Submission Number: NAT-4-11-12

Abstract Number: 433

INTERACTIVE REGULATION OF THE LOW-DENSITY LIPOPROTEIN RECEPTOR (LDL-R) GENE PROMOTER BY LH AND IGF-I/INSULIN IN PRIMARY CULTURES OF PORCINE GRANULOSA-LUTEAL CELLS.

Sekar Natesampillai* and Johannes D Veldhuis*

Division of Endocrinology, Dept. of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908 ¹

Abstract:
Insulin-like growth factor type I (IGF-I) and insulin can amplify gonadotropin-stimulated progesterone biosynthesis by selectively augmenting the expression of key steroidogenic genes in (porcine) granulosa-luteal cells; viz., LDL-R, StAR and P450scc, but not SCP-2. Here, we investigate LDL-R promoter regulation by insulin/IGF-I and/or LH in transiently transfected primary cultures of porcine granulosa-luteal cells. LH and LH and insulin or IGF-I (each 100 ng/ml) increased expression of a full-length LDL-R promoter (-1076 bp) driving a firefly luciferase reporter by 8- to 20- fold, as normalized by Renilla luciferase coexpression. Analysis of multiple 5'-nested deletion constructs of the LDL-R promoter showed that deletion of -139 upstream of the translational start site virtually abolished LDL-R promoter responsiveness to LH and IGF-I/insulin. Further deletional analysis revealed that the joint hormone-responsive regions were localized between -255 and -139 bp of LDL-R promoter. Treatment with the non-LH agonists, 8 Br-cAMP (1 mM) or forskolin (10 M) with or without insulin/IGF-I also augmented LDL-R promoter expression. Since insulin and IGF-I also enhance LH-induced cAMP accumulation, we tested intracellular cAMP's possible role as a necessary mediator of LH-driven LDL-R promoter expression. Cotransfection of LDL-R promoter with a Protein Kinase Inhibitor (PKI) minigene driven by RSV markedly inhibited LH and/or IGF-I/insulin-stimulated luciferase activity. Cotransfection of an RSV/PKI mutant gene with the LDL-R promoter, restored 60-80% of maximal luciferase activity. Since LDL-R expression is regulated by the abundance of intracellular free cholesterol, we assessed the impact of 30-min pretreatment of LDL-R promoter (-1076 bp)-transfected cells with 25-hydroxycholesterol (1 and 10 mM). Exogenous sterol markedly attenuated bi-hormonally driven LDL-R promoter expression, thus affirming a feedback-sensitive and powerful sterol-repressive region in this gene. In summary, insulin and IGF-I can interact with LH via the PKA signaling pathway to up regulate LDL-R gene promoter trans-activation in granulosa-luteal cells in vitro.

Keywords: LDL-R promoter, granulosa, insulin/IGF-I, LH

This abstract is being presented at: 3:00 PM in session:
SESSION 16: GENE EXPRESSION: TISSUE SPECIFICITY AND REGULATION OF STEROIDOGENESIS