CRH suppressed TGFβ1-induced Epithelial–Mesenchymal Transition via induction of E-cadherin in breast cancer cells

Since its discovery in biopsies from breast cancer patients, the effect of corticotropin-releasing hormone (CRH) on carcinoma progression is still unclear. Transforming growth factorβ1 (TGFβ1) promotes Epithelial–Mesenchymal Transition (EMT) and induces Snail1 and Twist1 expressions. Loss of epithelial cadherin (E-cadherin) mainly repressed by Snail1 and Twist1, has been considered as hallmark of Epithelial–Mesenchymal Transition (EMT). Two breast cancer cell lines, MCF-7 and MDA-MB-231 were used to investigate the effect of CRH on TGFβ1-induced EMT by transwell chamber. And HEK293 cells were transiently transfected with CRHR1 or CRHR2 to explore the definite effects of CRH receptor. We reported that CRH inhibited migration of human breast cancer cells through downregulation of Snail1 and Twist1, and subsequent upregulation of E-cadherin. CRH inhibited TGFβ1-mediated migration of MCF-7 via both CRHR1 and CRHR2 while this inhibition in MDA-MB-231 was mainly via CRHR2. Ectopic re-expression of CRHR1 or CRHR2 respectively in HEK293 cells increased E-cadherin expression after CRH stimulation. Furthermore, CRH repressed expression of mesenchymal marker, N-cadherin and induced expression of Occludin, inhibiting EMT in MCF-7 & MDA-MB-231. Our results suggest that CRH may function as a tumor suppressor, at least partly by regulating TGFβ1-mediated EMT. These results may contribute to uncovering the effect of CRH in breast tumorigenesis and progression.     

Corticotropin-releasing hormone induces Fas ligand production and apoptosis in PC12 cells via activation of p38 mitogen-activated protein kinase.

Recent experimental findings involve corticotropin-releasing hormone (CRH) in the cellular response to noxious stimuli and possibly apoptosis. The aim of the present work was to examine the effect of CRH on apoptosis and the Fas/Fas ligand system in an in vitro model, the PC12 rat pheochromocytoma cell line, which is widely used in the study of apoptosis and at the same time expresses the CRH/CRH receptor system. We have found the following. CRH induced Fas ligand production and apoptosis. These effects were mediated by the CRH type 1 receptor because its antagonist antalarmin blocked CRH-induced apoptosis and Fas ligand expression. CRH activated p38 mitogen-activated protein kinase, which was found to be essential for CRH-induced apoptosis and Fas ligand production. CRH also promoted a rapid and transient activation of ERK1/2, which, however, was not necessary for either CRH-induced apoptosis or Fas ligand production. Thus, CRH promotes PC12 apoptosis via the CRH type 1 receptor, which induces Fas ligand production via activation of p38.     

Corticotropin-releasing hormone stimulates SGK-1 kinase expression in cultured hippocampal neurons via CRH-R1

Corticotropin-releasing hormone (CRH) has been shown to exhibit various functions in hippocampus. In the present study, we examined the effect of CRH on the expression of serum/glucocorticoid-inducible protein kinase-1 (SGK-1), a novel protein kinase, in primary cultured hippocampal neurons. A dose-dependent increase in mRNA and protein levels of SGK-1 as well as frequency of SGK-1-positive neurons occurred upon exposure to CRH (1 pmol/l to 10 nmol/l). These effects can be reversed by the specific CRH-R1 antagonist antalarmin but not by the CRH-R2 antagonist astressin 2B. Blocking adenylate cyclase (AC) activity with SQ22536 and PKA with H89 completely prevented CRH-induced mRNA and protein expression of SGK-1. Blockage of PLC or PKC did not block CRH-induced SGK-1 expression. Our results suggest that CRH act on CRH-R1 to stimulate SGK-1 mRNA and protein expression in cultured hippocampal neurons via a mechanism that is involved in AC/PKA signaling pathways.     

