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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Int J Cancer. Author manuscript; available in PMC Jun 25, 2009.
Published in final edited form as:
PMCID: PMC2701904
NIHMSID: NIHMS99129

Hepatitis C Virus Targets Over-Expression of Arginase I in Hepatocarcinogenesis

Abstract

Hepatitis C virus (HCV) infection is often associated with chronic liver disease, which is a major risk factor for the development of hepatocellular carcinoma (HCC). To study the HCV-host cell relationship on the molecular level, HepG2 and Huh7 cells were stably transfected with an infectious cDNA clone of HCV or with empty vector. Evidence for HCV replication was obtained in both culture systems. HCV also stimulated growth in vitro. To identify genes whose altered expression by HCV are important to the pathogenesis of infection, RNAs were isolated from HepG2-HCV and HepG2-vector cells, and subjected to microarray analysis. The results showed that arginase 1 mRNA and protein were elevated about 3-fold in HCV positive compared to negative cells (P < 0.01). Arginase 1 expression was elevated in more than 75% of HCV infected liver samples compared to paired HCC from the same patients (> 33% positive) and to uninfected liver tissues (0% positive). Arginase 1 specific siRNA inhibited the ability of HCV to stimulate hepatocellular growth in culture by > 70%, suggesting that the metabolism of arginine to ornithine may contribute to HCV mediated stimulation of hepatocellular growth. Introduction of arginase specific siRNA also resulted in increased nitric oxide synthase (iNOS) (>1.2 fold), nitric oxide (NO) production (> 3 fold) and increased cell death (>2.5-fold) in HCV positive compared to negative cells, suggesting that these molecules potentially contribute to hepatocellular damage. Hence, an important part of the mechanism whereby HCV regulates hepatocellular growth and survival may be through altering arginine metabolism.

Keywords: arginine, arginase 1, hepatocellular carcinoma, hepatitis C virus, nitric oxide

Introduction

Chronic hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma (HCV) (1-3), although the nature of this relationship at the cellular and molecular levels is not clear. HCV core, NS3 and NS5A have been shown to potentially contribute to hepatocarcinogenesis (2,4), but most of these experiments have used cell culture systems over-expressing each of these proteins. Interestingly, HCV core over-expressing transgenic mice develop steatosis and eventually hepatocellular carcinoma (HCC) (5,6), which is similar to what is observed in chronic human infections (7). However, during the pathogenesis of natural infection, both liver and HCC nodules replicate HCV at levels estimated to be 5-50 copies of HCV RNA molecules per cell (8). Therefore, if HCV contributes importantly to HCC, it will be important to do so in the context of liver cells replicating these levels of virus.

Several years ago, this laboratory developed a system in which an infectious clone of HCV was shown to replicate at physiological levels in HepG2 and Huh7 cells following stable transfection of virus cDNA (9). Importantly, HCV was shown to stimulate the growth of these cells in vitro, in soft agar, and accelerated the development of tumors in nude mice (9). These observations suggested that intact HCV promoted tumorigenesis, and that these cell culture systems could be used to learn more about the mechanisms of HCV associated hepatocarcinogenesis.

Arginase 1 is an enzyme highly expressed in the liver that converts L-arginine to L-ornithine plus urea (10). L-ornithine is the precursor for polyamines, which promote cell growth (11). Interestingly, arginase 1 expression is elevated in the serum of patients with colorectal (12,13), pancreatic (14), prostatic (15), and breast cancers (16,17), as well as in basal cell and squamous cancers of the skin (18). Elevated arginase expression has also been observed in the cytoplasm of dysplastic cells and gastric cancer cells from clinical samples as well as in gastric cancer cell lines (19), suggesting that it could be made by both preneoplastic and tumor cells. Likewise, elevated arginase has been found in intestinal tissues obtained from patients with inflammatory bowel disease and adenomas, as well as colorectal carcinomas (20), suggesting that arginase may play an important role in tumor pathogenesis. Elevated serum arginase has also been detected among patients with cirrhosis and HCC (21). Independent observations have shown that increased serum arginase levels were associated with chronic liver disease (CLD) (22). This was consistent with microarray analysis, showing the arginase was most highly up-regulated in nontumor liver compared to tumor among HCV infected HCC bearing patients (23,24). Given a possible role for elevated arginase expression in HCV associated HCC, experiments were designed to test the hypothesis that arginase was up-regulated by HCV, and if so, that it contributed importantly to the mechanism whereby HCV stimulated hepatocellular growth and tumorigenesis.

