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β-Estradiol results in a proprotein convertase subtilisin/kexin type 9-dependent increase in low-density lipoprotein receptor levels in human hepatic HuH7 cells.

ABSTRACT: The lower risk of coronary artery disease in premenopausal women than in men and postmenopausal women implicates sex steroids in cardioprotective processes. β-Estradiol upregulates liver low-density lipoprotein receptor (LDLR), which, in turn, decreases circulating levels of low-density lipoprotein, which is a risk factor for coronary artery disease. Conversely, LDLR protein is negatively regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9). Herein, we investigated PCSK9 regulation by β-estradiol and its impact on LDLR in human hepatocarcinoma HuH7 cells grown in the presence or absence of β-estradiol. Immunoblot analysis showed upregulation of LDLR at 3 μm β-estradiol (140%), and the upregulation reached 220% at 10 μm β-estradiol; only at the latter dose was an increase in LDLR mRNA detected by qPCR, suggesting post-translational regulation of LDLR. No changes in PCSK9 mRNA or secreted protein levels were detected by qPCR or ELISA, respectively. β-estradiol-conditioned medium devoid of PCSK9 failed to upregulate LDLR. Similarly, PCSK9 knockdown cells showed no upregulation of LDLR by β-estradiol. Together, these results indicate a requirement for PCSK9 in the β-estradiol-induced upregulation of LDLR. A radiolabeling assay showed a significant, dose-dependent decrease in the ratio of secreted phosphoPCSK9 to total secreted PCSK9 with increasing β-estradiol levels, suggesting a change in the functional state of PCSK9 in the presence of β-estradiol. Our results indicate that the protein upregulation of LDLR at subtranscriptionally effective doses of β-estradiol, and its supratranscriptional upregulation at 10 μm β-estradiol, occur through an extracellular PCSK9-dependent mechanism.

SUBMITTER: Starr AE

PROVIDER: S-EPMC5008176 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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β-Estradiol results in a proprotein convertase subtilisin/kexin type 9-dependent increase in low-density lipoprotein receptor levels in human hepatic HuH7 cells.

Starr Amanda E AE Lemieux Valérie V Noad Jenny J Moore Jasmine I JI Dewpura Thilina T Raymond Angela A Chrétien Michel M Figeys Daniel D Mayne Janice J

The FEBS journal 20150518 14

The lower risk of coronary artery disease in premenopausal women than in men and postmenopausal women implicates sex steroids in cardioprotective processes. β-Estradiol upregulates liver low-density lipoprotein receptor (LDLR), which, in turn, decreases circulating levels of low-density lipoprotein, which is a risk factor for coronary artery disease. Conversely, LDLR protein is negatively regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9). Herein, we investigated PCSK9 regulation ...[more]

PMID: 25913303

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Project description:Background: The low-density lipoprotein receptor (LDLR) in the liver plays a crucial role in clearing low-density lipoprotein cholesterol (LDL-C) from the bloodstream. This process takes place mainly in the liver. Under atherogenic conditions, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), secreted by the liver, binds to LDLR on hepatocytes, preventing its recycling and enhancing its lysosomal degradation. This process reduces LDL-C clearance, promoting hypercholesterolemia. Epsins, a family of ubiquitin-binding endocytic adaptors, are key regulators of atherogenesis in lesional cells, including endothelial cells and macrophages. Given epsins' canonical role in regulating endocytosis of cell surface receptors, we aimed to determine whether and how liver epsins contribute to PCSK9-mediated LDLR endocytosis and degradation, thereby impairing LDL-C clearance and accelerating atherosclerosis. Methods: Liver-specific epsin knockout (Liver-DKO) atherosclerotic models were generated in ApoE-/- and PCSK9-AAV8-induced atheroprone mice fed on a Western diet. We utilized single-cell RNA sequencing, along with molecular, cellular, and biochemical analyses, to investigate the physiological role of liver epsins in PCSK9-mediated LDLR degradation. Additionally, we explored the therapeutic potential of nanoparticle-encapsulated siRNAs targeting epsins 1 and 2 in ApoE-/- mice with established atherosclerosis. Results: Western diet (WD)-induced atherosclerosis was significantly attenuated in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, as well as in PCSK9-AAV8-induced Liver-DKO mice compared with PCSK9-AAV8-induced wild-type (WT) mice accompanied by reductions in blood cholesterol and triglyceride levels. Mechanistically, single-cell RNA sequencing of hepatocytes and aortas isolated from atherosclerotic ApoE-/- and ApoE-/-/Liver-DKO mice revealed epsin-deficient Ldlrhi hepatocytes with diminished lipogenic potential. Notably, pathway analysis of hepatocytes showed increased LDL particle clearance and enhanced LDLR-cholesterol interactions under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls, correlating with decreased plasma LDL-C levels. Furthermore, pathway analysis of the aortas showed attenuated inflammation and endothelial activation, coupled with reduced lipid uptake, and enhanced cholesterol efflux under WD treatment in ApoE-/-/Liver-DKO mice compared with ApoE-/- controls. Moreover, the absence of liver epsins led to an upregulation of LDLR protein expression in hepatocytes. We further demonstrated that epsins bind LDLR via the ubiquitin-interacting motif (UIM), enabling PCSK9-mediated LDLR degradation. Depleting epsins abolished this degradation, thereby preventing atheroma progression. Lastly, targeting liver epsins with nanoparticle-encapsulated epsins siRNAs effectively ameliorates dyslipidemia and inhibits atherosclerosis progression. These results are consistent with findings showing an increased epsin1 and epsin2 expression in atherosclerotic cardiovascular disease patients. Conclusions: Liver epsins drive atherogenesis by promoting PCSK9-mediated LDLR degradation, thereby elevating circulating LDL-C levels and heightening lesional inflammation. As such, targeting epsins in the liver represents a promising therapeutic strategy to mitigate atherosclerosis by preserving LDLR and enhancing LDL-C clearance in the liver.

Dataset Information

β-Estradiol results in a proprotein convertase subtilisin/kexin type 9-dependent increase in low-density lipoprotein receptor levels in human hepatic HuH7 cells.

Publications

β-Estradiol results in a proprotein convertase subtilisin/kexin type 9-dependent increase in low-density lipoprotein receptor levels in human hepatic HuH7 cells.

Similar Datasets

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