Abstract
We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum oxysterol-binding protein implicated in cellular lipid homeostasis. We now investigated its action in hepatic cells in vivo and in vitro. Adenoviral overexpression of ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum (−34%, −26%, −37%, respectively) and liver tissue (−40%, −12%, −24%), coinciding with reduction of nuclear (n)SREBP-1 and -2 and mRNA levels of their target genes. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing by RNA interference moderately suppressed or induced the expression of SREBP-1 and SREBP-2 target genes, respectively. In accordance, cholesterol biosynthesis was reduced by ORP8 overexpression and enhanced by ORP8 silencing in [3H]acetate pulse-labeling experiments. ORP8, previously shown to bind 25-hydroxycholesterol, was now shown to bind also cholesterol in vitro. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation analyses revealed the nuclear pore component Nup62 as an interaction partner of ORP8. Co-localization of ORP8 and Nup62 at the nuclear envelope was demonstrated by BiFC and confocal immunofluorescence microscopy. Furthermore, the impact of overexpressed ORP8 on nSREBPs and their target mRNAs was inhibited in cells depleted of Nup62. Our results reveal that ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism, and provide clues of an entirely new mode of ORP action.
Highlights
Cellular cholesterol biosynthesis and uptake, as well as fatty acid biosynthesis, are controlled by transcription factors named sterol regulatory element binding proteins (SREBPs) and their sterolsensing accessory factor, the SREBP cleavage activating protein (SCAP) [1,2,3]
The plasma total cholesterol, triglyceride (TG), and choline-containing phospholipid (PL) concentrations were determined for the mice at 5 days after adenovirus encoding ORP8 (AdORP8) injection and revealed a significant reduction of cholesterol (234%) and TG (237%), as well as tendency of reduced PL (226%) as compared to a control virus encoding GFP (AdGFP)-transduced animals (Fig. 1B)
In order to elucidate the underlying mechanism we analyzed by Western blotting the amount of SREBP-1 and -2 in nuclear fractions prepared from the liver of the transduced animals, as well as the precursor SREBPs in liver total protein preparations
Summary
Cellular cholesterol biosynthesis and uptake, as well as fatty acid biosynthesis, are controlled by transcription factors named sterol regulatory element binding proteins (SREBPs) and their sterolsensing accessory factor, the SREBP cleavage activating protein (SCAP) [1,2,3]. When the cellular cholesterol level is low, SREBP-SCAP complexes move to the Golgi apparatus, where SREBPs undergo a two-step proteolytic processing, leading to the release of an N-terminal fragment, basic helix-loop-helix leucine zipper transcription factor. These factors enter the nucleus where they bind to sterol regulatory elements (SRE) in the promoter regions of a number of genes whose products mediate the synthesis of cholesterol and fatty acids. Large proteins carrying a classical nuclear localization signal (NLS) are transported into the nucleus through the following multistep process: The NLS is recognized by the adaptor protein importin-a (karyopherin-a). The FG domains have an unfolded structure and are responsible for interaction with importin-cargo complexes moving through the pore [6,7]
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