HBx Up-regulating CD147 Promotes Liver Fibrosis In Hepatic Stellate Cells

Liver fibrosis results from chronic liver injury during a long-term wound-healing response, which causes increasing excessive accumulation of extracellular matrix(ECM) proteins and eventually leads to fibrogenesis and later cirrhosis. The hepatic stellate cells(HSCs) are the main ECM-producing cells during this process, and they activate and differentiate from quiescent vitamin A-storing cells into proliferative myofibroblasts in response to fibrogenic liver injury. Activated HSCs express many ECM proteins including collagen type I, α-smooth muscle actin(α-SMA), transforming growth factor-β1(TGF-β1), matrix metalloproteinase(MMP), and tissue inhibitors of metalloproteinases, which contribute to liver fibrosis.Clinical studies suggest that hepatitis B virus(HBV) chronic infection is the most important cause of liver cirrhosis and hepatocellular carcinoma(HCC) in human patients. As one of encoded-proteins by HBV, the HBV X protein(HBx) is a multifunctional regulatory protein that plays a crucial role in transcription, signal transduction, cell cycle progress, protein degradation pathways, apoptosis, and genetic stability in liver disease progression. TGF-β1 is considered a key mediator of liver fibrogenesis by activating HSCs. TGF-β1 predominance is detected in HBV-related liver fibrogenesis and HSCs exposed to conditioned medium from HBx-expressing hepatocytes show increased expression of TGF-β1. HBx directly activates transcription of the TGF-β1 gene in transfected hepatoma cells through the Egr-1 binding sites.CD147, a typical membrane protein, is a memberof the immunoglobulin super family. CD147 is widely expressed on numerous cells in carcinomas and inflammation tissues, while expressed negatively or at a very low level in normal tissues. Our previous study and other’s reveal that prominent expression of CD147 in perisinusoidal area in human liver cirrhosis, which indicate that this molecule may play a role in liver fibrosis and cirrhosis. CD147 expression in HSCs is elevated by TGF-β1 stimulation, but the regulating mechanism is not uncovered.In this study, we hypothesized a direct role of HBx in the development of liver fibrosis by the paracrine activation of HSCs through TGF-β1-CD147 signaling loop. We here showed that HBx induced the active TGF-β1 release from hepatocytes, which exerted on HSC activation by directly transcriptional regulation of CD147 through TGF-β1/Smad signaling pathway. The CD147 upregulating contributed to HBV-associated liver fibrosis progression.Part 1: HBx upregulated CD147 expression to activate HSCs through paracrine TGF-β1. HBx-transfected human non-tumour hepatic L02 cell line, named L02-HBx was established and showed HBx expression by western blot analysis. The ectopic expression of HBx significantly induced the elevation of total and active TGF-β1 levels in L02-HBx cells(P<0.001). We next tested the effects of HBx on CD147 expression in human HSC cell line(LX-2) after exposure to conditioned medium from L02-HBx cells or a non-contact co-culture system. We observed that CD147 was significantly increased in LX-2 cells either incubation with L02-HBx conditioned medium or co-cultured with L02-HBx cells. This up-regulation was inhibited with a selective antagonist of TGF-β1 type-1 receptor SB431542, which demonstrated that TGF-β1 signaling transduction was involved in CD147 expression by a paracrine way. We then evaluated the levels of CD147 and fibrosis-related genes in response to different doses of TGF-β1 in LX-2 cells. The mRNA and protein levels of CD147, α-SMA, α1(I) collagen, and MMP-2 were significantly up-regulated with TGF-β1 stimulation in dose-dependent manners. We then transfeced CD147 gene into LX-2 cells, and found TGF-β1, α-SMA, and α1(I) collagen mRNA were significantly upregulated. Furthermore transient over-expression of CD147 promoted LX-2 cell migration and contraction as detected by wound healing assay and collagen-based cell contraction assay. Collectively, our data suggested that HBx was capable of inducing the secretion of TGF-β1 in hepatocytes that resulted in paracrine activation of HSCs by CD147-TGF-β1 positive feedback mechanism transduction.Part 2: CD147 was upregulated and promoted liver fibrogenesis in HBV-tg mice. We used HBV-tg mice(C57BL/6J-Tg(Alb1HBV)44Bri/J) for assessing the role of CD147 in liver fibrogenesis. We found that the livers from 12-month-old HBV-tg mice appeared irregular shape and noticeable regenerative nodules with significantly higher weight compare to C57BL/6 control mice. H&E staining of liver tissues from HBV-tg mice demonstrated intense polymorphonuclear leukocyte and macrophage infiltration with severe centrilobular necrosis. Masson’s trichrome staining of the liver was performed to assess the collagen distribution and showed that extensive collagen deposition and pseudolobular formation was evident in liver tissues from HBV-tg mice compared with normal control, which was confirmed by sirius red staining. These results verified that liver fibrosis was spontaneously developed in HBV-tg mice. Accordingly, western blotting and/or immunohistochemistry showed that α-SMA, MMP-2, and α1(I) collagen were remarkably increased in livers from HBV-tg mice, and HBx was uniquely expressed. We then checked the expression of key molecules of TGF-β1-CD147 positive feedback loop in liver tissues. Immunohistochemistry analysis displayed a stronger staining of CD147 and TGF-β1 in liver tissues from 6-month-old HBV-tg mice. Double immunofluorescece detected that both TGF-β1 and CD147 colocalized with α-SMA-positive cells in fibrotic liver tissues, respectively, which suggests that TGF-β1-CD147 signaling in activated HSCs is involved in HBV-related liver fibrosis.Part 3: Smad signaling was involved in TGF-β1-induced CD147 expression in HSC. We treated LX-2 cells with TGF-β1, the protein levels of Smad2, Smad3, and Smad4 showed no changes. However the phosphorylations of Smad2(p-Smad2) and Smad3(p-Smad3) were elevated in a TGF-β1-dose-dependent manner(the same results were tested in HBV-tg mice),and Smad4 aggregated from cytoplasm to nucleus. Then, LX-2 cells were treated with si-Smad2, si-Smad3, and si-Smad4, respectively, the corresponding Smad expressions were silenced, coupled with the fact that the TGF-β1-upregulated CD147 was depressed. These findings suggested that TGF-β1 induced expression of CD147 in HSCs through activation of Smad signaling by the cooperation of Smad2, Smad3, and Smad4. To determine whether Smad4 directly regulated CD147 expression at a transcriptional level, we co-transfected CD147 promoter(-1761/+37) with pReceiver-M02-Smad4 and pGL3-Basic plasmids into LX-2 cells. Dual-luciferase reporter assay showed a Smad4 dose-dependent increase in luciferase activity. We further identified the CD147 promoter core region and analyzed one candidate SBE sequence located between positions-444 and-449. We then generated a MutCD147 construct that contained mutations in the SBE domain by site-directed mutagenesis. We found that the promoter activity was completely abolished upon mutation of the Smad4-binding site in SBE, suggesting that SBE is required for activation of the CD147 promoter by Smad4. To show direct binding of Smad4 to the putative binding site within the critical CD147 promoter region, Ch IP assay was performed in LX-2 cells. The PCR-amplified fragment of the CD147 promoter region containing the SBE was retrieved from the immunoprecipitates using an anti-Smad4 antibody. These results show that Smad4 activates CD147 transcription by direct interaction with the SBE domain that is located in the proximal promoter of the CD147 gene.