Intracellular calcium ions (Ca2+) regulate steroidogenesis in the placenta, adrenal gland, testis, and ovary. Earlier data indicate that Ca2+/calmodwlin-dependent protein kinase (CamK) may mediate Ca 2+-dependent up-regulation of CYP11A (cholesterol side-chain cleavage). To examine this notion further, we assessed the expression and actions of isotype-specific CamK on in vitro transcription of the swine CYP11A gene promoter in primary cultures of ovarian granulosa-luteal cells. RT-PCR and oligodeoxyeucleotide sequencing identified gene transcripts encoding CamKII and IV in granulosa and theca cells and corpora lutea. DNA sequence homology with the cognate human and rat genes was 97 and 94% (CamKII) and 96 and 88% (CamKIV), respectively. SDS-PAGE and isoform-specific immunoblotting corroborated expression of CamKII (-52 kDa) and CarnKIV (∼60 kDa) proteins. To monitor transcriptional control, granulosa-luteal cells were transfected transiently with a putative 5′-upstream regulatory region of the homologous CYP11A gene -2320 to +23 bp from the transcriptional start site driving luciferase (CYP11A/luc). Coexpression of constitutively active CamKIV elevated basal transcription by 3.5 ± 0.2-fold (P < 0.001), whereas inactive mutant CamKIV and native CamKII had no effect. Forskolin, an activator of adenylyl cyclase, stimulated expression of CYP11A/luciferase by 4.5 ± 0.9-fold (P < 0.001) and did not enhance transcriptional drive by exogenous CamKIV. Preliminary promoter-deletional analyses showed that a proximal 5′-fragment -100 to +23 bp, but not -50/+23 bp, retained full responsiveness to CamKIV (4.5 ± 0.4-fold; P < 0.001). Threefold cotransfection of -100/+23 bp CYP11A/luciferase, active CamKIV, and a dominant-negative mutant of the cAMP-responsive element binding protein (10, 100, and 250 ng) inhibited CamKIV-stimulated transcriptional activity by 17, 47, and 48% (pooled SEM± 2%) [P < 0.01]. The dominant-negative mutant of the cAMP-responsive element binding protein also repressed forskolin's stimulation of -100/+23 CYP11A/luciferase by 12,38, and 52% (P < 0.01). Based on these ensemble outcomes, we postulate that endogenous CamKIV may serve as a Ca2+-dependent effector mechanism to maintain basal CYP11A gene expression in ovarian granulosa-luteal cells.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism