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 20 microM 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.
Wang
Xing Jia
XJ
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. xingjia.wang@ttmc.ttuhsc.edu
Dyson
Matthew T
MT
Jo
Youngah
Y
Eubank
Darrell W
DW
Stocco
Douglas M
DM
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001095:Arachidonic Acids / Q000378:metabolism*; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D017931:DNA Primers; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D007700:Kinetics; D007984:Leydig Cell Tumor; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D016254:Mutagenesis, Insertional; D010051:Ovarian Neoplasms; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D000098682:Steroidogenic Acute Regulatory Protein
D001095:Arachidonic Acids; D017931:DNA Primers; D006893:Hydroxyeicosatetraenoic Acids; D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D000098682:Steroidogenic Acute Regulatory Protein; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D003994:Bucladesine; D000242:Cyclic AMP; D001094:Arachidonate 5-Lipoxygenase; D008156:Luciferases