Materials and Methods

Recombinant plasmids and cell transfection

The clone used for this was a full-length, HCV cDNA of genotype 1b that was shown to replicate in cell culture (25) and was infectious in chimpanzees (26). This cDNA was previously sequenced, cloned into the mammalian expression vector pRc/CMV2, and then stably transfected into the human hepatoblastoma cell line, HepG2 and the human hepatoma cell line, Huh-7, as described (9). Parallel transfections were performed with the empty expression vector, pRc/CMV2. The resulting cultures were propagated as described (27), without the selection of individual G418 resistant colonies, for further analysis. pRc/CMV-HCV included the hepatitis delta virus ribozyme that cleaved the nascent HCV RNA to yield the authentic 3′ end of the virus genome. In addition, although the HCV cDNA was cloned downstream of the CMV promoter in the pRc/CMV plasmid, the 5′ end of the HCV RNA mapped to the authentic 5′ end of HCV (9), showing that the virus RNA made from the integrated template was wild type. This system produced both negative and positive HCV RNA species in ratios and intracellular levels characteristic of infected liver, was positive for HCV core and virus encoded RNA dependent RNA polymerase (the NS5B protein), and secreted HCV RNA (but not expression plasmid sequences) that banded in gradients at the density expected for HCV particles obtained from the blood of infected patients (9).

Growth curves

Growth curves for each of the cell cultures under different experimental conditions were determined as described (9). Briefly, for each experiment, 2 × 105 cells were seeded into triplicate cultures overnight in cell culture medium containing 10% fetal bovine serum (10% FBS) or in serum free medium (SFM). Cells were then cultured in 10% FBS or in SFM for 4-5 days, depending upon the experimental design. Viability was estimated daily by the modified tetrazolium salt (MTT) assay (Cell Titer 96 Non-radioactive Cell Proliferation Assay, Promega, Madison, WI).

Microarray

mRNA was extracted from HepG2-HCV and HepG2-vector cells using the Oligotex mRNA Mini Kit (Qiagen, Valencia, CA) according the enclosed instructions. mRNA from each extraction was labeled with Cy3 or Cy5 and then hybridized to microarray slides obtained from the Delaware Biotechnology Institute (DBI) using the DBI Microarray Kit according to enclosed instructions. The microarray was performed in duplicate, and the data obtained analyzed by GeneSpring software (version 4.2).

Western blotting

For the preparation of whole cell lysates, cells were rinsed with 1 x PBS twice and then lysed with RIPA buffer (150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L/Tris-HCl, pH 7.5) for 15~30 mins at 4 °C and centrifuged at 10,000 x g at 4°C for 10 minutes. Samples were quantified and an average of 50-100μg of total protein was analyzed by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis. Following electrophoretic transfer of the separated proteins to Immobilon P membranes, western blotting was performed with anti-arginase 1, anti-iNOS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or anti-β-actin (Sigma-Aldrich Co., St. Louis, MO). All antibodies were used at a dilution of 1:200. Binding was visualized by enhanced chemiluminescence (ECL) using a protocol supplied by the manufacturer (Pierce Biotechnology, Inc., Rockford, IL).

Clinical specimens

Tumor and surrounding nontumor liver tissue specimens were collected at the time of surgical biopsy or liver transplantation from 22 patients with HCV associated HCC treated at the University of Pittsburg Medical Center. Another 9 cases of HCV associated HCC were collected from the Rabin Medical Center, Petach-Tikva, Israel. These tissues were snap frozen within 30-40 minutes after their removal from patients in the surgical suite. Five normal liver tissues from as many uninfected individuals who died of unrelated causes were also collected in the U.S. as controls. In the latter group, liver tissues were removed within 30-40 minutes of death and immediately snap frozen. HCC was histologically confirmed in all the test cases. Among the tumor bearing patients, 19 also had histological evidence of cirrhosis while 16 had steatosis. All test patients were positive for anti-HCV while all controls were from patients who were anti-HCV negative. All liver tissue samples were paraffin embedded and used retrospectively for these studies. Permission to use these as de-identified patient samples was approved by the Human Subjects Committee or Institutional Review Board of all the sites participating in this work.

