PCSK9 AND INFLAMMATION: IN VITRO STUDY ON HEPATOCYTES AND MACROPHAGES
Creators: name: RICCI, CHIARA
Publisher: name: Universit� degli Studi di Milano
Year: 2018

description: TNF-alpha induces proprotein convertase subtilisin kexin type 9 (PCSK9) expression in hepatic HepG2 cell line, through the activation of suppressor of cytokine signaling 3 (SOCS3): Background. The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by pro-inflammatory cytokines, including the tumor necrosis factor-? (TNF-?). SOCS3 is also implicated in hypertriglyceridemia associated to insulin-resistance (IR). Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) levels are frequently found to be positively correlated to IR and plasma very low-density lipoprotein-triglycerides (VLDL-TG) concentrations. Aim. The present study aimed to investigate the possible role of TNF-? and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. Methods and results. TNF-? induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein levels, with no effect on its promoter activity. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels while an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fatty-acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD-1), and apo-B. These responses were associated with significant increase of SCD-1 protein, activation of SREBP-1, accumulation of cellular TG and secretion of apoB. HepG2SOCS3 show lower phosphorylation levels of IRS-1 Tyr896 and Akt Ser473 in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, FAS and apoB mRNA. Conclusions. Our data candidate PCSK9 as a gene involved in lipid metabolism regulated by pro-inflammatory cytokine TNF-?, in a SOCS3 dependent manner. Proprotein subtilisin/kexin type 9 (PCSK9) induces pro-inflammatory response in macrophages: Background. Intraplaque release of inflammatory cytokines and chemokines from macrophages is directly implicated in atherogenesis, by inducing the proliferation and migration of media smooth muscle cells (SMCs) to the neointima. PCSK9 is present and released by SMCs within the atherosclerotic plaque but its role within the vascular wall is still unknown. Aim. In the present study, we tested the hypothesis of a pro-inflammatory effect of PCSK9 on macrophages. Methods and results. The pro-inflammatory effect of PCSK9 was assessed on THP-1-derived macrophages, exposed to different concentrations (0.250 � 2.5 �g/ml) of human recombinant PCSK9 (hPCSK9). After exposure for 24h to 2.5 �g/ml PCSK9, a significant induction of IL-1? (8.17�2.88 fold), IL-6 (36.4�19.3 fold), TNF-? (67.4�25.9 fold), CXCL2 (42.6�0.0 fold), and MCP1 (17.0�6.8 fold), were observed. Importantly, physiological concentration of PCSK9 (0.250 �g/ml) also elicited a significant pro-inflammatory effect. Similar results were observed in human primary macrophages, where 2.5 �g/ml of hPCSK9 increased IL-1? (14.61�2.47 fold), IL-6 (4.17�0.86 fold), TNF-? (4.51�2.08 fold), CXCL2 (2.58�0.26 fold), and MCP-1 (1.76�0.07 fold) gene expression and the released of TNF-? (+82.3%) and IL-6 (+41.8%) in cultured media, as determined by ELISA assay. Co-culture experiments of HepG2 overexpressing hPCSK9 and THP1 macrophages also showed the induction of mRNA TNF-? (1.89�0.35 fold), IL-1? (2.03�0.29 fold), MCP-1 (4.82�1.26 fold) and CXCL2 (5.40�0.61 fold). Finally, the effect of hPCSK9 on TNF-? mRNA in murine LDLR-/- bone marrow macrophages (BMM) was significantly impaired as compared to wild-type BMM (5.44�0.28 fold vs 35.4�2.7 fold for LDLR-/- and wild-type, respectively). Conclusions. The present study provided evidences of a pro-inflammatory action of PCSK9 on macrophages, mainly dependent by the LDLR. The pathophysiological relevance of this effect still needs to be determined.