Inhibition of Thromboxane A Synthase Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Abstract

The cyclooxygenase-2 (COX2)-dependent inhibition of Leydig cell steroidogenesis has been demonstrated. To understand the mechanism for this effect of COX2, the present study examined the role of an enzyme downstream of COX2, namely thromboxane A synthase (TBXAS), in steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate induced a concentration-dependent increase in cAMP-induced steroidogenic acute regulatory (StAR) protein in MA-10 mouse Leydig cells. The increase in StAR protein occurred concomitantly with a significant increase in steroid hormone production. Similar results were obtained in StAR promoter activity assays and RT-PCR analyses of StAR mRNA levels, suggesting that inhibition of TBXAS activity enhanced StAR gene transcription. These observations were corroborated when TBXAS expression was specifically inhibited by RNA interference. Although the RNA interference reduced mRNA levels of TBXAS, it increased StAR mRNA levels, StAR protein, and steroidogenesis. Additional studies indicated that inhibition of TBXAS activity reduced DAX-1 protein, a repressor in StAR gene transcription. In the absence of cAMP, inhibition of TBXAS activity did not induce a significant increase in steroid hormone and StAR protein. However, addition of a low level of cAMP analogs dramatically increased steroidogenesis. Lastly, inhibition of protein kinase A activity essentially abolished the steroidogenic effect of the TBXAS inhibitor. Thus, the results from the present study suggest that a minimal level of protein kinase A activity is required for the steroidogenic effect of the TBXAS inhibitor and that inhibition of TBXAS activity or its expression increase the steroidogenic sensitivity of MA-10 mouse Leydig cells to cAMP stimulation.