Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: Possible convergence of protein kinase A, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways

Insulin and insulin-like growth factor I(IGF-I) can amplify gonadotropin-st imulated steroidogenesis by augmenting the expression of key sterol regulat ory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, st eroidogenic acute regulatory protein, and P450 cholesterol side-chain cleav age enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal int eractions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act v ia concerted transcriptional control of promoter expression. To this end, w e transiently transfected primary monolayer cultures of porcine granulosalu teal cells with a reporter vector containing the putative 5 ' -upstream ful l-length (pLDLR1076/luc) regulatory region(-1076 to +11 bp) of the homologo us LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH a nd insulin (or IGF-I) stimulated LDL receptor transcriptional activity maxi mally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luc iferase. Further analysis of multiple 5 ' -nested deletional constructs of the LDL receptor gene promoter showed that deletion of - 139 bp upstream of the transcriptional start site virtually abolished basal expression and pr omoter responsiveness to LH and insulin/IGF-I. In contrast, full basal acti vity and 60-80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other ti ssues is negatively regulated by the abundance of intracellular free choles terol, we assessed the impact of concomitant pretreatment of granulosa-lute al cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 muM). Excess sterol markedly (50-70%) attenuated bihormonally and, in lesse r measure, LH-stimulated and basal LDL receptor promoter expression, thus a ffirming a feedback-sensitive sterol-repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM) , and forskolin (10 muM) with or without insulin/IGF-I costimulation likewi se augmented LDL receptor promoter expression with similar strong dependenc y on the -255 to -139 bp 5 ' -upstream region. To assess more specific PKA- dependent mediation of LH's contribution to combined hormonal drive, the LD L receptor (- 1076 to +11 bp) reporter plasmid was cotransfected with a ful l-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven con stitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal tra nscriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To inve stigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected gr anulosa-luteal cells were pretreated for 30 min with two specific inhibitor s of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 mu M), or of mitogen-activated protein kinase kinase, PD 98059 (50 muM), U0126 (10 muM), or the latter's inactive derivative, U0124 (10 muM). Both classe s of antagonists impeded the ability of insulin or IGF-I to enhance LH-stim ulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in p orcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA r egions located between -255 and -139 bp 5 '- upstream of the transcriptiona l start site, 2) without abrogating sterol-sensitive repressive of this pro moter, and 3) by way of intracellular mechanisms that include the PKA, phop hatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pa thways.