HCV-activated NLRP3-inflammasome regulates lipid ...

JBC Papers in Press. Published on December 23, 2015 as Manuscript M115.694059 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.694059 HCV-activated NLRP3-inflammasome regulates lipid metabolism

Hepatitis C virus-induced NLRP3-Inflammasome Activates the Sterol Regulatory Element Binding Protein (SREBP) and Regulates Lipid Metabolism

Steven McRae1, Jawed Iqbal1, Mehuli Sarkar-Dutta1, Samantha Lane1, Abhiram Nagaraj1, Naushad Ali2, and Gulam Waris1#

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Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Rosalind Franklin University of Medicine and Science, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL, United States of America 2

Department of Medicine, Section of Digestive Diseases and Nutrition, University of Oklahoma, Oklahoma City, Oklahoma, United States of America

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Corresponding author Phone: 847 578 8839 Fax: 847 578 3349

[email protected]

1 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Running Title: HCV-activated NLRP3-inflammasome regulates lipid metabolism

HCV-activated NLRP3-inflammasome regulates lipid metabolism

INTRODUCTION Chronic liver disease resulting from HCV infection represents a major global health problem. HCV infection often leads to chronic hepatitis in up to 60-80% of infected adults and progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (1). The HCV genome is a 9.6kb positive-sense single stranded RNA molecule containing a 5’ untranslated region (UTR), a single

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open reading frame, and a 3’UTR (2). The 5’UTR contains an internal ribosome entry site (IRES), which directs cap-independent translation of a polyprotein precursor of ~3000 amino acids that is cleaved by viral proteases and host cell signal peptidases into mature structural proteins (core, E1, E2, p7) and nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (2). The majority of HCV infected individuals develop persistent infection that promotes chronic inflammation which is considered to be the primary catalyst for progressive liver disease and development of HCC. Our recent work highlights a mechanism of chronic inflammation through the activation of the NLRP3-inflammasome in HCVinfected hepatoma cells (3). In addition, previous studies have also shown the activation of NLRP3inflammasome in hepatic macrophages and monocytes (4-7). Activation of the inflammasome is a major mechanism of inflammation leading to the production of proinflammatory interleukin1β (IL-1β) and IL-18 cytokines via caspase-1 activation (8). Most inflammasomes consist of a member of the NOD-like receptor (NLR) family of cytosolic receptors that either directly interact with caspase-1 or are indirectly coupled to it by the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), and procaspase-1 (8). Activated caspase-1 processes pro-IL-1β and IL-18 into their mature forms. In chronic HCV infection, induction of proinflammatory molecules including IL-1β plays a central role in the pathogenesis of HCV (9, 10). In addition to their role in IL-1β and IL-18 regulation, NLRP3, ASC and caspase-1 are increasingly being recognized to have inflammasome/cytokine-independent functions (11-15). Recent studies demonstrated that inflammasome-independent NLRP3 augments TGF-β1 signaling in kidney epithelium and cardiac fibroblasts (12, 13). NLRP3 is also known to interact with ubiquitin ligase associated protein SGT1, heat-shock protein 90 (HSP90), and thioredoxin (TRX)-interacting protein (TRIXP) (16, 17). Typically, caspase-1 mediates the maturation of the IL-1β and IL-18 in immune and non-immune cells (18). However, studies have shown that several proteins associated with the glycolytic pathway are cleaved by caspase-1 which is suggestive of a broader role of caspase-1

ABSTRACT Hepatitis C virus (HCV) relies on host lipids and lipid droplets (LDs) for replication and morphogenesis. The accumulation of LDs in infected hepatocytes manifests into hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study demonstrates the activation of the inflammasome complex in HCVinfected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that HCV-activated NLRP3-inflammasome is required for the upregulation of lipogenic genes such as HMGCS, FAS, and SCD. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, ASC, and caspase-1), we further show that the activation of the NLRP3-inflammasome plays a critical role in lipid droplets formation. The NLRP3inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of Insig proteins. This subsequently leads to the transport of SCAP-SREBPs complex from the ER to the Golgi followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits NLRP3-inflammasome to activate SREBPs and host lipid metabolism leading to liver disease pathogenesis associated with chronic HCV.


