Abstract: 5

THE DISTRIBUTION AND FUNCTION OF NPC1 PROTEIN IN CHOLESTEROL TRAFFICKING IN HUMAN GRANULOSA-LUTEIN CELLS.

H Watari¹ , EJ Blanchette-Mackie² , NK Dwyer² , PG Pentchev² , JM Glick¹ , JF Strauss, III¹
Center for Research on Reproduction and Women and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA ¹
National Institutes of Health, Bethesda, MD 20892 ²

Human steroidogenic cells utilize LDL cholesterol for hormone synthesis. LDL taken up by cell surface receptors is moved to the lysosomes where LDL cholesterol esters are hydrolyzed. Free cholesterol exits from lysosomes and is moved to sites of utilization. Mobilization of lysosomal free sterol is mediated by the Niemann-Pick type C1 protein (NPC1), encoded by a gene which when mutated produces a lysosomal storage disease. We analyzed the distribution of NPC1 in human granulosa cells and its role in the utilization of LDL cholesterol for steroidogenesis. NPC1 was localized to punctuate organelles stained for the lysosomal glycoprotein, LAMP-2. Treatment with U18666A or progesterone, compounds that block NPC1 function caused accumulation of LDL free cholesterol, identified by filipin staining, in NPC1 positive, LAMP-2 positive vesicles. These treatments increased the free cholesterol content of the cells. U18666A suppressed LDL-supported progesterone production, but not progesterone production driven by 22-hydroxycholesterol. The inhibitor of acyl-CoA cholesterol acyltransferase, 58035, reduced cellular cholesterol ester levels but did not affect the distribution of NPC1. The cholesterol side-chain cleavage inhibitor, aminoglutethimide, inhibited progesterone synthesis and caused accumulation of cholesterol esters in Nile Red-positive droplets. NPC1 protein remained in LAMP-2 positive vesicles. Neither 58035 nor aminoglutethimide caused accumulation of filipin-positive lysosomes. We conclude that NPC1 is localized to lysosomes in human steroidogenic cells; that disruption of its function results in the accumulation of free cholesterol in lysosomes and inhibition of LDL-supported steroidogenesis; and that blockade of post-lysosomal cholesterol metabolism does not affect NPC1 distribution nor put back pressure on the lysosomal apparatus.

    This abstract is being presented on Saturday, July 31 at 2:30 PM at Todd 116.