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?-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. ?-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 ?-estradiol and its impact on LDLR in human hepatocarcinoma HuH7 cells grown in the presence or absence of ?-estradiol. Immunoblot analysis showed upregulation of LDLR at 3 ?m ?-estradiol (140%), and the upregulation reached 220% at 10 ?m ?-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. ?-estradiol-conditioned medium devoid of PCSK9 failed to upregulate LDLR. Similarly, PCSK9 knockdown cells showed no upregulation of LDLR by ?-estradiol. Together, these results indicate a requirement for PCSK9 in the ?-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 ?-estradiol levels, suggesting a change in the functional state of PCSK9 in the presence of ?-estradiol. Our results indicate that the protein upregulation of LDLR at subtranscriptionally effective doses of ?-estradiol, and its supratranscriptional upregulation at 10 ?m ?-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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Project description:Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a causal risk factor for cardiovascular disease. The mechanisms underlying Lp(a) clearance from plasma remain unclear, which is an obvious barrier to the development of therapies to specifically lower levels of this lipoprotein. Recently, it has been documented that monoclonal antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) can lower plasma Lp(a) levels by 30%. Since PCSK9 acts primarily through the low density lipoprotein receptor (LDLR), this result is in conflict with the prevailing view that the LDLR does not participate in Lp(a) clearance. To support our recent findings in HepG2 cells that the LDLR can act as a bona fide receptor for Lp(a) whose effects are sensitive to PCSK9, we undertook a series of Lp(a) internalization experiments using different hepatic cells, with different variants of PCSK9, and with different members of the LDLR family. We found that PCSK9 decreased Lp(a) and/or apo(a) internalization by Huh7 human hepatoma cells and by primary mouse and human hepatocytes. Overexpression of human LDLR appeared to enhance apo(a)/Lp(a) internalization in both types of primary cells. Importantly, internalization of Lp(a) by LDLR-deficient mouse hepatocytes was not affected by PCSK9, but the effect of PCSK9 was restored upon overexpression of human LDLR. In HepG2 cells, Lp(a) internalization was decreased by gain-of-function mutants of PCSK9 more than by wild-type PCSK9, and a loss-of function variant had a reduced ability to influence Lp(a) internalization. Apo(a) internalization by HepG2 cells was not affected by apo(a) isoform size. Finally, we showed that very low density lipoprotein receptor (VLDLR), LDR-related protein (LRP)-8, and LRP-1 do not play a role in Lp(a) internalization or the effect of PCSK9 on Lp(a) internalization. Our findings are consistent with the idea that PCSK9 inhibits Lp(a) clearance through the LDLR, but do not exclude other effects of PCSK9 such as on Lp(a) biosynthesis.

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

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