PCSK9 and Lipid Metabolism

Abstract

Purpose of review:

The purpose of this review is to highlight the recent findings of one of the most promising therapeutic targets in low-density lipoprotein (LDL) cholesterol management, proprotein convertase subtilisin/kexin type 9 (PCSK9).

Recent findings:

Endoplasmic reticulum (ER) cargo receptor, surfeit locus protein 4 (SURF4) interacts with PCSK9 and regulates its exit from ER and its secretion. Once secreted, PCSK9 binds to heparin sulfate proteoglycans on the hepatocyte surface and this binding is required for PCSK9–LDL receptor (LDLR) complex formation and LDLR degradation. Post-transcriptionally, recent work has shown that PCSK9 gets degraded in the lysosomes by activation of the glucagon receptor signaling, providing more data on the hormonal regulation of PCSK9. Finally, human studies with PCSK9 inhibitors offered more evidence on their benefits and safe use.

Summary:

Recent work on the regulation of PCSK9 has enhanced our understanding of its biology, which may provide important information for future PCSK9-based therapies.

INTRODUCTION

Atherosclerotic cardiovascular disease (CVD) is the leading cause of death worldwide [1]. Large population studies demonstrated a positive correlation between elevated levels of low-density lipoprotein cholesterol (LDL-C) and atherosclerosis and lowering of LDL-C is the primary target of therapy [2]. Introduced in 1987, statin drugs were a major development in the treatment of hyperlipidemia worldwide [3]. By inhibiting Hmg-CoA-reductase, the rate-limiting enzyme in the mevalonate pathway, statins lower cholesterol synthesis. This decrease in cholesterol in liver activates sterol regulatory element-binding protein-2 (SREBP-2), which upregulates low-density lipoprotein receptor (LDLR) expression and thus decreases plasma LDL-C. Meta-analyses of randomized clinical trials of statin therapy revealed that each 1 mmol/L reduction in LDL-C results in ~20% reduction of global cardiovascular risk and decreases all-cause mortality by ~10% [4]. Despite their widespread use, approximately 10–20% of patients are noted to be “statin-intolerant” with myalgia and other musculoskeletal complaints noted as common side-effects warranting statin discontinuation [5]. Moreover, growing evidence suggests that statin use may increase the risk of new-onset type 2 diabetes in patients with established risk factors [6, 7]. These results prompted researchers to identify novel molecules that may be targeted safely and can be used as an alternative therapy for LDL-C management.

The discovery of the ninth member of the proprotein convertase family, proprotein convertase subtilisin/kexin type 9 (PCSK9) in 2003 led to a major change in the approach to CVD treatment [8]. PCSK9 is a liver-secreted protein that has the peculiar ability to interfere and block the natural recycling of LDLR, resulting in impaired LDL-C clearance from the plasma [9]. Mutations in the PCSK9 gene were first diagnosed in a French family and have been identified as the third genetic cause of familiar hypercholesterolemia, making PCSK9 protein a great target for CVD treatment [10]. Humans carrying loss-of-function mutations in the PCSK9 gene present with low LDL-C plasma levels and decreased CVD risk, while gain-of-function mutations of PCSK9 are associated with high LDL-C levels and increased risk for CVD [10, 11].

Structure and function of PCSK9

Located on chromosome 1, PCSK9 gene encodes a 692-amino acid secreted protein, composed of signal peptide (aa 1–30), prosegment (aa 31–152), catalytic (aa 153–454) and C-terminal cysteine-histidine-rich (aa 455–692) domains. PCSK9 protein is abundantly expressed in the liver [8]. Besides liver, PCSK9 is expressed in small intestine and kidney and recent work has suggested that PCSK9 is also expressed in human adipocytes and its mRNA levels in adipose tissue positively correlates with body mass index values [12].

