Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol

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PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice ( Surf4 fl/fl Alb-Cre + ) to investigate the physiologic role of SURF4 in vivo. Surf4 fl/fl Alb-Cre + mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by ∽60% in Surf4 fl/fl Alb-Cre + mice, with a corresponding ∽50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B. Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4 fl/fl Alb-Cre + mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.

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  1. Evaluation Summary:

    By the use of liver specific gene knock-out approaches Tang et al have clearly documented an important role for an endoplasmic reticulum sorting receptor, SURF4, in the efficient secretion of PCSK9, a protein in circulation that binds to and enhances the intracellular uptake and degradation of the low density lipoprotein receptor. As a consequence of liver specific knock-outs, adult mice survive well with an exceptionally lower level of circulating cholesterol, triglycerides and various lipoproteins. These compelling results reinforce the prospect for the development of therapeutic approaches in cholesterol and LDL reduction by targeting the intracellular association of PCSK9 and the SURF4 receptor. The work is elegant and complete with the only concern that some of the work and results overlap work published previously by some of these authors.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Tang et al. in this report investigate the effects of deleting Surf4 in mouse liver by generating three different mouse models. Previously this group has shown that Surf4 functions as a cargo receptor that facilitates the secretion of PCSK9 in cultured cells. Here they have deleted the gene in hepatocytes and find that there is a significant reduction in plasma PCSK9 levels with a resulting increase in LDLR protein and lowering of plasma cholesterol levels. Surf deletion in hepatocytes using albumin-Cre had no deleterious effects in liver. What was found was a 60% reduction in plasma PCSK9 with no change in PCSK9 mRNA levels. These results were confirmed using Cas9 mice in which Surf4 was acutely deleted. Consistent with the known function of PCSK9, the reduction in plasma PCSK9 was associated with a significant increased in liver LDLR protein levels. In addition to dramatically lower plasma cholesterol levels in all lipoprotein fractions, they also find reduced plasma TG levels they show was due to a marked reduction in apoB and TG secretion. Interestingly, there was no defect in intestinal lipid absorption. Combined the studies are well done and convincing show the role of Surf4 in facilitating PCSK9 and apoB secretion from liver. Interesting remaining questions would be to address whether Sruf4 plays a similar role in intestine and whether it is required for fat absorption from the gut.

  3. Reviewer #2 (Public Review):

    Multiple approaches are used to selectively deplete the Surf4 cargo receptor in mouse liver revealing that serum cholesterol and triglyceride levels are substantially reduced due to inefficient transport of APOB-containing lipoproteins from the ER of hepatic cells. The work also demonstrates that serum levels of proprotein convertase subtilisin/kexin type 9 (PCSK9) are reduced in Surf4-deficient mice building on a previous report from this group showing that Surf4 facilitates ER export and secretion of PCSK9 in cultured cells (Emmer et al., 2018). The study convincingly confirms and extends other recent reports showing hepatic deficiency of Surf4 markedly reduces plasma lipid levels and protects mice from atherosclerosis. Collectively these findings are important for understanding lipid metabolism and may inform treatments for cardiovascular disease.

    Strengths of this study include tissue specific approaches in the mouse model to deplete Surf4 from liver for comprehensive analysis of physiological consequences. Cre-mediated excision of SURF4 fl/fl in liver is the primary model in addition to acute inactivation of SURF4 in adult mice using a CRISPR/Cas9 system or liver-targeted siRNAs. All three approaches reduced serum PCSK9 levels after Surf4 depletion in support of the model that Surf4 acts as a cargo receptor for ER export and secretion of PCSK9. Further characterization of liver-specific Surf4 depletion shows moderately increased hepatic LDLR as expected for decreased PCSK9 levels. Striking decreases in serum cholesterol, triglycerides and APOB protein were also observed, beyond what would be expected for complete PCSK9 deficiency. These findings are consistent with two other recent reports showing dramatically reduced APOB-containing lipoproteins in serum after hepatic inactivation of Surf4 in mouse. The evidence indicates lipoprotein secretion is reduced because APOB lipoprotein (VLDL) depends on the Surf4 cargo receptor for efficient transport from ER in liver.

    Other strengths from the current study of Surf4-deficient mice include a thorough analysis of glucose levels, fatty acid metabolism and lipid adsorption, which are largely unchanged when Surf4 is inactivated. Assessments of body mass (no difference), liver mass (slight increase) and hepatic fat content (small increase) are reported. Surprisingly, levels of cholesterol, triglyceride and APOB in liver are not significantly increased and there is no apparent hepatic dysfunction or steatosis. mRNA seq analysis of Surf4-deficient liver reveals significant down regulation of genes involved in lipid biosynthesis and upregulation of selected ERAD machinery, potentially in response to ER accumulation of APOB-containing lipoprotein.

    Limitations of the study are that there are likely to be other secretory proteins in liver that depend of Surf4 for efficient secretion and how these factors contribute to lipid metabolism and physiology are not known. In addition the authors point out discrepancy with one of the earlier reports (B. Wang et al., 2021) where no changes in PCSK9 levels were detected after Surf4 depletion. This difference is proposed to be related to differences in mouse genetics or husbandry, although the current study does not provide information on light/dark cycle and composition of mouse diet as was provided in the B. Wang et al., 2021 report.

    Overall the findings reported in the current study are comprehensive, rigorous, informative and support their primary conclusions