Abstract
Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.
Highlights
Hormone-sensitive lipase (HSL) is a multifunctional enzyme that is critically involved in regulating energy homeostasis
We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity
MA-10 cells treated with Bt2cAMP (0.5 mM) for 6 h resulted in 5.3 Ϯ 0.5- and 2.4 Ϯ 0.3-fold increases in phosphorylation of HSL (P-HSL) at Ser-660 and Ser-563 over untreated cells, respectively (Fig. 1A)
Summary
Hormone-sensitive lipase (HSL) is a multifunctional enzyme that is critically involved in regulating energy homeostasis. Oxysterols act as ligands for the liver X receptors, LXR␣ (NR1H3) and LXR (NR1H2), which are members of the nuclear receptor superfamily of transcription factors [23, 24] These receptors form obligate heterodimers with retinoid X receptors (RXRs), and have been shown to regulate the transcription of a number of genes involved in cholesterol utilization and metabolism, including SREBP-1c, ABCA1, apoE, and StAR [23,24,25]. Utilizing MA-10 mouse Leydig tumor cells (a cell line that closely resembles its normal counterpart and has been widely used in studying physiological functions) as an in vitro model, the present studies provide evidence that HSL is primarily involved in catalyzing cholesteryl ester hydrolysis and, by so doing, plays an important role in the regulation of cAMP-mediated StAR expression and steroidogenesis through modulation of LXR pathways
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