After synthesis, PCSK9 proprotein is directed to the endoplasmic reticulum (ER) where the signal peptide (1–30) is removed. Different from all other members of proprotein convertase family, PCSK9 is unique in a way that the autocatalytically cleaved 14 kDa inhibitory prosegment noncovalently binds to the 60 kDa mature form of PCSK9 in the ER, which results in its trafficking to the Golgi and secretion as an inactive dimer complex. Given that PCSK9’s self-cleavage in the ER is critical for its function, conditions that interfere with ER function largely affect PCSK9’s fate. For example, ER stress-causing agents reduce PCSK9 secretion through retaining it in the ER [13]. Of note, recent work has shown that loss-of-function PCSK9 mutants accumulate in the ER without activating the ER stress response or apoptosis, raising the possibility that reducing PCSK9 secretion, via ER retention, may serve as a novel approach to lower plasma PCSK9 and LDL-C [14].

Coat protein complex II (COP II) is essential for the transport of many secreted proteins from the ER to the Golgi and recently surfeit locus protein 4 (SURF4) was identified as an ER cargo receptor that actively recruits PCSK9 into COPII vesicles and promotes the efficient ER exit and secretion of PCSK9 [15]. The absence of SURF4 results in accumulation of PCSK9 in the ER and impaired PCSK9 secretion, suggesting SURF4 as a novel therapeutic target for the treatment of hypercholesterolemia. Within the secretory pathway PCSK9 can interact with and degrade LDLR, which is prevented by PCSK9’s binding to glucose-regulated protein 94 (GRP94) in the ER [16]. Upon secretion, the catalytic domain of PCSK9 interacts with the epidermal growth factor precursor homology repeat-A (EGFA) region of LDLR at the cell surface. Asp residues in the ligand-binding repeat domain of LDLR also contribute to LDLR’s binding to PCSK9 [17]. The PCSK9-LDLR complex enters the cells in clathrin-coated vesicles, which results in the degradation of LDLR in the lysosome [18]. It is noteworthy to mention that PCSK9 binding to LDLR has tissue specificity and liver is the most responsive tissue to this regulation, suggesting the requirement of a co-receptor specific for liver. A recent study has shown that heparin sulfate proteoglycans (HSPG), highly present on the surface of the hepatocytes, capture PCSK9 and this binding is required for PCSK9-LDLR complex formation and LDLR degradation [19]. Heparan-sulfate mimetics such as dextran sulfate, pentosane sulfate and suramin bind PCSK9 and inhibit PCSK9-mediated LDLR degradation. Heparin-like molecules can also interact with LDL-C and play an important role in LDL-C-induced inhibition of PCSK9 uptake into the cells [20].

Although the best-known function of PCSK9 is to target LDLR for lysosomal degradation in hepatocytes, which increases LDL-C and atherosclerotic CVD, PCSK9 may affect atherosclerosis development through its effects on endothelial and vascular smooth muscle cells [21, 22]. Moreover, platelet activation and blood clotting factor VIII (FVIII) levels were also shown to be regulated by PCSK9, suggesting that PCSK9 contributes to CVD etiology through regulating many different pathways [23]. Interestingly, several lines of evidence suggest that PCSK9 may have other pleiotropic properties like improving septic shock outcome [24]; however, these results were not supported by others and need further investigation [25].

