Egr-1 Induction in Rat Granulosa Cells by Follicle-Stimulating Hormone and Luteinizing Hormone: Combinatorial Regulation By Transcription Factors Cyclic Adenosine 3′,5′-Monophosphate Regulatory Element Binding Protein, Serum Response Factor, Sp1, and Early Growth Response Factor-1

Abstract

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within -164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at -64/-46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the -164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE-131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

FSH signaling pathways in immature granulosa cells that regulate target gene expression: Branching out from protein kinase A

“…2, and not by alternate CREB kinases downstream of Akt, ERK, p38 MAPK, or PKC. However, cAMP response elements have been identified in only a small subset of FSH-regulated genes, namely inhibin-α [36], aromatase [37], GIOT-1 [12], Egr-1 [9], and c-fos [38]. Thus, CREB is not sufficient to activate the majority of FSH target genes.…”

“…1. Activated target genes in mural granulosa cells encode proteins such as G-protein-coupled receptors (GPCRs) like that for luteinizing hormone (LH) [4]; intracellular signaling proteins such as the type II beta regulatory subunit (RIIβ) for protein kinase A (PKA) [5], phosphodiesterase (PDE) 4D [6], serum glucocorticoid kinase (SGK) [7], and the A kinase anchoring protein (AKAP) microtubule-associated protein (MAP) 2D [8]; transcription factors such as early growth response factor (Egr)-1 [9], liver receptor homolog (LRH)-1 [10,11], and gonadotropininduced ovarian transcription factor-1 (GIOT-1) [12]; immediate early gene products such as c-Fos and c-Jun [13] and c-Myc [14]; autocrine factors such as epiregulin [15] and vascular endothelial growth factor (VEGF) [16]; the alpha and beta subunits of the heterodimeric hormone inhibin [17,18]; cell cycle proteins such as cyclin D 2 [19]; the extracellular matrix protein cartilage link protein (Crtl1) [20]; and rate-limiting enzymes that regulate steroidogenesis, such as P-450 aromatase for estrogen production [21] and P-450 cholesterol side chain cleavage (SCC) for progesterone production [22].…”

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

“…FSH binds its GPCR, leading to the activation of PKA via adenylyl cyclase-mediated cAMP production (18). Transcriptional responses to FSH are dependent upon activation of PKA, based on the ability of the catalytic inhibitor PKI or H89 to inhibit target gene expression in cultured GCs (9,13,19,20). Likewise, lentiviral expression of a constitutively active PKA catalytic subunit mutant (PKA-CQR) closely mimics gene expression patterns of FSH (21), suggesting that PKA is necessary and sufficient for most transcriptional actions of FSH.…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…The gonadotrophs of these mice are atrophic and do not synthesize LH. Egr-1 is involved in GnRH-dependent stimulation of LHβ gene expression as was shown by several laboratories (Tremblay and Douin, 1999;Dorn et al, 1999;Russell et al, 2003;Mouillet et al, 2004). Although GnRH neurons were detected in the Egr-1 knockout mice, the function of these neurons was not examined.…”