Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells.
CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.
Westphal
Nicole J
NJ
University of Michigan, Neuroscience Program, Molecular and Behavioral Neuroscience Institute, Ann Arbor, Michigan 48108, USA.
Seasholtz
Audrey F
AF
D051698:Activating Transcription Factor 2 / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction; D015854:Up-Regulation
D051698:Activating Transcription Factor 2; D051792:Basic Helix-Loop-Helix Proteins; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D007987:Gonadotropin-Releasing Hormone; D020929:Mitogen-Activated Protein Kinase Kinases; D009687:Nuclear Proteins; D012333:RNA, Messenger; C075668:Tcf3 protein, mouse; C067133:corticotropin releasing factor-binding protein