PCSK9 regulation

The proximal promoter of PCSK9 gene contains a functional sterol regulatory element (SRE), which is required for sterol-responsive element binding protein-2 (SREBP-2) to induce PCSK9 transcription [26]. SREBP-2 is a master regulator of cholesterol homeostasis in cells [27]. By inhibiting HMG-CoA reductase, statins induce SREBP-2 activity and paradoxically stimulate the expression of PCSK9 mRNA, which attenuates their LDL-C–lowering effect [28]. PCSK9 gene also contains a hepatocyte nuclear factor 1α (HNF1α) binding motif in the proximal region of its promoter (Figure 1A). HNF1α levels are upregulated by statin treatment and downregulated by the plant-derived cholesterol-lowering compound berberine and activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway [26, 29]. Furthermore, NAD-dependent deacetylase SIRT6, through its interaction with FOXO3, modulates histone acetylation of Pcsk9, thereby suppressing its transcription [30]. Recently, E2F1, a transcription factor controlling cell cycle, has been shown to bind the Pcsk9 promoter and regulate Pcsk9 mRNA levels. E2F1 deficient mice have lower PCSK9 in liver, which results in lower plasma LDL-C levels supporting the hypothesis that E2F1 might be a promising therapeutic target for plasma LDL-C management [31]. Besides these transcription factors, recent work has shown that thyroid-stimulating hormone (TSH) increases PCSK9 mRNA expression in HepG2 cell line (Figure 1A). Moreover, circulating TSH levels in subclinical hypothyroid patients correlate with plasma LDL-C and PCSK9, suggesting a role for TSH in cholesterol metabolism [32]. Finally, the major metabolite of berberine, berberrubine, has been proposed to exert potent inhibitory effect on PCSK9 through a pathway involving extracellular signal regulated kinase (ERK), which results in higher LDLR levels in cellular models, awaiting in vivo testing [33]. Post-transcriptionally, miR-191, miR-222 and miR-224 has been recently shown to directly interact with the 3’-UTR of PCSK9 mRNA and regulate PCSK9 expression (Figure 1B) [34]. Post-translationally, PCSK9 gets regulated via Furin and PC5/6. Furin-cleaved shorter fragment of PCSK9 has less LDLR-binding capacity and is unable to degrade it [35]. In addition to this regulation, recent work has suggested that PCSK9 gets phosphorylated on its Ser 688 residue, which is decreased by chronic statin treatment; however, the significance of this finding remains to be investigated [36].

Figure 1. Molecular mechanisms of PCSK9 regulation.

(A) Statins, berberine, insulin, mTORC1, SIRT6, thyroid stimulating hormone (TSH), E2F1 and berberrubine regulate PCSK9 mRNA expression through various mechanisms. (B) miR-191, miR-222 and miR-224 directly interact with the 3’-UTR of PCSK9 mRNA and contribute to PCSK9 regulation. (C) Glucagon receptor signaling, thorugh its downstream effectors, Epac2 (exchange protein activated by cAMP 2) and Rap1 (Ras-related protein 1), enhances lysosomal degradation of PCSK9.

Besides these, PCSK9 has been shown to be hormonally regulated by insulin and estrogens, mostly at the mRNA level [37-39]. Interestingly, PCSK9 levels are reduced by fasting; however, in-depth mechanistic information is lacking [40]. Glucagon is the major fasting hormone that contributes to energy metabolism through regulating the hepatic glucose production during hypoglycemia. Glucagon also participates in the regulation of cholesterol metabolism as glucagon administration lowers plasma LDL-C and glucagon receptor antagonists developed for type 2 diabetes management increase LDL-C [41-45]. Consistent with this, recent work describing an inactivating glucagon receptor mutation with nearly complete inhibition of glucagon action in a pediatric case reported elevated LDL-C levels [46]. However, the underlying mechanism(s) hasn’t been fully uncovered. In this context, our recent findings shed some light on this mechanism and showed that glucagon regulates plasma LDL-C through PCSK9 [47]. Silencing of hepatic glucagon receptor or inhibition of glucagon action resulted in increased plasma PCSK9 and LDL-C; whereas, glucagon treatment of WT mice had the opposite effects. Mechanistically, we showed that glucagon, acting through its downstream mediators EPAC2 (exchange protein activated by cAMP 2) and RAP1 (Ras-related protein 1), enhanced lysosomal degradation of PCSK9 without an effect on Pcsk9 mRNA levels (Figure 1C). Our results provided novel insights into the molecular interplay between hepatic glucagon signaling and LDL-C metabolism and described a new posttranscriptional mechanism of PCSK9 regulation. It is important to note that previous work reported the involvement of lysosomal degradation in PCSK9 regulation and glucagon has been shown to stimulate lysosomal degradation pathways [48-50]. The mechanism(s) linking glucagon receptor signaling to lysosome-mediated degradation of PCSK9 remains to be elucidated.

