Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene.

A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.

Tissue distribution and hormonal regulation of messenger ribonucleic acid for regulatory and catalytic subunits of adenosine 3',5'-monophosphate-dependent protein kinases during ovarian follicular development and luteinization in the rat.

cDNA probes specific for the regulatory (R) subunits [RII51; (mol wt, 51,000) and RI (mol wt, 49,000)] and a catalytic (C alpha) subunit of cAMP-dependent protein kinases were used to analyze the hormonal regulation, tissue distribution, and content of mRNAs for these kinase subunits in the rat ovary. Filter hybridization assays demonstrated that mRNA specific for RII51 increased in both thecal and granulosa cells of preovulatory (PO) follicles, then declined precipitously (less than 10-fold) in both cell types within 7 h after an ovulatory (10-IU) dose of hCG and remained low in corpora lutea. Dose-response studies showed that doses of FSH greater than 2 micrograms were required to increase RII51 mRNA in granulosa cells of hypophysectomized (H) rats, whereas doses of 0.5-1.0 micrograms were effective in granulosa cells of H estradiol-treated (HE) rats. At all doses (0.5-50 micrograms) of FSH administered, RII51 mRNA was 4 times higher in granulosa cells of HE rats than in H rats. Solution hybridization assays demonstrated that the concentration of RII51 mRNA increased 6-fold from 20 molecules/granulosa cell in H rats to 120 molecules/cell in H rats treated with estradiol and FSH (HEF). Transcription assays using nuclei of granulosa cells demonstrated further that the 5- to 10-fold increases in RII51 mRNA content in estradiol-/FSH-induced granulosa cells was associated with increased transcription of the RII51 gene. In thecal cells of small antral (SA), PO, and luteinizing follicles, changes in the content of mRNA for RI and C alpha kinase subunits showed a pattern similar to that for RII51. However, in granulosa cells, mRNA specific for RI and C alpha was highest in SA follicles, declined in PO follicles, and remained unchanged during luteinization. The content of mRNA for RI (45-70 molecules/cell) and C alpha (10 molecules/cell) also changed less than 2-fold in granulosa cells of H, HE, and HEF rats. In summary, these results indicate that the content of RII51 mRNA is hormonally regulated in both thecal cells and granulosa cells during follicular development and luteinization. Low concentrations of gonadotropins increase RII51 mRNA, whereas an ovulatory dose of hCG causes mRNA for RII51 to decrease rapidly. In granulosa cells, induction of mRNA for RII51, but not that for RI and C alpha is induced by the actions of estradiol and FSH, and involves increased transcription of the RII51 gene.

Cholesterol side-chain cleavage P450 messenger ribonucleic acid: evidence for hormonal regulation in rat ovarian follicles and constitutive expression in corpora lutea.

Using an affinity-purified antibody against cholesterol side-chain cleavage P450 (P450scc) and a human P450scc cDNA probe, 11 rat P450scc cDNA clones were identified and isolated from our rat granulosa cell lambda gt11 cDNA expression library. Two clones were plaque purified and subcloned into pBR322. One of these P450scc cDNA clones, approximately 1.2 kilobases (kb) in size, was used as a probe for Northern and filter hybridization assays to analyze the tissue distribution and hormonal regulation of P450scc mRNA in rat ovarian follicles and corpora lutea. Northern transfers revealed a single P450scc mRNA species about 2.0 kb in size. Filter hybridization assays showed that P450scc mRNA was low in granulosa cells and thecal cells of small antral follicles, was increased in both tissues of preovulatory follicles, and was rapidly (within 7 h) and maximally increased (30-fold) during hCG-induced luteinization. P450scc enzyme and mRNA were also elevated in corpora lutea isolated from pregnant rats (days 4-22 of gestation) and rats 1 day after parturition (day 23). The elevation of P450scc enzyme and mRNA was maintained despite the marked decline in serum progesterone concentrations between days 19-22, suggesting that once P450scc mRNA is induced in luteal tissue it may be constitutively expressed. Administering hormones to granulosa cells in culture and to hypophysectomized immature rats in vivo demonstrated that the induction of P450scc mRNA by FSH in granulosa cells was time, dose, and estradiol dependent. High doses of FSH acting on estradiol-primed cells gave the greatest response. The increase in P450scc mRNA in cultured granulosa cells was also stimulated by forskolin and was directly associated with increased synthesis of cAMP and progesterone accumulation. Thus, whereas the induction of P450scc mRNA in granulosa cells was dependent on hormones and cAMP, the maintenance of P450scc mRNA and P450scc protein in corpora lutea appears to involve constitutive expression of P450scc mRNA.