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

Abstract

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 30 bp (−67/−96) sequence of the StAR promoter. Also, HPLC was used to identify AA metabolites involved in StAR gene transcription. It was found that 1 mM 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 0.2 mM dbcAMP addition of 20 μM 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.