In previous reports we showed that PCSK9 is efficiently secreted from HepG2 cells . Herein, we show that changes in secreted PCSK9 reflect changes in intracellular PCSK9 expression in a HepG2 cell model (Figs 2 and 4). This direct link between maturation of proPCSK9 to mPCSK9 and its secretion further suggests, along with our published association of 'loss of function' PCSK9 variants with reduced levels of plasma PCSK9 , that plasma levels of PCSK9 can serve as a measure of regulation of this physiologically relevant PCSK in humans.
This study demonstrates that plasma PCSK9 levels are significantly modified by two common lipid therapies; fibrates and statins (Tables 1 and 2 and Figs 1 and 3). The effect of fibrate therapy on plasma PCSK9 levels has not been previously reported. Fibrates (peroxisome proliferator activated receptor alpha (PPARα) agonists) are a class of drug that is used to primarily treat hypertriglyceridemia and low HDLC. They induce the PPARα pathway and upregulate genes that encode proteins that increase HDL production and secretion, while reducing very low-density lipoprotein (VLDL) triglyceride secretion . Fibrates have variable effects on circulating LDLC levels through an unknown pathway . Indeed in our cohort of patients (n = 19); 8 individuals showed an increase, while 11 individuals showed a decrease in LDLC levels following fibrate therapy (Fig 1). Likewise, the levels of plasma PCSK9 both increased and decreased following fibrate treatment; overall, however there was a significant increase in plasma PCSK9 levels of 17% (Table 1 and Fig 1). As well, changes in plasma PCSK9 were significantly inversely correlated to changes observed in LDLC following fibrate therapy (Fig 1).
Our cell culture data indicated that fenofibrate (up to 200 μM) does not significantly change the expression of PCSK9 or the LDLR (Fig 2). A recent report suggests that PCSK9 mRNA expression can be significantly repressed in HepG2 cells by fenofibrate at concentrations >200 μM . Similar to this study we also see non-significant downregulation of PCSK9 protein levels at 200 μM but not at lower concentrations. It is important to note that based on the pharmacokinetics for gemfibrozil  and fenofibrate  in humans, the subjects in our study received 100 μM and 30 μM, respectively, well below that reported to repress PCSK9 transcription ex vivo. Taken together with our data from human studies (Fig 1), it is likely that therapeutic doses of fibrates exert their effect on plasma levels of PCSK9 indirectly, in response to changes in cholesterol levels. We suggest that in the case of increased levels of circulating LDLC, intracellular levels of sterols increase, downregulating genes activated by the SREBP pathway (including PCSK9) and vice versa.
PCSK9 is upregulated by cellular cholesterol depletion through the sterol regulatory element binding protein-2 (SREBP-2) pathway . The SREBP family includes three transcription factors, SREBP-1a, -1c and -2. SREBP-2 preferentially activates genes involved in cholesterol biosynthesis and metabolism, such as HMGCoA synthase and HMGCoAR. SREBPs -1a and -1c preferentially activate genes involved in fatty acid biosynthesis, such as acetyl CoA carboxylase and ATP citrate lyase . The role of SREBP-2 in PCSK9 transcriptional regulation is well documented [12–14, 31] while several reports suggest that PCSK9 is also regulated by SREBP-1c [12, 13, 32]. Statins inhibit HMGCoAR, a rate-limiting enzyme in cholesterol biosynthesis and have been shown to significantly increase PCSK9 mRNA in HepG2 cells and primary human hepatocytes through activation of the SREBP-2 pathway .
In this report, we measured plasma PCSK9 levels in response to low-dose atorvastatin therapy (10 mg/day) and show an average increase of 7.4% in circulating levels of PCSK9 in individuals following treatment (effective dosage 0.0217 μM). Another group showed that 40 mg/day atorvastatin further increased plasma PCSK9 by 34% . However, they did not report the significant inverse correlation between percent change in LDLC and PCSK9 that we observed (Fig 3). This may be because their study group was smaller (n = 12 versus n = 40). The results we report here for protein upregulation of PCSK9 by simvastatin in HepG2 cells (Fig 4; 40% at 0.05 μM) was comparable to that observed in their study undergoing atorvastatin therapy (34% at 0.08 μM). Collectively, our cell culture and human studies suggest that in response to statins, increases in PCSK9 mRNA levels are reflected in circulating levels of PCSK9 (Fig 3).
Statins reduce LDLC levels through a second mechanism; they increase LDLR levels through the SREBP-2 pathway, further decreasing circulating LDLC. PCSK9, which can degrade the LDLR, is also upregulated by statins in vivo (Fig 3) and ex vivo (Fig 4). When we measured the effect of simvastatin on the LDLR protein expression in the HepG2 cell line, it showed significant upregulation at 1 μM (Fig 4). However, LDLR expression was more highly upregulated than that of PCSK9 (2.0× versus 1.5× at 1 μM and 2.6× versus 1.5× at 10 μM simvastatin, respectively). This suggests that although PCSK9 is a negative regulator of the LDLR and, in the case of statins, is upregulated in concert with the LDLR by the SREBP-2 pathway, the increase in PCSK9 at the protein level is not sufficient to completely negate upregulation of the LDLR.
In our study the average increase in plasma PCSK9 was greater following fibrate therapy (17%) than following statin therapy (7%). These two post-hoc study groups are exclusive in terms of participants; therefore, we cannot directly compare differences in responses between the two therapies. However, we do know that an increase in PCSK9 may limit the efficacy of therapies in terms of lowering circulating LDLC levels.
In both fibrate and statin cohorts, the inverse correlation between changes in plasma PCSK9 and LDLC was significant in men, and not in women, although the numbers of women were low and therefore may not have been significantly powered. Previously we reported a gender dichotomy in the direct relationship between LDLC levels and plasma PCSK9 in men and not women , suggesting that gender-specific hormones such as estrogens and/or testosterone may also affect PCSK9 transcription and/or expression. This is an avenue we are pursuing.