Abstract
This study was conducted to examine the mechanism for arachidonic acid (AA) regulation of steroidogenic acute regulatory (StAR) protein expression and the relationship between AA and cAMP in hormone-induced steroidogenesis. Dibutyryl cyclic AMP (Bt(2)cAMP)-stimulated MA-10 Leydig cells were treated with AA and/or the phospholipase A(2) inhibitor, dexamethasone. Dexamethasone significantly reduced Bt(2)cAMP-stimulated progesterone production, StAR promoter activity, StAR mRNA, and StAR protein. The inhibitory effects of dexamethasone were reversed by the addition of 150 microm AA to MA-10 cells. In addition, MA-10 cells were treated with the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), the 5-lipoxygenase inhibitor, AA861, the epoxygenase inhibitor, miconazole, and the cyclooxygenase inhibitor, indomethacin. Both NDGA and AA861 inhibited progesterone production and StAR protein expression. AA861-inhibited progesterone synthesis and StAR protein were partially reversed by addition of the 5- lipoxygenase metabolite, 5(S)-hydroperoxy-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid. Inhibition of epoxygenase activity inhibited progesterone production significantly, but StAR protein was only slightly reduced. Indomethacin enhanced StAR protein expression and significantly increased progesterone production. Inhibition of AA release or lipoxygenase activities did not affect protein kinase A activity, whereas inhibition of protein kinase A activity using H89 reduced Bt(2)cAMP-induced StAR protein. AA alone did not induce StAR protein expression nor steroid production. These results demonstrate the essential role of AA in steroid biosynthesis and StAR gene transcription and suggest the possible involvement of the lipoxygenase pathway in steroidogenesis. This study further indicates that AA and cAMP transduce signals from trophic hormone receptors to the nucleus through two separate pathways and act to co-regulate steroid production and StAR gene expression and indicates that both pathways are required for trophic hormone-stimulated steroidogenesis.
Highlights
The steroidogenic acute regulatory protein (StAR)1 plays a critical role in trophic hormone-stimulated steroid biosynthesis by facilitating the transfer of cholesterol, the substrate for all steroid hormones, to the inner mitochondrial membrane where it is converted to pregnenolone by the P450 side chain cleavage enzyme (P450scc) [1,2,3,4]
Effects of Arachidonic Acid on StAR Protein Expression and Steroid Production—To determine the effects of arachidonic acid (AA) release on StAR protein expression and steroid hormone production, Bt2cAMP-stimulated MA-10 mouse Leydig cells were treated with the PLA2 inhibitor, dexamethasone, which inhibits AA release from phospholipids
In order to confirm that the inhibitory effects of dexamethasone were due to the inhibition of AA release, 150 M AA was added to the Bt2cAMP-stimulated MA-10 cells treated with 1.0 M dexamethasone
Summary
StAR, steroidogenic acute regulatory protein; Bt2cAMP, dibutyryl cyclic AMP; AA, arachidonic acid; NDGA, nordihydroguaiaretic acid; PKA, protein kinase A; PLA2, phospholipase A2; LH, luteinizing hormone; bp, base pair; 5-HPETE, (6E,8Z,11Z,14Z)eicosatetraenoic acid; PBS, phosphate-buffered saline; RIA, radioimmunoassay. Since previous studies suggested that AA and its metabolites act at the ratelimiting step of steroid biosynthesis, cholesterol transfer to the inner mitochondrial membrane [14, 15], we reasoned that the StAR protein might be involved in the role of AA in the regulation of steroidogenesis. This hypothesis was proven correct by our recent studies, which indicated that AA release is essential for StAR protein expression [16]. We once again demonstrate that both pathways are required for trophic hormonestimulated steroid production and StAR gene expression
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