumor growth increasing angiogenesis but impairs cell migration scavenging the chemokine .
raf / mek / erk and pi3k / akt pathway dysregulation is a common alteration responsible of tumor initiation and progression . while the pathways are classically activated by growth factors , cross - talks and transactivation mechanisms with neuropeptide - cytokine - ck / gpcrs have been increasingly recognized .
this cross - talk activates erk1/2 , which is directly responsible for cell growth and differentiation , depending on the cellular context and represents one of the major proliferative pathways in cancer [ 168 , 169 ] .
the overexpression or constitutive activation of receptors for growth factors , cytokines , and cks potentiates the activation of ras / raf / mek / erk pathway also in pituitary adenoma .
mapk phosphorylation is relevant in different pituitary cell types such as att20 cells , where it regulates crh - induced pomc transcription , gonadotrophs for gnrh signaling , gh - secreting cells for ghrh - dependent cyclin d1 expression , and gh4c1 somatotroph cell line in which the gsp oncogene impairs ras / erk1/2-dependent prl gene regulation .
pi3k / akt / mtor pathway , activated by a variety of growth factors and hormones , when dysregulated , leads to aberrant growth of pituitary adenoma cells .
akt is overexpressed and hyperphosphorylated in nfpa , in a mouse model of tsh - oma , and in gh3 cells in which the inhibition of pi3k / akt signaling by octreotide increases the expression of the tumor suppressor gene zac1 [ 176 , 177 ] .
similarly to cell proliferation , survival mechanisms also sustain pituitary development and tumorigenesis : in particular the balance of pro- and antiapoptotic factors , physiologically contributing to normal pituitary cell plasticity , when unbalanced , favors pituitary cell transformation .
for example , in pituitary adenomas , antiapoptotic mediators , such as the bcl-2 protein family , are upregulated , while fas , a major apoptotic factor in different cell types , activates apoptosis in both normal rat lactotrophs and somatotrophs   and in pituitary adenoma cell lines .
several pituitary - related genes may exert a role in apoptosis of secretory pituitary cells , as the developmental factor pitx2 and the transcription factor pit-1 [ 183 , 184 ] , although their mechanisms are not yet fully understood .
recent studies , however , highlighted that , when considering the complexity of regulatory pathways involved in pituitary cell survival and proliferation , it should take into account not only apoptosis but also senescence , an alternative process acting during tumor - suppressive cell fate .
importantly , senescence is gaining biological significance also in pituitary adenomas , whose typical benign nature could result from protective antiproliferative mechanisms .
several transformation events ( e.g. , dna damage , loss of tumor - suppressor gene , oncogene activation , and growth factor overexpression ) induce preventive cellular senescence , characterized by cell cycle exit and subsequent irreversible proliferation arrest .
thus , pituitary tumors may be more prone to activate senescence - associated pathways , maintaining their benign behavior , preventing malignant transformation , and regulating their development .
interestingly , il-6 has been shown to participate in oncogene - induced senescence in pituitary gp130 overexpressing tumor cells . moreover ,
angiogenic and apoptotic processes cooperate in determining tumor aggressiveness , and this regulation might also be involved in the pathogenesis of pituitary transformation . the pituitary gland is highly vascularized but , unlike other solid malignancies , conflicting results are available on angiogenic factors associated with pituitary adenoma progression and recurrence [ 186 , 187 ] .
vegf was proposed as pituitary proangiogenic factor and possible therapeutic target , and the hypoxia - inducible factor- ( hif- )  , a key molecule in hypoxic pathways triggering vessel formation , was detected in pituitary tumor tissues [ 189 , 190 ] and may favor hemorrhage in pituitary macroadenomas .
cks , particularly cxcl12 signaling via cxcr4 and cxcr7 , represent candidate mediators of the above described intracellular pathways , determining proliferative , antiapoptotic , and angiogenic signals , thus possibly concurring to pituitary tumor development and aggressiveness .
pituitary adenomas are common intracranial tumors of the adenohypophysis causing serious morbidity , due to excessive hormonal secretion , mass effects , and local invasion of surrounding structures . at present , the understanding of biological and molecular pathogenesis and mechanisms of progression of these tumors is largely incomplete ( for review see [ 14 , 192 , 193 ] ) . emerging evidence reports that multiple factors might contribute to pituitary tumorigenesis , such as frequently altered gene expression , genetic ( aryl hydrocarbon receptor interacting protein , aip ; multiple endocrine neoplasia syndrome type 1 , men1 ; guanine nucleotide - activating alpha subunit , gnas , ) , and epigenetic ( cyclin - dependent kinase inhibitor 2a , cdkn2a , or p16 ; fgfr2/melanoma associated antigen -mage-3 pathway )   mutations , and abnormal micrornas .
the improvement of this knowledge is even more helpful taking into account the peculiar properties of pituitary adenomas as compared to other malignancies : they commonly grow slowly but with local invasive behavior and occasionally develop into high aggressive tumors .
this often prevents the efficacy of surgical and systemic medical treatments , the latter hampered by the lack of definite mechanisms underlying pituitary cell transformation and potential therapeutic targets .