Expression of urocortin and its receptors in the rat epididymis

Urocortin (UCN; 40 aa) is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated by two types of G-protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of the peptides are mediated and modulated not only by CRH receptors but also by a highly conserved CRH-binding protein (CRHBP). The aim of the present study was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot, RT-PCR and real-time RT-PCR in the rat epididymis. Urocortin, CRHR1 and CRHR2, but not CRHBP, were expressed in all segments of the rat epididymis. Specifically, UCN- and CRHR2-immunoreactivities (IRs) were distributed in epididymal epithelial cells of the caput, corpus and cauda. CRHR1-IR was found in the fibromuscular cells surrounding the epididymal duct and in the smooth musculature of the blood vessels throughout the organ. UCN and CRHR2 mRNA expression levels were higher in the caput and corpus than in the cauda, while CRHR1 mRNA level was higher in the cauda than those in the caput and corpus. In summary, UCN, CRHR1 and CRHR2 are expressed in the rat epididymis. It is suggested that CRH-related peptides might play multiple roles in the maturation and storage of spermatozoa.     

Differential Expression of CRH,UCN, CRHR1 and CRHR2 in Eutopic and Ectopic Endometrium of Women with Endometriosis

Endometriosis is considered as a benign aseptic inflammatory disease, characterised by the presence of ectopic endometrium-like tissue. Its symptoms (mostly pain and infertility) are reported as constant stressors. Corticotropin releasing hormone (CRH) and urocortin (UCN) are neuropeptides, strongly related to stress and inflammation. The effects of CRH and UCN are mediated through CRHR1 and CRHR2 receptors which are implicated in several reproductive functions acting as inflammatory components. However, the involvement of these molecules to endometriosis remains unknown. The aim of this study was to examine the expression of CRHR1 and CRHR2 in endometriotic sites and to compare the expression of CRHR1 and CRHR2 in eutopic endometrium of endometriotic women to that of healthy women. We further compared the expression of CRH, UCN, CRHR1 and CRHR2 in ectopic endometrium to that in eutopic endometrium of women with endometriosis. Endometrial biopsy specimens were taken from healthy women (10 patients) and endometrial and endometriotic biopsy specimens were taken from women with endometriosis (16 patients). Τhe expression of CRH, UCN, CRHR1, and CRHR2 was tested via RT-PCR, immunohistochemistry and Western blotting. This study shows for the first time that CRH and UCN receptor subtypes CRHR1β and CRHR2α are expressed in endometriotic sites and that they are more strongly expressed (p<0.01) in eutopic endometrium of women with endometriosis compared to healthy women endometrium at the mRNA and protein level. CRH, UCN, CRHR1 and CRHR2 mRNA were also more highly expressed in ectopic rather than eutopic endometrium (CRH, UCN, CRHR2α: p<0.01, CRHR1β: p<0.05) and protein (CRH and UCN: p<0.05, CRHR1 and CRHR2: p<0.01) in women with endometriosis. These data indicate that CRH and UCN might play an immunoregulatory role in endometriotic sites by affecting reproductive functions such as decidualization and implantation of women with endometriosis.     

Corticotropin-releasing hormone induces proliferation and TNF-alpha release in cultured rat microglia via MAP kinase signalling pathways