EXPERIMENTAL PROCEDURES Plasmids and reagents–The infectious HCV J6/JFH-1 cDNA (genotype 2a) and the replicationdefective HCV JFH-1/GND constructs were obtained from Dr. C. Rice (Rockefeller University, NY). Recombinant IL-1β were purchased from R&D systems (Minneapolis, MN). The wild-type human pFLAG-NLRP3 expression vector was obtained from Dr. J. Tschopp (University of Lausanne, Switzerland). MG132, ALLM, and inhibitors of caspase-1 (z-YVAD-FMK), and caspase-3 (DEVD) were from EMD Millipore (Massachusetts, MA). BODIPY (4, 4-difluoro-4bora-3a, 4a-diaza-s-indacene) was purchased from Invitrogen (Carlsbad, CA). Antibodies-The following antibodies were used according to the manufacturer’s protocols: HCV NS3 (Virogen, Watertown, MA), actin and β-tubulin (Sigma), NLRP3 for western blotting from Abcam (Cambridge, MA); for immunofluorescence (IFA) from ProSci (Atlanta, GA), ASC (MBL, Woods Hole, MA), caspase-1 (Invitrogen), SREBP-1, SREBP-2, Insig-1, Insig-2 and SCAP (Santa Cruz Biotechnology, Dallas, TX), and albumin (Thermo Scientific, Rockford, IL). The rabbit polyclonal antibody against HCV

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proteins (29). Insig-1 and Insig-2 are membranebound proteins that reside in the ER and play a central role in the regulation of SREBPs activation (30). When the cells are depleted in cholesterol, SCAP transports SREBPs from the ER to the Golgi, where Site-1 (S1P) and Site-2 proteases (S2P) act specifically and sequentially to release the active forms of SREBP which actively translocate into the nucleus and binds to the sterol response element (SRE) of the target genes. In this study, we investigated the mechanism of increased lipid biosynthesis in cells infected with HCV. Our studies show that HCVinduced NLRP3-inflammasome activates SREBPs and stimulates lipogenic gene expression and formation of LDs. Our results demonstrate that the proteolytic activation of SREBPs in HCV-infected cells is mediated by the interaction of NLRP3inflammasome with SCAP in the ER. We also demonstrate that caspase-1 activity is critical for SREBP activation. Collectively, these observations provide insight into the novel role of NLRP3-inflammasome in lipid homeostasis during chronic HCV infection.

in addition to maturation of IL-1β and IL-18 (19). Activation of caspase-1 leads to pyroptosis of the cells infected with intracellular bacteria (20). In contrast, the ability of caspase-1 in preventing hepatocyte death during redox stress by upregulating beclin 1 expression signifies its protective function in non-immune cells (11). Caspase-1 has also been shown to regulate the expression of NF-κB target genes through caspase-7 mediated cleavage of PARP1 (21). In addition, recent studies implicate the role of caspase-1 in cell survival by facilitating membrane biogenesis and cellular repair via regulation of lipid metabolism (22). A unique feature of HCV is its absolute reliance on host lipids in the various stages of the viral life cycle (23). To favor its proliferation, HCV alters cellular lipid metabolism by stimulating lipogenesis, impairing mitochondrial β-oxidation and cellular lipid export, and promoting a lipid-rich intracellular environment (23, 24). This alteration of lipid homeostasis results in the intracellular accumulation of cellular lipid storage organelles termed ‘lipid droplets’ (LDs) that play crucial roles in HCV life cycle, hepatic steatosis, and HCC (24-26). Sterol regulatory element binding proteins (SREBPs) are the master regulators of lipid homeostasis which activate the transcription of genes encoding enzymes involved in the biosynthesis of cholesterol, triglycerides, phospholipids and fatty acids (27). Previously, we have shown the activation of SREBPs in HCVinfected human hepatoma cells (28). However, the underlying mechanism by which HCV activates SREBP is not clearly understood. To be active, SREBPs must be cleaved to produce the active/mature forms. There are three SREBP isoforms, designated SREBP-1a, SREBP-1c and SREBP-2 (27). SREBP-1a activates all SREBP target genes whereas SREBP-2 and SREBP-1c activate genes involved in cholesterol and fatty acid synthesis, respectively (27). SREBPs are synthesized as endoplasmic reticulum (ER)membrane-bound precursors and exist in complex with SREBP cleavage-activating protein (SCAP) (27). SCAP is both an escort for SREBPs and a sensor of sterol. The retention of the SCAPSREBP complex in the ER is mediated by the binding of SCAP to Insig (insulin induced gene)