Immunohistochemical staining

Paraffin-embedded human liver sections (5 μm thick) were heated at 60°C, deparaffinized, and then rehydrated. Endogenous peroxidase activity was blocked by 3% H2O2. An avidin/biotin blocking kit (Vector laboratories, Burlingame, CA) was then used to prevent nonspecific binding. Staining was performed using the Vectastain ABC kit (Vector laboratories) according to instructions provided by the manufacturer. The primary antibody, rabbit anti-arginase 1 (Santa Cruz Biotechnology), was incubated on slides for 1 hour at room temperature. Slides were then incubated with the secondary antibody, biotinylated goat anti-rabbit IgG, followed by horseradish peroxidase-conjugated avidin. Product was revealed using the DAB peroxidase substrate kit (Vector laboratories). As a control, primary antibody was replaced with isotype-matched control IgG (Santa Cruz Biotechnology) in the reaction mixture. Sections were counterstained with hematoxylin and then dehydrated, sealed, and analyzed.

RNA interference

To examine the effects of arginase 1 over-expression upon hepatocellular growth, cells were transfected with arginase 1 specific or control siRNAs. Briefly, cells were seeded in 96-well (2×104/well) or 6-well (2×105/well) plates and cultured in minimal essential medium (MEM) containing 10% FBS until they became 60-80% confluent. Cultures using 6-well plates were then transiently transfected with 60 pmols total per well of two pooled arginase 1 specific siRNAs (Arg 1 and Arg 2, Dharmacon, Lafayette, CO), two HCV specific siRNAs (GGUCAUCGAUACCCUCAdTdT and CGGGGUAGGUCGCGUAAUUdTdT) or two unrelated control siRNAs. In experiments using 96-well plates, 3 pmol of each siRNA was used for each well. Transient transfections were performed using the siRNA transfection protocol supplied by the manufacturer (Dharmacon). Briefly, transfections were carried out for 6 hours at 37°C in a CO2 incubator. Medium containing 2 times the concentration of serum and antibiotics was added to the transfection medium without removal of the original transfection reagent, and the cultures incubated for 18-24 hours. The medium was aspirated and replaced with growth medium containing 10% FBS or with SFM. Cells were then analyzed for growth and for both arginase 1 and iNOS expression levels after 24, 48 and 72 hours. The number of viable cells was determined by the modified MTT assay (Cell Titer 96 Non-radioactive, Promega).

Arginase assay

The arginase assay kit was purchased from Bioassay Systems (Hayward, CA). Lysates were prepared from 1 × 106 cells according to enclosed instructions and then assayed for arginase activity in a 96-well protocol as outlined by the manufacturer.

Nitric oxide (NO) detection

Huh7-HCV cells were transfected with arginase 1 or control siRNAs and cultured in SFM as described above. NO concentration in cell culture medium was detected using a Nitric Oxide Colorimetric Assay Kit (Biomol, Plymouth Meeting, PA). NO was analyzed at 24, 36 and 48 hours.

Flow cytometry

Cell cycle analysis was conducted exactly as described (28).

Statistics

Cell growth, real-time RT-PCR detection of HCV RNA, and NO detection were evaluated using the Student t test. A P value < 0.05 was considered significant.

Results

Relationship between arginase 1 expression, HCV stimulated hepatocellular growth, and HCV replication/expression

Earlier results showed that HCV promoted the growth of HepG2 cells in culture, in soft agar, and accelerated subcutaneous tumor formation in nude mice (9). When the human hepatoma cell line, Huh7, was stably transfected with the same full-length HCV cDNA (or vector), HCV was also shown to stimulate growth of Huh7 cells in culture (Fig. 1A). HCV also promoted anchorage independent growth of Huh7 cells in soft agar and accelerated tumor formation in nude mice (unpublished data). These observations confirmed the results obtained in HCV positive compared to negative HepG2 cells (9). Hence, HCV stimulated the growth of hepatoblastoma and hepatocellular carcinoma cells.