We have previously demonstrated that corticotropin-releasing hormone (CRH) receptor 1 (CRH-R1) is functionally expressed in rat microglia. In the present study, we show that CRH, acting on CRH-R1, promoted cell proliferation and tumour necrosis factor-alpha (TNF-alpha) release in cultured rat microglia. Exogenous CRH resulted in an increase in BrdU incorporation compared with control cells, which was observed in a range of concentrations of CRH between 10 and 500 nm, with a maximal response at 50 nm. The effect of CRH on BrdU incorporation was inhibited by a CRH antagonist astressin but not by a cAMP-dependent protein kinase inhibitor H89. Exposure of microglial cells to CRH resulted in a transient and rapid increase in TNF-alpha release in a dose-dependent manner. In the presence of astressin, the effects of CRH on TNF-alpha release were attenuated. CRH effects on TNF-alpha release were also inhibited by specific inhibitors of MEK, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) (PD98059) or p38 mitogen-activated protein kinase (SB203580), but not by H89. Furthermore, CRH induced rapid phosphorylation of ERK and p38 kinases. Astressin, PD98059, and SB230580 were able to inhibit CRH-induced kinase phosphorylation. These results suggest that CRH induces cell proliferation and TNF-alpha release in cultured microglia via MAP kinase signalling pathways, thereby providing insight into the interactions between CRH and inflammatory mediators.     

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.     

Getting closer to affective disorders: the role of CRH receptor systems

Depressive disorders are a leading cause of morbidity and mortality worldwide. Current antidepressant drugs targeting monoamine neurotransmitter systems have a delayed onset of action, and fewer than 50% of the patients attain complete remission after therapy with a single antidepressant. A large body of preclinical and clinical evidence points to a key role of the corticotropin-releasing hormone (CRH) receptor 1 subtype (CRHR1) in mediating CRH-elicited effects in anxiety, depressive disorders and stress-associated pathologies. Genetic modification of CRHR1 function in mice by the use of conventional and conditional knockout strategies enables further analysis of specific elements in the CRH circuitry. The recent characterisation of several selective small-molecule CRHR1 antagonists offers new possibilities for the treatment of anxiety and depression.     

V1b and CRHR1 receptor heterodimerization mediates synergistic biological actions of vasopressin and CRH

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.     

Involvement of CRH receptors in urocortin-induced hyperthermia

The actions of individual corticotropin-releasing hormone (CRH) receptor (CRHR1 and CRHR2) were studied on the hyperthermia caused by urocortin 1, urocortin 2 and urocortin 3 in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, up to 6h. In order to study the possible role of CRH receptors, the animals were treated with a urocortins together with the urocortin receptor inhibitors CRF 9-41, antalarmin and astressin 2B to influence the action of urocortins in initiating hyperthermia. Urocortin 1 at a dose of 2microg caused an increase in colon temperature, maximal action being observed in body temperature at 3h. CRH 9-41 and antalarmin, CRHR1 receptor antagonists, prevented the urocortin-induced increase in colon temperature while astressin 2B (CRHR2 receptor antagonist) was ineffective. Urocortin 2 at a dose of 2microg showed a byphasic action in increase in colon temperature having the first peak between 30 min and 1h and the second peak at 4h following treatment. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 2. Urocortin 3 in a dose of lmicrog increased colon temperature; the maximal effect was observed at 2h. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 3. The results demonstrated that urocortin 1, 2 or 3 when injected into the lateral brain ventricle caused increases in body temperature is mediated by urocortin receptors. The action of urocortin 1 is mediated by CRHR1 receptor, while in the action of urocortin 2 and urocortin 3 CRHR2 receptor is involved.     

A Transcriptome-Wide Screen for mRNAs Enriched in Fetal Leydig Cells: CRHR1 Agonism Stimulates Rat and Mouse Fetal Testis Steroidogenesis