PCSK9 inhibition as a therapy for lowering LDL-C

The great interest in PCSK9 research over the past 15 years is mainly due to its rapid and efficient translation from basic science to clinical practice. Fully human monoclonal antibodies against PCSK9 (alirocumab and evolocumab) that disrupt the binding of extracellular PCSK9 to LDLR are now approved by US Food and Drug Administration for use in patients with familial hypercholesterolemia and in patients with clinical atherosclerotic CVD who require additional lowering of LDL-C levels [51]. Clinical trials have demonstrated that treatment with these anti-PCSK9 antibodies results in ~60% lowering of LDL-C and protects against cardiac ischemic events (including myocardial infarction) and induces atheroma regression when combined with statins [52, 53]. Moreover, in patients with homozygous and heterozygous familial hypercholesterolemia who respond poorly to high-dose statins, treatment with PCSK9 antibodies further lowered LDL-C suggesting the idea that PCSK9 inhibition may improve cardiovascular health and life expectancy of these patients who have very high LDL-C levels [54, 55].

Both alirocumab and evolocumab are well-tolerated and current data indicate that PCSK9 inhibitors do not result in increased risk for adverse events such as an increase in new-onset type 2 diabetes, liver toxicity or impairment in cognitive function even in patients whose LDL-C levels were lowered less than 20 mg/dl [56, 57]. These data suggest that extremely low plasma LDL-C with PCSK9 inhibition is safe. Interestingly, anti-PCSK9 antibodies led to significant dose-related reductions in plasma levels of Lipoprotein (a) [Lp(a)], which contains apolipoprotein B-100 (apoB) covalently bound to a plasminogen-like glycoprotein, apolipoprotein (a) [58]. Lp (a) is encoded by the LPA gene and is now recognized as an independent cause of atherosclerotic cardiovascular disease [59]. Although atorvastatin was shown to paradoxically increase plasma Lp(a), PCSK9 neutralization led to ~30% lowering of circulating Lp(a), which further supports a beneficial role for PCSK9 inhibition in CVD prevention [60]. Despite these beneficial effects of anti-PCSK9 antibodies, the need for monthly subcutaneous injections and their high-cost have limited their wide-spread use [61]. In this context, recent work using long-acting small interfering RNA directed against hepatic PCSK9 have shown significant reductions in LDL-C comparable to anti-PCSK9 antibody treatment [62]. Moreover, this RNA interference approach is more durable and has the potential to improve adherence without serious side effects [63]. However, long-term follow-up on the effectiveness and safety of this approach is needed. Currently, alternative approaches like small molecule inhibitors of PCSK9, PCSK9 vaccination or gene editing are under development to target PCSK9, which may bring alternative modalities to lower LDL-C if translated into humans [64-67]. Recent identification of a targetable groove adjacent to the EGF(A)-binding site of PCSK9 provided the proof of concept that future small molecule inhibitors of PCSK9 is feasible [64].

CONCLUSION

PCSK9 is an important player in cholesterol homeostasis. By binding to hepatic LDLR and promoting its degradation, PCSK9 reduces LDL-C uptake, leading to an increase in plasma LDL-C concentrations. The exciting new findings on its biology and function has broaden our knowledge; however, more mechanistic data is needed, which could potentially be harnessed for therapeutic intervention and could guide the use of anti-PCSK9 therapies in different patient populations. Recent data on the efficacy and tolerability of PCSK9 inhibitors is promising and accumulating evidence suggest that PCSK9 inhibition has many beneficial effects besides LDL-C management like lowering Lp(a) levels. Long-term safety data on anti-PCSK9 therapies in clinical practice will provide a therapeutic option for managing dyslipidemia.

Key points:

• LDL-C management is protective against atherosclerotic CVD.

• PCSK9 is a novel therapeutic target that degrades LDLR and contributes to LDL-C homeostasis.

• Inhibition of PCSK9 not only lowers LDL-C but also reduces atherogenic Lp(a) in the plasma.

• Current data suggest that PCSK9 inhibition via monoclonal antibody injection and RNA interference is safe.