Corticotropin-releasing hormone (CRH) and urocortins (UCN) bind to two types of GPCRs (CRH-R1 and CRH-R2), activating a plethora of signalling cascades, including the mitogen-activated protein kinases (MAPKs) pathways. Although previous studies have identified that CRH directly regulates adrenal steroidogenesis1, the second messenger systems and downstream effectors remain poorly defined. The adrenal cell line H295R, which express both CRH-Rs1 was used to investigate CRH-R signalling characteristics and UCN-I effects on enzymes involved in adrenal steroidogenesis. Confocal analysis identified receptor hotspots on the plasma membrane and diffuse, low level specific staining throughout the cytoplasm, which may represent unprocessed receptors or cytoplasmic CRH-R variants. Functional coupling of the activated receptors to Gs/adenylyl cyclase resulted in a 34 fold increase in cAMP levels following treatment of cells with 100 nM UCN-I. Using phospho-specific antibodies for ERK1/2 we observed a transient increase in ERK1/2 activation (maximal response 47 fold above basal at 510 min) in response to UCN-I (100 nM). However, UCN-I did not induce any significant activation of p38 MAPK. Confocal microscopy studies showed that active ERK1/2 was exclusively localised to the cytoplasm at the time points measured (030 min). UCN-I treatment for 4 h significantly induced StAR mRNA and protein levels, and appeared to involve ERK1/2 activation since pretreatment with the specific MEK1/2 inhibitor (U0126) abolished this effect. Real-time PCR experiments also showed that treatment of H295R cells with 100 nM UCN-I for 24 h significantly up-regulated CYP11A, CYP17 and 3βHSD. However, ERK1/2 activation was found to be important for CYP17 and 3(HSD but not CYP11A up-regulation. We conclude that H295R cells express functional CRH-Rs, which couple to both cAMP and ERK1/2 signalling pathways. UCN-I can induce expression of specific enzymes involved in adrenal steroidogenesis through an ERK1/2-dependent pathway.
(1) Willenberg HS et al., Neuroendocrinology 2005 (82) 274281.
03 - 07 May 2008
European Society of Endocrinology