Fetal testis steroidogenesis plays an important role in the reproductive development of the male fetus. While regulators of certain aspects of steroidogenesis are known, the initial driver of steroidogenesis in the human and rodent fetal testis is unclear. Through comparative analysis of rodent fetal testis microarray datasets, 54 candidate fetal Leydig cell-specific genes were identified. Fetal mouse testis interstitial expression of a subset of these genes with unknown expression (Crhr1, Gramd1b, Itih5, Vgll3, and Vsnl1) was verified by whole-mount in situ hybridization. Among the candidate fetal Leydig cell-specific factors, three receptors (CRHR1, PRLR, and PROKR2) were tested for a steroidogenic function using ex vivo fetal testes treated with receptor agonists (CRH, PRL, and PROK2). While PRL and PROK2 had no effect, CRH, at low (approximately 1 to 10) nM concentration, increased expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star in GD15 mouse and GD17 rat testes, and in conjunction, testosterone production was increased. Exposure of GD15 fetal mouse testis to a specific CRHR1 antagonist blunted the CRH-induced steroidogenic gene expression and testosterone responses. Similar to ex vivo rodent fetal testes, ≥10 nM CRH exposure of MA-10 Leydig cells increased steroidogenic pathway mRNA and progesterone levels, showing CRH can enhance steroidogenesis by directly targeting Leydig cells. Crh mRNA expression was observed in rodent fetal hypothalamus, and CRH peptide was detected in rodent amniotic fluid. Together, these data provide a resource for discovering factors controlling fetal Leydig cell biology and suggest that CRHR1 activation by CRH stimulates rat and mouse fetal Leydig cell steroidogenesis in vivo.     

Synergistic role of cAMP and IP(3) in corticotropin-releasing hormone-induced cell shape changes in invertebrate immunocytes

Immunocytes from the mollusc Mytilus galloprovincialis express corticotropin-releasing hormone (CRH) receptor subtype (CRH-R1 and CRH-R2)-like mRNAs. Using computer-assisted microscopic image analysis, we have found that exogenous CRH provokes changes in the cellular shape of immunocytes, and that this response is extracellular Ca(2+)-dependent. The various inhibitors of transduction signaling pathways, i.e. suramin sodium, 2', 5'-dideoxyadenosine, neomycin sulfate, calphostin C, H-89, and wortmannin, completely or partially inhibit these changes. The present findings demonstrate that PKA, PKC, and PKB/Akt are involved in CRH-induced cell shape changes in immunocytes, and that the cellular effect of CRH needs the synergistic action of the two second messengers, cAMP and IP(3).     

Behavioral and cardiac responses after intracerebroventricular corticotropin-releasing hormone (CRH) administration: role of adrenal cortical hormones.

Intracerebroventricularly (icv) administered corticotropin-releasing hormone (CRH) produces a dose-dependent increase in heart rate in association with behavioral activation. The present study was designed to investigate whether these CRH-induced responses are dependent on adrenal function. The effects of adrenalectomy (ADX) and subsequent corticosterone replacement were studied. Administration icv of 300 ng of CRH failed to produce behavioral activation and tachycardia in ADX rats. Corticosterone replacement restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. This latter result may be related to the fact that the baseline heart rate of ADX animals appeared to be significantly higher than that of corticosterone-treated ADX animals. It is concluded that circulating adrenal corticosterone in ADX rats is involved in the expression of the behavioral and cardiac effect of central CRH.     

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.     

Urocortin in the lateral septal area modulates feeding induced by orexin A in the lateral hypothalamus

The intermediate portion of the lateral septum (LSi) contains high levels of urocortin (UCN) peptide and type 2 corticotropin-releasing hormone (CRH) receptor (CRHR2) and has anatomic and functional connections with the lateral hypothalamus (LH). We tested the effect of UCN in the LSi on feeding. Injection of 10 or 30 pmol UCN into LSi significantly decreased feeding in food-deprived rats for 24 h without producing conditioned taste aversion (CTA). Pretreatment with a CRH receptor antagonist, alpha-helical CRH (alpha-hCRH), blocked the inhibitory effect of UCN on deprivation-induced feeding at 1 and 2 h postinjection. Furthermore, UCN in the LSi significantly decreased feeding induced by LH-injected orexin A at 2 and 4 h postinjection, and addition of alpha-hCRH blocked the inhibitory effect of UCN on orexin A-induced feeding. In conclusion, UCN significantly inhibits feeding induced by deprivation and LH-injected orexin A without producing a CTA, an effect that is mediated by CRHR2. These data define the LSi as an important site for UCN-induced anorexia and indicate that LSi UCN may influence orexin A feeding signals in the LH.