however , few studies addressed the potential role of components of this peptide family in regulating human pituitary tumorigenesis .
cxcl8 mrna was identified in a small percentage of anterior pituitary adenomas [ 199 , 200 ] , altogether with the expression of cxcr2 , the cxcl8 receptor that also binds other cxc cks ( cxcl1 , cxcl7 ) , confirming the potentiality of autocrine stimulation in pituitary adenomas .
indeed , a consistent release of cxcl8 was observed in primary cultures derived from human somatotroph adenomas , induced by stimulation with interleukin-1 and inhibited by gh releasing hormone ( ghrh ) .
thus , a further assessment of the possible role of this ck in the pathogenesis of pituitary tumors is required , likely being based on cxcl8 ability to recruit active neutrophil within the adenoma , influencing the inflammatory response or acting as mitogen for normal and transformed cells . however , the majority of studies focused on the analysis of cxcl12 and cxcr4 expression in human neoplastic pituitary tissues and their role in adenoma cell proliferation [ 128 , 129 , 203205 ] .
cxcr4 mrna is expressed in almost all gh - secreting pituitary adenomas and in the great majority of nfpas , whilst cxcl12 was identified in about 2/3 of these tumors .
notably , most cxcl12-positive cells also express cxcr4 strongly suggesting an autocrine / paracrine regulation of tumor cell proliferation [ 129 , 205 ] .
this hypothesis was further confirmed measuring the in vitro basal secretion of cxcl12 by human pituitary adenoma primary cultures resulting in an autocrine constitutive stimulation of dna synthesis   ( figure 2 ) and , indirectly , by the absence of cxcr4 activating mutations in gh - secreting and nfpa able to sustain adenoma cell proliferation .
this observation was confirmed by a high percentage of different types of secreting pituitary adenomas showing expression of both cxcl12 and cxcr4 [ 129 , 203 , 204 ] .
finally , the evaluation of cxcr4 and cxcl12 expression in invasive and noninvasive pituitary adenoma specimens , by flow cytometry and immunohistochemical staining , demonstrated that the percentage of cxcr4- and cxcl12-positive cells was significantly higher in invasive pituitary adenomas .
thus , the correlation of cxcr4 and cxcl12 expression levels and tumor invasiveness was proposed to be exploited as potential early diagnostic biomarkers , one of the major challenges in diagnosis and treatment of invasive tumors . among the mechanisms that occur in pituitary tumorigenesis
interestingly , while controversial findings on the role of vegf were reported [ 207209 ] , cxcl12 has been proposed as a better defined proangiogenic and proliferative factor in pituitary adenomas .
in fact , cxcl12 and cxcr4 are concomitantly upregulated in hypoxic foci within pituitary tumor tissues , and one of the main cxcl12 effects in pituitary adenomas is to mobilize cd34- ( and cxcr4- ) expressing endothelial progenitors and promote their homing in ischemic foci activating the proangiogenic program .
moreover , in gh3 rat pituitary adenoma cells , hypoxia - activated cxcl12-cxcr4 signaling interacts with the endocrine pathways resulting in upregulation of gh synthesis and secretion and cell proliferation .
thus , in pathological conditions ( i.e. , hypoxia ) , on one hand , increased cxcl12 and cxcr4 expression and signalling may promote neoangiogenesis by recruiting endothelial progenitor cells and/or inducing proliferation of endothelial cells and , on the other , directly favouring hormone hypersecretion and pituitary cell proliferation .
interestingly , gh4c1 rat pituitary adenoma cell line expresses cxcr4 but not cxcl12 , thus it was proposed as a suitable model to characterize the molecular pathways regulated by this receptor in pituitary adenomas , without the interference of the endogenously released ck [ 127 , 211 , 212 ] . in these cells
cxcl12 exerts a powerful secretagogue and mitogenic activity , as well as promotes cell migration .
interestingly , these effects are induced by different and independent intracellular mechanisms , although all of them were ptx - dependent .
gh secretion is a ca - dependent event , in which increased ion concentration resulted from ip3-mediated ca release from intracellular stores .
conversely , gh4c1 proliferation is induced by cxcl12 through the activation of erk1/2 through the  classical  mek - dependent pathway and via the activation of the cytosolic ca - dependent tyrosine kinase , pyk2 , that , in turn , activates the large - conductance ca - activated k channels ( bkca ) [ 95 , 127 , 211 , 212 ] . similarly ,
, the role of cxcl12/cxcr4 as potential pharmacological target in acromegalic patients has been scantily investigated .
it was shown that a synthetic antagonist of cxcr4 , d - arg3fc131 , is able to inhibit the growth of gh3 tumor cells and trigger apoptosis both in vitro and in mice xenografts .
similar results were obtained using phidianidine a , an indole alkaloid isolated from the marine opisthobranch mollusc phidiana militaris , which reduced gh4c1 proliferation , migration , and erk1/2 phosphorylation .
cxcr7 , the second cxcl12 receptor , was reported to be expressed in the att20 mouse corticotroph pituitary adenoma cell line , but the characterization of its possible role in pituitary adenoma development or progression will require further evaluation .
