| StAR is the rate-limiting step in steroid production and is transcriptionally down regulated by toxin exposure. StAR transports cholesterol across the mitochondria membrane for metabolism into steroids. We cloned the entire coding region of largemouth bass (LMB) StAR and used this sequence to develop a real-time PCR assay to quantify StAR mRNA levels in LMB ovarian follicle cultures. Exposure to dbcAMP and TGF-beta, two potent signaling molecules known to regulate mammalian steroidogenesis, modulate LMB StAR. TGF-beta down regulates and dbcAMP upregulates StAR mRNA. A polyclonal antibody specific to LMB StAR was developed to measure protein levels by western blot. To further analyze the regulation of LMB StAR, a 3 kb portion of the promoter was cloned. In silico analysis of this segment with other StAR promoters available in the database showed potential conserved regulatory sites that imply control by a wide range of transcription factors. The 3 kb promoter segment was transfected into Y-1 cells, a mouse adrenalcortical cell line and tested with dbcAMP and TGF beta. The 3 kb construct responded positively to dbcAMP but was not significantly impacted by TGF-B exposure compared to the 1.8 kb length promoter. Mutation of potential regulatory sites in the promoter, including ERE (estrogen response elements), ROR (retinoic acid related receptor), and COUP-TF (chicken ovalbumin upstream promoter) sites were tested for their role in cAMP and TGF-beta signaling. Together, these data suggest that one way toxins may repress steroid synthesis, and more specifically StAR, is through TGF-beta signaling. |
