Abstract 419: StarD5: Role in a Novel Pathway of Intracellular Cholesterol Transport

Abstract

Introduction: How cholesterol is moved within the cell via a non-vesicular means and is mobilized against a gradient to the plasma membrane has never been clearly understood. StarD5, a member of the StarD4 subfamily of START (steroidogenic acuteregulatory lipid transfer) domain proteins, is able to bind cholesterol and can be induced by ER stress. Its function, however, has remained a mystery. The objective of this study was to attempt to define the role of StarD5 in intracellular cholesterol metabolism by following its response to ER stress. Methods: StarD5 knockdown cells were obtained by using siRNA, while infection with an adenovirus encoding StarD5 was used to selectively overexpress the protein. Immunoblots were performed to determine knockdown and overexpression of StarD5. Filipin staining and immunocytochemistry were used to determine the localization of free cholesterol within the cells. Apoptosis during ER stress was determined by staining cells with Alexa Fluor-488 Annexin V. Results: Overexpression of StarD5 in THP-1 macrophages led to a rapid redistribution of StarD5 and cholesterol from the Golgi/ER to the plasma membrane. StarD5 knockdown led to a marked increase in free cholesterol within the Golgi and ER in 3T3-L1 cells. Furthermore, thapsigargin-induced apoptosis (which leads to an abnormal accumulation of cholesterol in the ER) was markedly increased with StarD5 knockdown. Conclusion: StarD5 has the ability to respond to different stresses with a directed movement, mobilizing cholesterol between the ER/Golgi to the plasma membrane in order to establish a needed cholesterol membrane concentration gradient amongst intracellular organelles. These findings demonstrate a previously unexplored pathway of intracellular cholesterol transport whose baseline function is induced by ER-stress; likely as a means to stabilize the cell during periods of stress by mobilizing excess cholesterol from the ER and stabilizing the plasma membrane through cholesterol induced changes in permeability and fluidity.