Abstract
The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). As such, loss-of-function (LOF) PCSK9 variants that fail to exit the endoplasmic reticulum (ER) increase hepatic LDLR levels and lower the risk of developing CVD. The retention of misfolded protein in the ER can cause ER stress and activate the unfolded protein response (UPR). In this study, we investigated whether a variety of LOF PCSK9 variants that are retained in the ER can cause ER stress and hepatic cytotoxicity. Although overexpression of these PCSK9 variants caused an accumulation in the ER of hepatocytes, UPR activation or apoptosis was not observed. Furthermore, ER retention of endogenous PCSK9 via splice switching also failed to induce the UPR. Consistent with these in vitro studies, overexpression of PCSK9 in the livers of mice had no impact on UPR activation. To elucidate the cellular mechanism to explain these surprising findings, we observed that the 94-kDa glucose-regulated protein (GRP94) sequesters PCSK9 away from the 78-kDa glucose-regulated protein (GRP78), the major activator of the UPR. As a result, GRP94 knockdown increased the stability of GRP78-PCSK9 complex and resulted in UPR activation following overexpression of ER-retained PCSK9 variants relative to WT secreted controls. Given that overexpression of these LOF PCSK9 variants does not cause UPR activation under normal homeostatic conditions, therapeutic strategies aimed at blocking the autocatalytic cleavage of PCSK9 in the ER represent a viable strategy for reducing circulating PCSK9.
Highlights
The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR)
Cells transfected with PCSK9Q152H exhibited 6.2-fold greater levels of endoplasmic reticulum (ER)-resident pro-PCSK9 than that of endogenously expressed PCSK9 found in empty vector (EV)–transfected controls and ϳ3-fold greater levels than PCSK9WT-transfected controls
To investigate the effect of PCSK9 variants on unfolded protein response (UPR) activation, HuH7 cells cotransfected with the ER activated indicator (ERAI) plasmid in addition to plasmids encoding a variety of PCSK9 variants were examined via immunoblotting of GRP78 and CCAAT/enhancer– binding protein homologous protein (CHOP)
Summary
The proprotein convertase subtilisin/kexin type-9 (PCSK9) plays a central role in cardiovascular disease (CVD) by degrading hepatic low-density lipoprotein receptor (LDLR). The phenotype associated with such LOF mutations occurs due to the inability of the ER-resident nascent pro-PCSK9 to undergo autocatalytic cleavage and maturation in the ER, thereby leading to its retention This was found to drive LDLR expression and accounts for the low circulating LDL levels found in patients harboring such mutations [10, 12]. The UPR signaling cascade consists of three major transducers located at the ER membrane that are activated upon dissociation of GRP78 from their intra-ER domains These “arms of the UPR” include (a) inositol-requiring protein-1␣ (IRE1␣), which splices and activates X-box– binding protein-1 (XBP1), (b) activating transcription factor-6 (ATF6), and (c) protein kinase RNA (PKR)–like ER kinase (PERK), which promotes the expression of the proapoptotic CCAAT/enhancer– binding protein homologous protein (CHOP). ER PCSK9 retention was shown to cause ER stress/UPR activation following knockdown of GRP94
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