studies on human pituitary adenoma cells derived from postsurgical specimens are limited , mostly due to their low proliferative activity in vitro . however ,
such generalized cxcr4 and cxcl12 overexpression in human pituitary adenomas , as compared to normal pituitary , strongly suggests that , in conditions of deregulation , this receptor system could be a relevant factor for pituitary adenoma development and/or progression .
however , cxcl12 effects of on cell proliferation were directly evaluated in vitro on a small number of primary cultures of adenoma cell derived from ghoma , nfpa , and acth - secreting adenomas . to avoid interference with the cxcl12 released from fibroblasts a specific protocol to obtain adenoma cell cultures highly purified   was used .
cxcl12 induced a statistically significant increase in dna synthesis in the majority ( 65% ) of the adenoma tested .
interestingly , in few adenomas , the blockade of cxcr4 with amd3100 , a known cxcr4 antagonist , caused , beside the abolishment of cxcl12-mediated increase in cell proliferation , also a reduction of basal dna synthesis . measuring cxcl12 levels in the culture medium , it was shown that these tumors retained in vitro a significant basal secretion of cxcl12 causing a constitutive , autocrine stimulation of dna synthesis .
thus , these data suggest that pituitary adenoma cells over - express cxcl12 and cxcr4 as compared to normal tissue and that an autocrine activation of this pathway ( figure 2 ) , actually , occurs in vivo .
overall , these observations imply that cxcl12/cxcr4 axis might play an important biological role in pituitary adenoma as potential growth and angiogenic factor for pituitary cells .
the increased cxcr4 activation may result from either endocrine ( increased cxcl12 levels may reach pituitary through the blood stream or being released in the portal pituitary system from hypothalamus ) and/or autocrine / paracrine mechanisms ( figure 2 ) .
importantly , the latter mechanism seems to be active mainly in pituitary tumors rather than in normal gland , where most of the cxcr4-expressing normal cells do not express cxcl12 .
chemokines are key factors in cns physiology and pathology , being relevant mediators in cancer development . the cxcl12/cxcr4-r7 signaling pathway plays a unique role in the regulation of a variety of cell types , including embryonic and cancer cells . in particular , deregulation of this chemokinergic system
is strictly related to tumor initiation and progression , and the balance of its activity within the tumor microenvironment is highly complex phenomenon .
multiple factors cooperate to pituitary tumor pathogenesis , but , although to date not explored in depth , a pivotal role of ck and their receptors seems to be important .
several mechanisms by which cxcl12/cxcr4 modulates pituitary function and promotes adenoma cell proliferation and their target as potential therapeutic approach have been suggested .
cxcl12 is overexpressed in pituitary cells where it can directly influence adenoma formation , through autocrine mechanisms resulting in constitutive cxcr4 activation .
this pathway grants pituitary cells with a proliferative advantage that triggers clonal expansion of transformed cells and sustains tumor cell survival . increasing knowledge about pituitary cell origin and development might provide significant insights into deregulated pathways in pituitary tumorigenesis
. finally , cxcr4 is an easily druggable target and the characterization of its role in pituitary adenomas could pave the way for novel pharmacological approaches , especially for those adenoma subtypes , ( i.e. , tsh and acth secreting tumors , as well as nfpa ) still waiting for efficacious drugs .
 chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation , infection , and cancer . 
 several chemokines and cognate receptors are constitutively expressed in the central nervous system , not only in glial and endothelial cells but also in neurons , controlling neurogenesis , neurite outgrowth , and axonal guidance during development . in particular , the chemokine cxcl12 and its receptors , cxcr4 and cxcr7 , form a functional network that controls plasticity in different brain areas , influencing neurotransmission , neuromodulation , and cell migration , and the dysregulation of this chemokinergic axis is involved in several neurodegenerative , neuroinflammatory , and malignant diseases . 
 cxcr4 primarily mediates the transduction of proliferative signals , while cxcr7 seems to be mainly responsible for scavenging cxcl12 . 
 importantly , the multiple intracellular signalling generated by cxcl12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions , although a direct modulation of pituitary functioning via autocrine / paracrine mechanisms was also reported . both cxcl12 and cxcr4 
 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation , as well as hormonal hypersecretion . in this review 
 we focus on the physiological and pathological functions of immune - related cyto- and chemokines , mainly focusing on the cxcl12/cxcr4 - 7 axis , and their role in pituitary tumorigenesis . accordingly 
 , we discuss the potential targeting of cxcr4 as novel pharmacological approach for pituitary adenomas . 
1. Background
2. Chemokine Classification and Receptor Interactions 
3. Physiological Functions of CKs: Focus on the CXCL12/CXCR4-R7 Axis in the CNS
4. Chemokine Functions in Normal Pituitary 
5. CXCL12/CXCR4-R7 in Cancer Development and Progression: Autocrine/Paracrine Loops
6. A Network Map of Proliferative Signaling in Pituitary Tumor Development
7. Chemokines in Pituitary Tumorigenesis 
8. Conclusions 