RECK regulation of NASH and fibrosis

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PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) is a global epidemic, progresses to nonalcoholic steatohepatitis (NASH) and fibrosis, and results in the development of hepatocellular carcinoma and increased cardiovascular mortality. Unfortunately, no pharmacological therapies are available yet to treat NASH and fibrosis, necessitating the identification of novel targets and approaches. RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a unique membrane-anchored protein, has been shown to inhibit multiple mediators involved in inflammation and fibrosis. Our novel preliminary data demonstrate that hepatic RECK protein levels are markedly reduced with increasing severity of NASH and fibrosis in clinical patients and in our pre-clinical mouse model of western diet (WD)-induced NASH and fibrosis. Since RECK gene deletion is embryonically lethal, we generated RECK floxed (RECKfl/fl) and CAG-CATflox-RECK transgenic mice. Our proof-of-concept pilot studies demonstrate that while hepatocyte-specific RECK knockdown by AAV8-mediated Cre recombinase exacerbates NASH and fibrosis in short-term WD-fed mice, its overexpression in hepatocytes blunts liver inflammation, Kupffer cells (KC) and hepatic stellate cell (HSC) activation. Moreover, our preliminary in vitro data in primary mouse hepatocytes, KCs and HSCs in which RECK is either silenced or overexpressed support our in vivo studies. These preliminary studies suggest that sustaining RECK expression is hepatoprotective. Therefore, we hypothesize that Cre-Lox mediated RECK deletion specifically in hepatocytes enhances pro-inflammatory signaling by enhancing amphiregulin (AREG) cleavage by ADAM (A Disintegrin And Metalloproteinase domain-containing protein) 10- and 17, leading to increased epidermal growth factor receptor (EGFR) and HSC activation collectively contributing to worsening of long-term WD-induced NASH and fibrosis (Aim 1). Conversely, transgenic overexpression of RECK, specifically in hepatocytes, will be protective. We will also determine if rescuing RECK expression by ectopic overexpression in hepatocyte-specific RECK deficient mice with established WD-induced NASH and fibrosis can be reversed (Aim 1). Since KCs are the predominant resident liver macrophages and HSCs are considered the principal cell type responsible for hepatic fibrosis, we will establish the importance of RECK deletion and transgenic overexpression in Kupffer cells and HSCs on cellular injury and activation, extracellular matrix deposition, and fibrosis (Aim 2). In both Aims, molecular mechanisms underlying inflammation and fibrosis will be investigated in co-culture studies using primary hepatocytes, KCs and HSCs isolated from these gene-altered mouse models. Thus, our proposed genetic and interventional approaches will mechanistically establish RECK as a novel upstream regulator in the pathogenesis of both NASH and fibrosis with potential as a future therapeutic.