Insulin-like growth factor-I increases expression of the porcine P-450 cholesterol side chain cleavage gene through a GC-rich domain.

R J Urban,M A Shupnik,Y H Bodenburg

doi:10.1016/s0021-9258(18)47313-9

Abstract

The promoter/regulatory region of the porcine P-450 cholesterol side chain cleavage (P450scc) gene was cloned from a porcine genomic library. This gene contains a GC-rich region (-130-100) that mediates insulin-like growth factor I (IGF-I) and cAMP stimulation of gene expression in porcine granulosa cells. Stimulation of gene expression by cAMP occurs in 6 h, while IGF-I stimulation occurs in 24-48 h. This region is also responsive to insulin but not fibroblast growth factor. The effects of IGF-I and cAMP on gene expression in porcine granulosa cells are additive. In Y1 adrenal cells, the same region is responsive to cAMP but not to IGF-I. Gel shift assays using an oligonucleotide of this region and nuclear extract protein from porcine granulosa and Y1 adrenal cells identified three DNA-protein complexes (C1-C3). The binding activity of the complexes did not change with IGF-I or forskolin treatment of granulosa cells. Mutational analysis results were consistent with IGF-I regulating gene expression through the C2 DNA protein complex. Moreover, this complex binds differently in gel shift assay to mutant oligonucleotides with porcine and Y1 and nuclear extract protein. We conclude that IGF-I stimulates porcine P450scc gene expression through a distinct, cell-specific protein complex binding to a GC-rich domain.

Highlights

The promoter/regulatory region of the porcine P-450 factor I (IGF-I) is produced locally in the ovary and regulates cholesterol side chain cleavage (P45Oscc) genewas granulosa cell function and steroidogenes(i2s) .IGF-I increases cloned from a porcine genomic library
Gel shift assays using an oligonucleotideof this region and nuclear extract proteinfrom porcine granulosa andY1 adrenal cells identified three DNAaprotein complexes
Searching for known consensus binding sitesfor transcription factors showed a presumptive GC box from position -119/-109 (Fig. 1).This GC boxdiffered fromGC boxes found in thebovine and humanP45Oscc gene by only one G to A substitution [13, 17]

Summary

EXPERIMENTAL PROCEDURES

Materials-All restriction enzymes wereobtained from Bethesda Research Laboratories. Nitrocellulose filters were obtained from Micron Separations, Inc.(Westboro, MA). The 1.5-kb DNA fragment was sequenced by the dideoxy method of Sanger et al [28]using double-stranded DNA sequencingwith a Sequenase version 2 sequencing kit (United States Biochemical, Cleveland, OH) This DNAfragment contained 200 bpupstream of the transcriptional start site of porcineP45Oscc (see "Results").This 200 bp regiown as used to screen additional clones as described above. The control plasmid contained the mouse metallothionein I promoter with the humangrowth hormone gene [36] This promoter is expressed in Y1 adrenal cells [15, 16].The precipitate was removed after 24 h, and fresh medium was added with the appropriate hormones but without 15% horse serum. Probability values of 50.05 were consideredstatistically significant.Data are presented as mean S.E

RESULTS

PORCINE GRANULOSA CELLS

Data are presented as the mean

DISCUSSION