RAT CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME-P-450 (P-450SCC) GENE - STRUCTURE AND REGULATION BY CAMP INVITRO

The entire rat P-450scc gene has been cloned, positions/sequences of the exon-intron boundaries (I-IX) are described and 940 base pairs (bp) of 5'-flanking DNA have been sequenced, compared to mouse, bovine and human genes, and analyzed by functional assays. Primer extension analysis mapped the transcription start site 32 bp upstream of the initiator methionine codon. The rat P-450scc promoter was ligated to the human growth hormone (GH) gene yielding p-940RsccGH. This rat P-450scc fusion gene and a mouse gene (p-1500MsccGH) were transiently transfected into primary cultures of rat granulosa cells and Y1 adrenal cells. In the Y1 cells primer extension analysis showed that the rat P-450sccGH gene was expressed at lower basal levels than that of the mouse gene but showed greater stimulation (4-8-fold) in response to 8-bromo-cyclic AMP than the mouse (3-4-fold). Similar results were obtained when the fusion genes were transfected into primary cultures of rat granulosa cells and production of GH was measured in the media of the cells stimulated with forskolin. Furthermore, we document that gonadotropins (follicle-stimulating hormone/luteinizing hormone) can induce luteinization of granulosa cells in vitro, that this process is associated with constitutive maintenance of P-450scc mRNA in the absence of hormones/cAMP, that these events associated with luteinization are differentiation-stage specific and occur only in granulosa cells of preovulatory follicles but not in small antral follicles, that the process can be inhibited by cycloheximide if the protein synthesis inhibitor is present during the first 6 h of exposure to luteinizing hormone but not if added for short durations (3-5 h) later, and that once luteinization is induced P-450scc mRNA expression and progesterone biosynthesis are not strictly dependent on cAMP. Thus, the P-450scc gene is regulated by both cAMP-dependent and cAMP-independent mechanisms, each of which are associated with a specific stage of granulosa cell differentiation. The DNA domains involved in regulating these two diverse processes remain to be determined. Although there was remarkable sequence homology among rat, mouse, bovine, and human genes within 70 bp of the transcriptional start site, no other sequence similarities revealed conserved functional domains among the four genes. Although some cAMP-responsive element-like sequences are present in the rat gene, these were not conserved in the other species; including the mouse which showed high sequence homology with the rat throughout 900 bp of 5'-flanking DNA. Thus, the cAMP domains specific to this and other steroidogenic genes remain to be clearly identified.