Figure 1
A. Growth curves for Huh-HCV ([diamond]) and Huh7-vector (□) cells grown in medium containing 10% FCS. Results are the average of three independent experiments. B. Western blot results of endogenous arginase 1 in Huh-HCV cells (lane 1) and in ...

To discern the mechanism(s) whereby HCV stimulated growth, both pairs of cultures grown to late log phase were subjected to microarray analysis. Arginase 1 was found to be up-regulated 3.9 ± 0.4 fold in HepG2-HCV compared to HepG2-vector cells (P < 0.01) and 4.2 ± 0.5 fold in Huh7-HCV positive compared to negative cells (P < 0.01). When experiments were conducted to provide technical validation of this finding by northern blotting, arginase 1 was also shown to be up-regulated in HCV positive compared to negative cells. In HepG2-HCV compared to HepG2-vector cells, for example, arginase 1 mRNA expression was elevated some 3.2 ± 0.4 fold (P < 0.01), while in Huh7-HCV compared to Huh7-vector cells, arginase 1 expression was increased 3.7 ± 0.3 fold (P < 0.01) (unpublished data). When additional technical validation was conducted by western blotting of lysates, arginase 1 was shown to be elevated 3.4 ± 0.4 fold in Huh7-HCV cells compared to vector transfected cells (P < 0.01) (Fig. 1B). HCV also stimulated the growth of HepG2 cells in culture (Fig. 1C) and this was similarly associated with elevated levels of arginase 1 (Fig. 1D). These results suggest that arginase 1 expression is elevated at the RNA and protein levels in the presence of HCV.

Experiments were then designed to ask whether inhibition of HCV replication/expression also blocked the ability of HCV to stimulate arginase 1 expression. Accordingly, HepG2-HCV cells were transiently transfected with pooled HCV specific or control siRNAs. Real-time RT/PCR quantification showed a 78 ± 6% decrease in intracellular levels of HCV RNA by day 2 post-transfection, and a 92 ± 7% decrease by day 3 (Fig. 2A), confirming that the siRNAs against HCV are effective in suppressing virus replication/expression. Western blot analysis showed a 2.4 ± 0.3 fold increase in steady state levels of arginase 1 expression in HCV positive (Fig. 2B, lane 1) compared to negative cells (Fig. 2B, lane 2). When arginase specific siRNAs were pooled and transfected into HepG2-HCV cells, there was little decrease in the intracellular levels of HCV RNA compared to cells transfected with control siRNAs (Fig. 2A), even though the levels of arginase 1 protein was suppressed 78 ± 8.2% (P < 0.005) (Fig. 2B, lane 3). Inhibition of HCV replication/expression by addition of pooled HCV specific siRNAs reduced the levels of endogenous arginase 1 (Fig. 2B, lane 4), confirming the direct relationship between HCV replication/expression with the cellular levels of arginase 1. To determine whether the relative levels of arginase protein in Fig. 2B reflected the endogenous levels of arginase activity, cell lysates prepared in Fig. 2B were also evaluated for this activity. The results showed that relative levels of arginase activity and corresponding protein were similar (Fig. 2B), indicating that arginase activity and protein levels correlated in these cells. Similar results were obtained using Huh7-HCV cells (unpublished data). These observations suggest that HCV replication/expression is associated with the up-regulated expression and activity of arginase 1, but that elevated arginase 1 does not appear to support HCV replication/expression.

Figure 2
Relationship between HCV replication and arginase 1 expression. A. HepG2-HCV cells were transiently transfected with pooled control siRNAs (ctrl), HCV specific siRNAs (HCV), or arginase 1 specific siRNAs (arg). The intracellular levels of HCV RNA were ...

