Abstract 662: Human Membrane-associated Progesterone Receptor (Hpr6) is Involved in the Regulation of Cholesterol Synthesis
Hpr6 was originally identified as a putative human intracellular membrane-bound steroid receptor related to a porcine progesterone-binding protein. Hpr6 is homologous with Dap1p, a yeast protein that confers resistance to alkylating agents and stabilizes Erg11p (lanosterol demethylase) in the sterol synthesis pathway: Deletion of Dap1p impairs sterol synthesis and decreases the expression of lanosterol demethylase protein but not mRNA, suggesting that Dap1p regulates the translation or stability of this enzyme. Dap1p is a heme-binding protein with sequence similarity to cytochrome b5, an electron transfer heme-protein. These studies were undertaken to determine if Hpr6 was necessary for cholesterol synthesis in mammalian cells. Spectrophotometric analysis of purified Hpr6 demonstrated that it binds heme, and cellular immunofluorescence microscopy showed that expression of the protein in McARH7777 rat hepatoma cells results in localization to the endoplasmic reticulum, as has been seen in MCF-7 breast cancer cells. In knock-down experiments in several breast cancer cell lines (BT549; MDA231) Hpr6 RNAi attenuated by 30 – 40% the induction of cholesterol synthesis in cells incubated overnight in sterol-free media. Overexpression of Hpr6 in hepatoma cells increased cholesterol synthesis by approximately 25%, whereas expression of an Asp120Gly mutant of Hpr6, which is deficient in heme binding, reduced cholesterol synthesis by 50%, thus acting as a dominant negative. The pattern of accumulation of sterol intermediates in cells expressing either form of Hpr6 (wild-type or mutant), as well as in cells in which Hpr6 expression was reduced by RNAi, was not different from that of control cells, indicating that Hpr6 affected the overall cholesterol synthesis pathway. These results suggest that Hpr6 is in the regulatory pathway for cholesterol synthesis and are consistent with its known interaction with Insig-1, a key protein in the cholesterol regulatory pathway.
Supported by an NIH COBRE grant, P20 RR-15592, and by the University of Kentucky Office of the Executive Vice President for Research.