Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7

Abstract

BACKGROUND & AIMS The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor beta (TGFB) signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6, and another inhibitory SMAD (SMAD7), promote HCV infection in human hepatoma cells and hepatocytes. METHODS We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with JFH1 HCVcc; we measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. HCV entry in HepG2/miR122/CD81 cells, which support entry and replication of HCV, were transfected with small-interfering (si)RNAs and gene expression profiles were analyzed. Uptake of labeled low-density lipoprotein (LDL) and cholesterol were measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 mRNAs. RESULTS siRNA knockdown of SMAD6 blocked the binding and infection of cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased infection by HCV. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells following SMAD6 knockdown. SMAD6 knockdown also reduced transcription of gene encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR (VLDLR), and the scavenger receptor class B member 1 (SCARB1 or SR-BI) in hepatocytes; SMAD6 knockdown also reduced cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. HCV infection of cells increased expression of SMAD6, which required the activity of nuclear factor-kB (NF-kB), but not TGFB. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. Conclusions In studies of hepatoma cell lines and human primary hepatocytes, we found that infection with HCV leads to activation of NF-κB, leading to increased expression of SMAD6 and SMAD7. Upregulation of SMAD6 and SMAD7 lead to increased expression of HSPGs, such as SDC1, as well as LDLR, VLDLR, and SR-BI, which promote HCV entry and propagation, as well as cell uptake of cholesterol and lipoprotein.

Infection of Rat Osteoblasts with Junin Virus Promotes the Expression of Bone Morphogenetic Protein 6, an Osteogenic Differentiation Inducer

“…This agrees with the increased levels of BMP-6 expression observed after JUNV infection in OBCs. Similarly, upregulation of BMPs/Smad signaling pathway has been reported as necessary for viral entry in the case of infection of hepatocytes with hepatitis C virus [26]. In the case of human hepatitis B virus and human immunodeficiency virus, it has been described that an increase in viral transcription and replication is mediated by transforming growth factor-β/BMP signaling, iron or hepcidin either in human hepatocytes or human primary macrophages, and CD4+ T cells, respectively [27, 28].…”

SMAD About Hepatitis C Virus Cell Entry and Liver Disease

“…5 Transcriptomics analysis revealed that silencing of SMAD6 decreased the expression of HSPGs, with a corresponding reduction in cell surface expression of heparan sulfate. 3 Furthermore, SMAD6 knockdown decreased expression of cholesterol uptake receptors and HCV entry factors, including low-density lipoprotein receptor and scavenger receptor class B1 type I. In contrast, exogenous expression of SMAD6 was shown to up-regulate the expression of HSPGs and cholesterol uptake receptors.…”

“…Furthermore, a comprehensive identification of HCV host dependencies is crucial to better understand the pathogenesis of HCV-induced liver disease biology and to identify strategies for the prevention of hepatocellular carcinoma. In this issue of Gastroenterology , Zhang et al 3 characterize the role of SMAD6 in the regulation of HCV entry and uncover a new role for inhibitory SMADs (I-SMADs) in viral pathogenesis.…”

“…Although several host proteins have been identified as regulatory factors, the detailed regulation of the HCV entry process is only partially understood. 4 Zhang et al 3 follow up on their previous identification of SMAD6 as an HCV host factor. 5 Transcriptomics analysis revealed that silencing of SMAD6 decreased the expression of HSPGs, with a corresponding reduction in cell surface expression of heparan sulfate.…”

“…In contrast, exogenous expression of SMAD6 was shown to up-regulate the expression of HSPGs and cholesterol uptake receptors. 3 By modulating the expression of viral entry factors, SMAD6 regulates the ability of HCV to infect cells (Figure 1). …”