Expression of arginase 1 in vivo

Earlier studies showed that the expression of arginase 1 was up-regulated in the serum and in HCV infected liver from patients with HCC (21,22). Arginase 1 expression was also shown to be elevated in liver compared to tumor from HCV infected patients by proteomic analysis (23,24). However, the actual cells over-producing arginase 1 were not identified. Accordingly, when immunohistochemical staining was carried out for arginase 1, nontumor liver samples from 21 of 22 U.S. patients with HCC (95%) were moderately to strongly positive. The same was true in 7 of 9 samples from Israel (78%) (Table 1). Corresponding tumor samples from these same patients had weaker staining in fewer cells (Table 1). Among US patients, 14 of 22 had detectable arginase 1 staining in the tumor cells (65%), while among Israeli patients, only 3 of 9 were positive (33%) (Table 1). Having said this, there was only 1 US patient and 1 Israeli patient who had undetectable arginase 1 staining in both tumor and nontumor. Likewise, arginase 1 was below the limits of detection in all 5 uninfected liver samples, suggesting that up-regulated expression of arginase 1 was associated with HCV infected liver, and to a lesser extent in tumor, but not in uninfected liver. An example of arginase 1 expression patterns in liver and tumor is presented in Fig. 3A. Note the strong cytoplasmic staining in many hepatocytes (lower portion of the panel) compared to the HCC cells (upper portion of the panel). Use of preimmune rabbit serum in place of the primary antibody at the same dilution resulted in no discernable staining, thereby demonstrating specificity of the primary antibody (Fig. 3B). Higher power arginase 1 staining in a section from another patient showed modest but diffuse staining in the nontumor compartment compared to nearly undetectable staining within the adjacent tumor nodule (Fig. 3C). Lastly, arginase 1 staining of uninfected liver resulted in little or no signal (Fig. 3C). These results suggest that elevated arginase 1 staining is associated with chronic HCV infection in vivo. The finding that HCV is also associated with elevated arginase 1 expression in cell culture (Figs. (Figs.11 and and2)2) suggests that up-regulated expression of arginase 1 may be an early step whereby HCV contributes to tumor development, but that its up-regulated expression is no longer rate limiting during tumor progression.

Figure 3
Immunohistochemical staining for arginase 1 in HCV tumor/nontumor liver sections. (A) Tumor (top of panel) and nontumor liver (bottom of panel) stained with anti-arginase 1 (x100). (B) Consecutive section from the block in (A) stained with the same dilution ...
Table 1
Immunohistochemical staining for arginase 1 in HCV infected HCC patients

Impact of arginase 1 siRNAs upon the growth of HCV positive and negative cells

The findings that elevated arginase expression is characteristic of many tumor types (12-17,19-21), that arginase 1 expression is elevated in HCV positive cells (Fig. 1), and that HCV promotes hepatocellular growth and accelerates tumorigenesis (9), suggest that elevated arginase 1 expression may contribute to HCV mediated hepatocarcinogenesis. To test this hypothesis, HCV positive and negative Huh7 and HepG2 cells were transiently transfected with pooled arginase 1 specific or pooled control siRNAs, and cell growth determined by the MTT assay. In initial experiments, introduction of siRNAs into Huh7-HCV cells transiently reduced the protein levels of arginase 1 by up to 73% by 2-3 days post-transfection, while control siRNAs inhibited arginase 1 expression by only 9% (Fig. 4A). When the experiment was repeated, the growth of cells transiently transfected with arginase specific siRNAs was significantly inhibited compared to the same cells transfected with control siRNAs. Specifically, when Huh7-HCV cells were transfected with pooled arginase 1 specific siRNAs, growth was suppressed an average of 2.6 ± 0.2 fold at 72 hrs post-transfection (Fig. 4B) compared to Huh7-vector cells, which were suppressed 1.1 ± 0.2 fold at the same time point (P < 0.02) (Fig. 4C). At 96 hrs post-transfection, the growth of Huh7-HCV cells transfected with arginase 1 specific siRNAs was suppressed an average of 5.6 ± 0.5 fold (Fig. 4B) compared to Huh7-vector cells, which were suppressed an average of 2.0 ± 0.3 fold (P < 0.005) (Fig. 4C). Similar results were obtained with HepG2-HCV cells, where arginase 1 specific siRNAs suppressed growth at 72 hrs. by an average of 2.96 ± 0.35 fold (Fig. 4D) compared to 1.2 ± 0.2 fold for identically treated HepG2-vector cells (P < 0.01) (Fig. 4E). At 72 hrs, arginase 1 specific siRNAs suppressed the growth of HepG2-HCV cells by 4.3 ± 0.3 fold (Fig. 4D) while the same siRNAs suppressed HepG2-vector cells by 2.2 ± 0.28 fold (P < 0.01) (Fig. 4E). These results show that the growth of HCV positive cells are much more sensitive to inhibition by down-regulation of arginase 1 expression that HCV negative cells.

