Involvement of 5-lipoxygenase metabolites of arachidonic acid in cyclic AMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression

To understand the mechanism for the role of arachidonic acid (AA) in steroidogenic acute regulatory (StAR) gene transcription, sections of the -1/-966 StAR promoter were deleted to produce constructs of -1/-426, -1/-211, -1/-151, and -1/-110 and inserted into the PGL3 vector to drive luciferase expression. Results indicated that -1/-151 StAR promoter contains the elements that are most responsive to AA. Electrophoretic mobility shift assays using nuclear extracts from AA-treated MA-10 Leydig tumor cells showed that AA enhanced specific binding of the nuclear extract to a 30bp (-67/-96) sequence of the StAR promoter. Also, HPLC was used to identify AA metabolites involved in StAR gene transcription. It was found that 1mM N6,2-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) significantly increased the 5-lipoxygenase metabolites, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Moreover, in the presence of 0.2mM dbcAMP addition of 20μM 5-HPETE or 5-HETE significantly enhanced StAR protein expression and progesterone production (P<0.05). Similar results were obtained for StAR gene transcription with StAR mRNA levels and StAR promoter activities being significantly increased (P<0.05) when 5-HPETE was added to MA-10 cell cultures. In summary, the present studies demonstrated that cyclic AMP (cAMP) stimulated the production of the AA metabolites, 5-HPETE and 5-HETE, and showed that these metabolites enhanced StAR gene expression and steroid hormone production. The results further suggested that the AA-responsive element resides in the -67/-96 region of the StAR promoter.