STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

Jeanne Garbarino,Jan L Breslow,Frederik W Lund,Meihui Pan,Harvey F Chin,Frederick R Maxfield

doi:10.1194/jlr.m032227

Abstract

STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

Highlights

STARD4, a member of the evolutionarily conserved StAR-related lipid transfer (START) gene family, has been implicated in the nonvesicular intracellular transport of cholesterol
The response of STARD4 to cholesterol deficiency was examined in HepG2 cells cultured for 20 h in medium containing 5% lipoprotein-deficient serum (LPDS), compared with 10% FBS
STARD4 is clearly induced at the mRNA (Fig. 1A) and protein levels (Fig. 1B) in HepG2 cells cultured in 5% LPDS

Summary

Introduction

STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. We observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.—Garbarino, J., M. The precise mechanism responsible for this intracellular cholesterol gradient is not well understood, but it is likely achieved through the concerted efforts of a number of different homeostatic mechanisms, including tightly controlled transport processes Both vesicular transport, an ATP-dependent process requiring an intact cytoskeleton, and nonvesicular transport, which involves carrier proteins and occurs independent of ATP, have been implicated in intracellular cholesterol movement [2].

Methods

Results

Conclusion