The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

Zhang J, Dudley-Rucker N, Crowley JR, Lopez-Perez E, Issandou M, Schaffer JE and Ory DS
Source: J Lipid Res
Publication Date: (2004)
Issue: 45(2): 223-231
Cells used in publication:
Species: hamster
Tissue Origin: ovarian
A novel class of potent cholesterol-lowering agents has been described that upregulates low-density lipoprotein receptor (LDLr) activity, leading to a marked reduction of both LDL cholesterol and triglycerides in vivo. The steroidal LDLr up-regulator, GW707, is known to inferfere with intracellular sterol trafficking. GW707 is structurally similar to the amphiphilic compound U18666A which induces a Niemann Pick type C (NPC) disease-like phenotype. In NPC mutants the sterol trafficking defect results in failure to suppress sterol regulatory element binding protein (SREBP)-dependent gene expression. The authors studied the inhibition of sterol trafficking by the steroidal analog GW707 and its similarity to mechanism of action of U18666A. To examine the effect of treatment with GW707 on SREBP-dependent gene transcription, the authors used a sterol regulatory element (SRE)-containing reporter construct as an indicator SREBP-dependent gene expression. Wild type CHO or NPC1-null cells (M12) were nucleofected with a luciferase reporter construct and with a TK-Renilla transfection control. Cells were grown under different lipoprotein conditions in the presence or absence of GW707 or U18666A. Lysates were harvested and luciferase and Renilla activity determined. The results revealed that GW707 prevents LDL-cholesterol suppression of SREBP-dependent gene expression.
Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.