Immunoprecipitation and western blot analysis–Cellular lysates from mock and HCVinfected cells were prepared by incubating in radioimmune precipitation (RIPA) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM sodium formate, 10 µl/ml protease inhibitor cocktail (Thermo Scientific) for 30 min on ice. Equal concentrations of cellular lysates were immunoprecipitated with indicated antibodies overnight at 4°C. The immune complexes were incubated with protein ASepharose (Invitrogen) for 1 h at 4 °C, washed 3-4 times with RIPA buffer, and boiled for 5 min in SDS-containing sample buffer. The samples were then subjected to SDS-PAGE. Gels were electroblotted onto a nitrocellulose membrane (Thermo Scientific) in 25 mM Tris, 192 mM glycine and 20% methanol. Membranes were incubated overnight in blocking buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5% nonfat dry milk], and probed with primary antibody of interest for 1 h at RT. The membranes were then washed three times for 10 min in TBS-T (trisbuffered saline with 1% tween-20) followed by incubation with secondary antibody for 45 min at RT. After an additional washing cycle with TBST, immunoblots were visualized by using the LICOR odyssey system. Laser-scanning confocal microscopy– Mock and HCV-infected cells on coverslips were washed with PBS, fixed with 4% paraformaldehyde for 10 min at RT, permeabilized for 5 min with 0.2% Triton X-100, and blocked for 45 min with 5% bovine serum albumin in PBS. The cells were next incubated with primary antibody against the specific protein for 1 h at RT or overnight at 4○C, followed by incubation with Alexa fluor-labelled secondary antibodies (Invitrogen) for 1 h. After washing with PBS, cells were mounted with anti-fade reagent containing DAPI (4, 6-diamidino-2 phenylindole) (Invitrogen) and observed under a laser scanning confocal microscope (FLUOVIEW, FV10i). Immunohistochemistry (IHC)–Liver biopsies from normal and HCV-associated cirrhosis and HCC (no history of HBV, HIV infection, and fatty liver) were obtained from Liver Tissue Cell Distribution System (LTCDS), University of Minnesota, Minneapolis,

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NS5A was a kind gift of Dr. Craig Cameron (Pennsylvania State University, PA). The organelle localization immunofluorescence (IF) antibody sampler kit was from Cell Signaling Technology (Danvers, MA). Cell culture–The human hepatoma cell line, Huh-7.5 was obtained from Dr. C. Rice (Rockefeller University, NY) (31). Huh-7.5 cells were cultured at 37oC in a humidified atmonsphere containing 5% CO2 with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 U of penicillin/ml, and 100 μg of streptomycin sulfate/ml. HCV cell culture infection system–Fifteen micrograms of in vitro transcribed J6/JFH-1 RNA was delivered into Huh-7.5 cells by electroporation as described previously (3, 28, 32). Cells were passaged every 3-5 days; the presence of HCV in these cells and the corresponding supernatants were determined as described previously (33). The cell-free virus was propagated in Huh7.5 cell culture as described previously (32, 33). The expression of HCV protein in HCV-infected cells was analyzed by western blotting. The HCV cell culture supernatant was collected at appropriate time points and was used to infect naïve Huh7.5 cells at moi of 1 for 5-6 h at 37°C and 5% CO2 (32, 33). The viral titer in cell culture supernatant was expressed as focus forming unit (ffu) per ml, which was determined by the average number of HCV-NS5A-positive foci detected at the highest dilutions as described previously (33). The cell culture supernatant collected from Huh7.5 cells expressing JFH-1/GND (replication defective virus) was used as a negative control. Preparation of nuclear extracts‒ Nuclear lysates were prepared from mock and HCVinfected cells. Cells were lysed in hypotonic buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM Na3VO4, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 3 mg/ml aprotinin, 1 mg/ml pepstatin, 20 mM NaF and 1 mM DTT with 0.2 % NP-40) on ice for 15 min. After centrifugation at 4○C (13,000 rpm) for 1 min, the nuclear pellet was resuspended in high salt buffer (hypotonic buffer with 20% glycerol and 420 mM NaCl) at 4○C by rocking for 30 min following centrifugation. The supernatant was collected and stored at –80 ○C in aliquots.


media along with 20µl of the HiPerfect transfection reagent, the solution was allowed to incubate at RT for 10 min. The transfection solution was then added to the cells and the cells were harvested at different time points. Quantitative RT-PCR–Total cellular RNA was extracted from mock and HCV-infected cells using TRIzol (Invitrogen) and treated with RQ1 RNase-free DNase prior to cDNA synthesis. The cDNA was reverse-transcribed from 1μg of total RNA using reverse transcription kit (Life Technologies). Quantitative RT-PCR was carried out using SYBR green master mix (Life Technologies) and specific primers as described previously (3, 28, 31). Amplification reactions were performed under the following conditions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles for 10 s at 95 °C, and 1 min at 60 °C. Relative transcript levels were calculated using ∆∆Ct method as specified by the manufacturer. Cell viability assay–Mock (Huh7.5), HCV-infected cells, and HCV-infected cells transfected with various siRNA or treated with caspase-1 and caspase-3 inhibitors were placed in 96 wells plate. The cells were lysed and ATP was quantitated as per manufacturer’s instruction using CellTitre-Glo Luminescent Cell viability Assay Kit (Promega). The percent viability was calculated considering 100% viability for mock cells. The values represent the means + SD of three independent experiments performed in duplicate. Statistical analysis–Error bars show the standard deviations of the means of data from three individual experiments. Two-tailed unpaired t-tests were used to compare experimental conditions to those of the respective controls. In all tests, p