DECIPHERING THE ROLE OF ADIPOKINES ON PCSK9 REGULATION: EXPERIMENTAL EVIDENCE

Background: Adipose tissue is an endocrine organ secreting active molecules, namely, adipokines. In a condition of dysfunctional visceral fat depots, adipokines may be detrimental for the cardiovascular system. The present study was aimed at evaluating some of the molecular mechanisms linking adipokines and the expression of proprotein convertase subtilisin/kexin 9 (PCSK9), the key regulator of low-density lipoprotein receptor and also involved in triglycerides (TG) metabolism. This latter evidence come from genetic studies reporting high levels of TG in patients with gain of function (GOF) mutations in PCSK9 gene while, in those carrying loss of function (LOF) mutations, LDL-C reduction associates with reduced fasting and post-prandial TG levels. Leptin is a cytokine-like hormone produced mainly by white adipose tissue and playing a role not only in satiety but also in regulating blood pressure, endothelial function, glucose homeostasis, insulin sensitivity. Resistin is an adipokine produced by adipose tissue in mice and by monocytes in human; it is involved in inflammation and insulin resistance. Both leptin and resistin are dysregulated in obesity. In order to study the complex biosystem of TG metabolism we have developed and characterized an in vitro tool allowing to study the impact of genetic variants of lipoprotein lipase (LPL), one of the major regulators of TG levels. We believe that this in vitro tool could be of interest to study the effect of PCSK9 on TG regulation. Materials: Human epatocarcinoma cell line (HepG2); silencing RNA (siRNA) anti-PCSK9 and anti-STAT3; qPCR; Western blot; enzyme-linked immunosorbent assay (ELISA); PCSK9 promoter luciferase reporter assay with plasmids pGL3-PCSK9-D4 (wild type), pGL3-PCSK9-SREmut (mutated on SRE element), pGL3-PCSK9-HNFmut (mutated on HNF element), pGL3-PCSK9-D1 (wild type) and pGL3-PCSK9-D1-STAT3mut (mutated on STAT3). C57BL6J mice, ob/ob mice (mice lacking leptin). Results: In a clinical setting, a positive association between circulating levels of leptin and PCSK9 (p=0.044 and β=0.295) was found in patients with BMI<25 Kg/m2. In the same cohort and according to BMI strata, no associations were found between resistin and PCSK9. When the link between leptin and PCSK9 was investigate in ob/ob mice lacking leptin, the hepatic expression of PCSK9 was significantly lower compared to the one of wild type mice. In C57BL6 mice a high-fat-diet resulted in raised levels of serum leptin and increased hepatic expression of PCSK9. In HepG2 cells, leptin and resistin induced (i) PCSK9 gene expression by +95% and +150%, respectively, and (ii) PCSK9 promoter activity via the involvement of Sterol Regulatory Element motif. Indeed, a mutation in HNF-1 motif did not alter leptin- and resistin-driven luciferase activity. The mandatory role of signal transducer and activator of transcription 3 (STAT3) in the relationship between adipokines and PCSK9 has been confirmed by silencing STAT3. After silencing, leptin- and resistin were not able to regulate the mRNA expression of PCSK9. Although not fully characterized, the involvement of STAT3 in the PCSK9 promoter activity has been tested by inserting a mutation on the STAT3 responsive element. Apolipoprotein (APOB) and microsomal triglycerides transfer protein (MTP) mRNA were increased by leptin (+57% and +60%, respectively) and resistin (+ 50% for both), an effect dependent on PCSK9, as demonstrated by using siRNA anti-PCSK9. As a further confirmation, a significant increment in APOB was found in response to PCSK9 overexpression. In order to study the complex biosystem of TG metabolism, which is also a target of PCSK9, we have initially developed and characterized an in vitro tool allowing to evaluate the impact of genetic variants of LPL, an enzyme involved in TG metabolism. This technique allowed to distinguished between LPL mutations leading to a reduced secretion or a reduced activity despite a normal synthesis. Conclusions This work describes the relationship between PCSK9 expression and the adipokines leptin and resistin. The molecular basis beyond these findings require the involvement of STAT3 pathway and the activation of SRE motif at PCSK9 promoter level. Overexpression of PCSK9 led to an increment of gene expression of APOB, whereas silencing PCSK9 corresponded to an opposite effect i.e. a decrement in gene expression. The tool developed to study LPL mutations could be of help in evaluating PCSK9 variants associated with raised TG levels.