REGULATION OF START DOMAIN PROTEINS STARD1, STARD4, and STARD6 IN HUMAN LUTEINIZED GRANULOSA CELLS

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Basic/Translational
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-379
Bo Shi*1, Richard J. Kordus1, Gail F. Whitman-Elia1, Douglas M. Stocco2, Steven R. King2 and Holly A. LaVoie1
1University of South Carolina School of Medicine, Columbia, SC, 2Texas Tech University Health Sciences Center, Lubbock, TX
Luteal cells actively manage their cholesterol supplies to ensure adequate production of progesterone (P4) to support pregnancy.  Cholesterol reaching the mitochondrion is transferred to the interior of the organelle through the actions of the steroidogenic acute regulatory protein (STARD1) and converted to steroid by cytochrome P450scc.  STARD1's ligand-binding pocket is characteristic of StAR-related transfer (START) domain family members, like STARD4 and STARD6, which are also implicated in cholesterol trafficking.  Recently, STARD4 and in this study, STARD6 were detected in human luteinizing granulosa cells (hGC).  We therefore investigated how cholesterol availability and progesterone output affect all three sterol-transfer related proteins in hGC.  hGC obtained from normal women undergoing oocyte retrieval were cultured for 4 days prior to incubation in serum-free media +/- cAMP analog (8Br-cAMP) or phorbol ester (PMA).  We manipulated intracellular cholesterol levels first by stimulating P4 synthesis or suppressing it with the P450scc inhibitor aminoglutethimide (AG) and second by preculturing cells in media containing 10% fetal calf serum (FCS) or lipoprotein-deficient serum (LPDS) prior to stimulation +/- human low-density lipoprotein (LDL). Protein, mRNA and P4 levels were ascertained respectively by quantitative PCR, western blot and ELISA.  Consistent with prior data, both 0.25 and 1 mM 8Br-cAMP increase STARD1 mRNA and protein and P4 over 24 h.  Nanomolar doses of PMA that stimulate protein kinase C activity in other cell types also raise STARD1 mRNA, protein and P4 levels.  8Br-cAMP and PMA also markedly increase STARD4 but not STARD6 mRNA.  Reducing P4 production with AG fails to alter STARD1 or STARD6 mRNA levels.  However, AG blocks increases in STARD4 mRNA elicited by 8Br-cAMP but not PMA.  While STARD1 mRNA levels are independent of type of serum pretreatment, the presence of LDL unexpectedly blunts their response to PMA in FCS-exposed cultures.  On the other hand, LPDS pretreatment raises basal STARD4 mRNA.  Co-incubation with LDL suppresses vehicle and 8Br-cAMP-stimulated levels of STARD4 mRNA regardless of FCS or LPDS pretreatment, whereas PMA stimulation is less affected by LDL.  In summary, stimulation of STARD1 and P4 production coincides with increased STARD4 expression in hGC.  Lipoprotein starvation also elevates STARD4.  The presence of factors that block the depletion of or enrich for cholesterol (i.e., AG and LDL) oppose the stimulatory effect of 8Br-cAMP on STARD4.  Exogenous LDL partially represses PMA stimulation of STARD1 mRNA.  STARD6 is generally unaffected by serum condition and steroidogenic stimulus.  Altogether, these data point to roles for STARD4 in P4 synthesis and complex cholesterol-sensitive regulatory mechanisms linked to steroidogenesis that impact STARD1 and STARD4 gene expression.

Nothing to Disclose: BS, RJK, GFW, DMS, SRK, HAL

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: The Laura W. Bush Institute for Women’s Health and University Medical Center Women’s Health Innovation Fund (SK); NIH R03 HD070126 (HL)