The HIV Matrix Protein p17 Promotes the Activation of Human Hepatic Stellate Cells through Interactions with CXCR2 and Syndecan-2

BackgroundThe human immunodeficiency virus type 1 (HIV-1) p17 is a matrix protein involved in virus life's cycle. CXCR2 and Syndecan-2, the two major coreceptors for the p17 protein, are expressed in hepatic stellate cells (HSCs), a key cell type involved in matrix deposition in liver fibrotic disorders.AimIn this report we have investigated the in vitro impact of p17 on HSCs transdifferentiation and function and underlying signaling pathways involved in these processes.MethodsLX-2 cells, a human HSC line, and primary HSC were challenged with p17 and expressions of fibrogenic markers and of p17 receptors were assessed by qRT-PCR and Western blot. Downstream intracellular signaling pathways were evaluated with qRT-PCR and Western blot as well as after pre-treatment with specific pathway inhibitors.ResultsExposure of LX2 cells to p17 increases their contractile force, reshapes the cytoskeleton fibers and upregulates the expression of transdifferentiation markers including αSMA, COL1α1 and endothelin-1 through the activation of Jak/STAT and Rho signaling pathways. These effects are lost in HSCs pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide. Confocal laser microscopy studies demonstrates that CXCR2 and syndecan-2 co-associate at the plasma membrane after exposure to p17. Immunostaining of HIV/HCV liver biopsies from co-infected patients reveals that the progression of liver fibrosis correlates with a reduced expression of CXCR2.ConclusionsThe HIV matrix protein p17 is pro-fibrogenic through its interactions both with CXCR2 and syndecan-2 on activated HSCs.

【研究背景】人类免疫缺陷病毒1型（HIV-1）p17是一种基质蛋白，参与病毒的生命周期。CXC趋化因子受体2（CXCR2）与多配体蛋白聚糖-2（Syndecan-2）是p17蛋白的两种主要共受体，二者均在肝星状细胞（hepatic stellate cells, HSCs）中表达；而肝星状细胞是肝纤维化疾病中基质沉积的关键细胞类型。
【研究目的】本研究旨在体外探究p17蛋白对肝星状细胞转分化及功能的影响，以及该过程所涉及的潜在信号通路。
【实验方法】本研究使用p17蛋白处理人肝星状细胞系LX-2以及原代肝星状细胞，通过实时荧光定量PCR（qRT-PCR）与蛋白质免疫印迹（Western blot）检测纤维化标志物及p17受体的表达水平；同时通过上述两种实验技术，以及特异性信号通路抑制剂预处理后的实验，对下游细胞内信号通路进行评估。
【实验结果】将LX-2细胞暴露于p17蛋白后，可通过激活Janus激酶/信号转导与转录激活因子（Jak/STAT）及Rho信号通路，增强细胞收缩力、重塑细胞骨架纤维，并上调包括α平滑肌肌动蛋白（αSMA）、Ⅰ型胶原α1链（COL1α1）及内皮素-1在内的转分化标志物的表达。若先用接种过p17肽疫苗的HIV阳性患者血清预处理肝星状细胞，则上述效应会完全消失。共聚焦激光显微镜研究显示，暴露于p17蛋白后，CXCR2与Syndecan-2会在细胞膜处发生共定位。对同时感染HIV与丙型肝炎病毒（HCV）患者的肝脏活检组织进行免疫染色后发现，肝纤维化进展与CXCR2表达下调呈显著相关。
【研究结论】人类免疫缺陷病毒基质蛋白p17可通过与活化肝星状细胞表面的CXCR2及Syndecan-2结合，发挥促纤维化作用。