PLATFORM SESSION 4: GONADAL GENE REGULATION AND FUNCTION
Chair: Zeleznik, Tony1, 1
Co-chair: Bowen, Jennifer1, 1
4:30 PM-6:30 PM
(24) REGULATION OF TRANSCRPTIONAL ACTIVITY OF THE CYTOCHROME P450 SIDE-CHAIN CLEAVAGE AND STEROIDOGENIC ACUTE REGULATORY PROTEIN GENES IN GENETICALLY MODIFIED STABLE PORCINE GRANULOSA CELLS.
Chedrese, Jorge1, Agostini, Maria1, Smida, Andrea1, Bartlewski, Pawel1, LaVoie, Holly2, 1 Dept of Obstetrics, Gynecology & Reproductive Sciences, University of Saskatchewan, Saskatoon, Canada2 University of South Carolina, Dept of Cell Biology & Neuroscience, School of Medicine, Columbia, SC
ABSTRACT- Immortalized porcine granulosa cells, the JC-410, were stably transfected with genomic constructs containing the cytochrome P450 side-chain cleavage (P450scc) or steroidogenic acute regulatory (StAR) protein genes linked to the luciferase (LUC) reporter gene. JC-410 cells were co-transfected with the 2320(bp)-P450scc-LUC or 1423(bp)-pStAR-LUC, and a selectable marker, pSV-neo. The transfected colonies were isolated after culturing with the neomycin analogue, Geneticin. The expression of LUC and responsiveness to the protein kinase-A activator, cholera toxin (CT), were determined by a luminometric assay. The cell lines with RLU>10 000 (light emission during 10 or 20 sec, for P450scc and pStAR, respectively) were used in the present experiments. CT (100 ng/ml) and estradiol-17 (E2; 30 M) increased (P<0.05) the transcription of P450scc after 24 h of incubation (1.3- and 1.7-fold, respectively). Insulin (5 g/ml) increased transcription by 2.3-fold (P<0.05), and potentiated the effects of E2 (1.9- vs. 4.2-fold) and CT (2.5- vs. 3.0-fold; -insulin vs. +insulin, respectively), after 48 h. The 24-h treatment with E2 (10 or 30 M) or CT (30 or 100 ng/ml) resulted in 1.5-fold increases (P<0.05) in the transcription of pStAR. Insulin had no effect (P>0.05), but it further enhanced the effects of 30 M E2 or CT (2.0-fold; P<0.05). In summary, both CT and E2 directly stimulated transcriptional activity of P450scc and pStAR in genetically modified JC-410 cells. Insulin increased the transcription of P450scc and had the additive effect on CT- and E2-induced transcription of P450scc and pStAR. The JC-410 cells stably transfected with P450scc and pStAR provide a useful tool for studying the function of steroidogenic genes and regulatory potencies of a broad range of steroidogenesis-modulating agents.
KEY WORDS: stable porcine granulosa cells, cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, gene function