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.