The loss-of-function PCSK9Q152H variant increases ER chaperones GRP78 and GRP94 and protects against liver injury
Paul F. Lebeau, Hanny Wassef, Jae Hyun Byun, Khrystyna Platko, Brandon Ason, Simon Jackson, Joshua Dobroff, Susan Shetterly, William G. Richards, Ali A. Al-Hashimi, Kevin Doyoon Won, Majambu Mbikay, Annik Prat, An Tang, Guillaume Paré, Renata Pasqualini, Nabil G. Seidah, Wadih Arap, Michel Chrétien, Richard C. Austin
Paul F. Lebeau, Hanny Wassef, Jae Hyun Byun, Khrystyna Platko, Brandon Ason, Simon Jackson, Joshua Dobroff, Susan Shetterly, William G. Richards, Ali A. Al-Hashimi, Kevin Doyoon Won, Majambu Mbikay, Annik Prat, An Tang, Guillaume Paré, Renata Pasqualini, Nabil G. Seidah, Wadih Arap, Michel Chrétien, Richard C. Austin
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Research Article Hepatology Vascular biology

The loss-of-function PCSK9Q152H variant increases ER chaperones GRP78 and GRP94 and protects against liver injury

Abstract

Individuals harboring the loss-of-function (LOF) proprotein convertase subtilisin/kexin type 9 Gln152His variation (PCSK9Q152H) have low circulating low-density lipoprotein cholesterol levels and are therefore protected against cardiovascular disease (CVD). This uncleavable form of proPCSK9, however, is retained in the endoplasmic reticulum (ER) of liver hepatocytes, where it would be expected to contribute to ER storage disease (ERSD), a heritable condition known to cause systemic ER stress and liver injury. Here, we examined liver function in members of several French-Canadian families known to carry the PCSK9Q152H variation. We report that PCSK9Q152H carriers exhibited marked hypocholesterolemia and normal liver function despite their lifelong state of ER PCSK9 retention. Mechanistically, hepatic overexpression of PCSK9Q152H using adeno-associated viruses in male mice greatly increased the stability of key ER stress-response chaperones in liver hepatocytes and unexpectedly protected against ER stress and liver injury rather than inducing them. Our findings show that ER retention of PCSK9 not only reduced CVD risk in patients but may also protect against ERSD and other ER stress–driven conditions of the liver. In summary, we have uncovered a cochaperone function for PCSK9Q152H that explains its hepatoprotective effects and generated a translational mouse model for further mechanistic insights into this clinically relevant LOF PCSK9 variant.

Authors

Paul F. Lebeau, Hanny Wassef, Jae Hyun Byun, Khrystyna Platko, Brandon Ason, Simon Jackson, Joshua Dobroff, Susan Shetterly, William G. Richards, Ali A. Al-Hashimi, Kevin Doyoon Won, Majambu Mbikay, Annik Prat, An Tang, Guillaume Paré, Renata Pasqualini, Nabil G. Seidah, Wadih Arap, Michel Chrétien, Richard C. Austin

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Figure 5

ER PCSK9 retention reduces ER stress–induced cytotoxicity in cultured immortalized hepatocytes.

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ER PCSK9 retention reduces ER stress–induced cytotoxicity in cultured im...
(A) HEK293 cells were transfected with plasmids encoding either human PCSK9 WT (PCSK9WT) or the ER-retained variant PCSK9Q152H for 48 hours and subsequently treated with the ER stress–inducing agent thapsigargin (TG; 100 nM) or tunicamycin (TM; 2 μg/mL) for an additional 24 hours. ER stress marker expression was assessed by real-time PCR. (B) The expression of ER stress markers p-PERK, IRE1α, and GRP94 was also assessed in HuH7 immortalized hepatocytes transfected with either PCSK9WT or PCSK9Q152H in the presence and absence of TG using immunoblots. (C and D) Cytotoxicity was examined in PCSK9-transfected HepG2 cells in the absence (C) or presence (D) of siRNA targeted against GRP94 (siGRP94) using an LDH release assay. (E) Cytotoxicity was also examined in HEK293, HuH7, and HepG2 cells transfected with siRNA targeted against PCSK9 (siPCSK9) in the presence and absence of TG. (F and G) The effect of increased GRP78 and GRP94 expression on cytotoxicity caused by ER stress–inducing agents TG (100 nM) and TM (2 μg/mL) was also examined. (H) LDH release assays were carried out in HepG2 cells cotransfected with WT arginine vasopressin (VP) or a naturally occurring variant known to cause ER stress and cytotoxicity (VPG17V) in the presence or absence of ER-retained PCSK9Q152H. (I and J) These findings were confirmed by staining of apoptotic DNA damage using a TUNEL assay. (K) Effective transfection in HepG2 cells was confirmed using immunoblots. Values are represented as mean ± SD. *P < 0.05; †P < 0.05 vs. group 1; ††P < 0.05 vs. group 2. ANOVA was used for all statistical comparisons (A–J). Scale bars: J, 200 μm.