Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: Promoter activation, messenger ribonucleic acid stability,and sterol feedback

Insulin-like growth factor I(TGF-I) and the gonadotropin, FSH, can synergiz e to stimulate progesterone production in primary cultures of maturing gran ulosa cells. These trophic hormones increase law density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne chol esterol by granulosa cells. To determine whether and how IGF-I and FSH cont rol the genomic expression of the LDL receptor, we evaluated their individu al and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-fr ee) cultures of porcine granulosa cells. Ribonuclease protection assays rev ealed that LDL receptor mRNA accumulation was increased by human recombinan t IGF-I(100 ng/ml), FSH (25 ng/ml NIDDK sFSH-20), or their combination by 2 .2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54-75% by the concurrent addi tion of LDL substrate (50 mu g/ml). The combination of FSH and IGF-I signif icantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5'-ends, PCB with adapter-lig ated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1 076 bp of the porcine LDL receptor gene upstream of the coding region. In t ransient transfection assays, with a pLDLR1076/luciferase plasmid construct , FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mM) treatment (but not IGF-I alon e) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of th e LDL receptor gone promoter increased and eventually surpassed hormone-sti mulated effects, but was suppressed by LBL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between -255 and -139 upstream o f the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa c ells Even in the presence of sterol negative feedback and can do so mechani stically by a combination of promoter activation and increased mRNA stabili ty.