GP73 Regulates Endoplasmic Reticulum Stress-induced Hepatic Steatosis

When the lumen of endoplasmic reticulum(ER) accumulated with unfolded proteins, it triggers the unfolded protein response(UPR), which is also called ER stress adaptive program. The aim of UPR is to restore protein‐folding homeostasis and to mitigate ER stress by either reducing protein synthesis, accelerating protein degradation, enhance protein folding or increase lipid synthesis. Accumulating evidences have demonstrated the important roles UPR plays in regulating metabolic homeostasis. Although the exact mechanism behind ER stress in hepatic steatosis is not fully understood, stress‐induced sterol regulatory element binding proteins(SREBPs) are key transcription factors linking ER stress to lipid synthesis and hepatic steatosis.SREBPs are believed to be master transcriptional regulators of lipid homeostasis. The inactive precursors of SREBPs reside in ER membranes bound with SREBP cleavage‐activating protein(SCAP). Sterols induce binding of SCAP to Insig, and prevent SREBP/SCAP exit from the ER. Upon cellular sterol depletion, the SREBP/SCAP complex is translocated to the Golgi via COP‐II‐coated vesicles. There SREBPs undergo two proteolytic processes, first by site‐1 serine protease(S1P) first, then site‐2 intramembrane metalloprotease(S2P). As a result, the active SREBP transcription factors are released and enter nucleus to regulate the transcription of the target genes. In the cholesterol and triglyceride biosynthesis pathways, the active SREBP transcription factors trigger the expression of genes that encode cholesterogenic or lipogenic enzymes, thus promoting lipid synthesis.GP73, which is also named as Golgi membrane protein(Golm1) or Golgi phosphoprotein 2(GOLPH2), was a type II transmembrane protein located on cis and medial‐Golgi. Immunohistochemical studies demonstrate that GP73 is preferentially expressed in normal epithelial cells of various human tissues, but not in hepatocyte. However, in diseased livers or upon virus infection, hepatocyte expression of GP73 is dramatically upregulated. The previous research proves that GP73 upregulated the expression of sterolrelated genes and GP73 may be a key transcription factorlinking ER stress to SREBPs and lipid synthesis.Here in this study, we demonstrated that:1. GP73 regulates the transcriptional activity of SREBP1 and lipogenesis: We tested correlation of GP73 and the SCAP‐SREBP1 cutting via utilizing IP, WB and q PCR techniques based on Hep G2 cell, and following results were obtained :(1) GP73 can interacte with SCAP and the SREBP1;(2) Overexpression of GP73 strongly induced SREBP‐2 and SREBP‐1a expression;(3) GP73 increases SCAP‐SREBP association and promotes its Trafficking to the Golgi;(4) The m RNA level ofacety‐Co A, FASN, HMGCS1, HMGCS2 and HMGR increased with GP73 overexpression.2. GP73 is a key factor in ER stress‐induced hepatic steatosis: To induce ER stress in vitro, we treated Hep G2 cells with four classical ER stressors: Tm, Tg, H2O2 and Cu SO4.Then GP73 si RNAi duplexes(si GP73), which were designed to knockdown GP73, were introduced into Hep G2. We found that:(1) Expression of GP73 is induced upon ER stress in vitro;(2) ER Stress can promote the expression of SCAP and SREBP1 as well;(3) The expression of SCAP‐SREBP1 was significantly down‐regulated in si GP73 Hep G2 cells.3. GP73 regulates steatosis under pharmacologic ER Stress in vivo: We treated mice with Tm, and then To explore pathophysiologic roles of GP73 in ER stress‐induced steatosis, si GP73, which were designed to knockdown GP73, were introduced into mice hepatocytes via hydrodynamic tail vein injection. The m RNA levels for GP73 at 24, 48 and 96 hours after injection were analyzed:(1) Treatments with Tm showed elevated m RNA abundance of CHOP in liver. Interestingly, treatment with Tm also induced the elevated m RNA abundance of GP73;(2) Triglycerides(TG) and cholesterol(CH) was detected in mice livers post challenge with Tm for 8 hrs. Moreover, plasma TG and CH level was progressively reduced upon Tm challenge;(3) Increased amount of GP73 protein were detected in the liver at 8, 12 and 24 hours after Tm challenge.(4) Tm‐induced hepatic TG and CH accumulation were also greatly reduced in si GP73 mice. On the other hand, the detected amount of circulating plasma triglycerides and cholesterol in the blood was increased;(5) Liver staining revealed large lipid droplets in Tm‐challenged mice, while GP73 knockdown liver had lipid droplets considerably decreased in size and numbe. In this study, we revealed an important role of the GP73 protein in regulating lipid homeostasis.GP73 expression triggered by ER stress increased the formation of SCAP‐SREBP complex, resulting in enhanced SREBPs activation and lipogenesis.