Figure 4
(A) Levels of arginase 1 and iNOS, as determined by western blotting, at the indicated times after transfection of pooled arginase specific siRNAs, pooled irrelevant siRNAs, or after mock transfection of Huh7-HCV cells. All values were normalized to β-actin, ...

Levels of iNOS and NO in arginase over-expressing cells

Given that arginine is a substrate for both arginase 1 and inducible nitric oxide synthase (iNOS), elevated arginase 1 levels may deplete the substrate for iNOS (10). In addition, the fact that iNOS converts arginine to nitric oxide (NO), and that NO may impact upon tumorigenesis (29), suggests that it will be important to determine iNOS and NO levels in the presence of constitutively elevated arginase 1. When pooled arginase 1 specific siRNAs were introduced into HCV positive Huh7 cells, arginase 1 levels were depressed and iNOS levels became elevated ~2.5-5 fold compared to cells transfected with control siRNA over the 72 hrs of evaluation (Fig. 4A). Parallel experiments showed that the intracellular levels of NO also increased over the 72 hr period post-transfection, reaching an average of 3.3 ± 0.3 fold higher in Huh7-HCV cells at 72 hrs, compared to a 2.2 ± 0.25 fold increase by the same point in Huh7-vector cells (P < 0.025) (Fig. 5). Similar results were observed with HepG2-HCV compared to HepG2-vector cells (unpublished data, but analogous to the data shown in Fig. 5). Hence, suppression of arginase 1 with specific siRNAs probably resulted in an accumulation of arginine, with subsequent induction of iNOS, and conversion of arginine to NO and citrulline. In the presence of HCV, however, the elevated levels of arginase 1 appear to compete for the common substrate, arginine, resulting in depressed levels of iNOS and NO.

Figure 5
Nitric oxide concentration in the tissue culture supernatant from (A) Huh7-HCV cells transiently transfected with pooled arginase 1 specific siRNAs ([diamond]) or with pooled control siRNAs (□). (B) Nitric oxide concentrations were also evaluated ...

Discussion

Elevated arginase 1 expression is associated with many tumor types (12-17,19,20), including HCC (21). This study is the first to show that arginase 1 levels are elevated in HCV positive compared to HCV negative cells, and that this is related to HCV associated stimulation of hepatocellular growth (Fig. 1). These observations suggest that either HCV stimulates arginase 1 expression and/or that arginase 1 up-regulation is a cellular response to HCV infection. Either way, the fact that arginase 1 metabolizes arginine to ornithine, and that ornithine is further metabolized to polyamines, which promote cellular proliferation, may partially explain the correlation between HCV, elevated arginase 1, and cell growth (Fig. 1). This is strengthened by the finding that arginase 1 siRNAs partially block HCV stimulated hepatocellular growth, and that this directly correlates with the levels of endogenous arginase 1 (Fig. 4). Preliminary observations suggest that the blockage in hepatocellular growth is not associated with the activation of caspase 3, nor with the accumulation of lactate dehydrogenase or alanine aminotransferase in the cell culture supernatants (data not shown), suggesting that the blockage in cell growth does not trigger cell death. Hence, up-regulated arginase 1 may contribute importantly to the mechanism whereby HCV promotes cell growth.

The observed distribution of elevated arginase 1 in vivo, predominantly within peritumor hepatocytes, and to a lesser extent in HCC cells (Fig. 3), is in agreement with earlier proteomic analysis using samples from HCV infected HCC and nontumor liver, which also show up-regulated arginase 1 in liver and relatively little arginase 1 expression in tumor (23,24). These combined results are consistent with the hypothesis that elevated arginase 1 promotes cell growth in preneoplastic liver. The finding that there is generally higher levels of HCV replication and expression in hepatocytes compared to HCC cells (30) is also consistent with the results of the siRNA experiments herein, which show a close correlation between HCV stimulated hepatocellular growth and elevated arginase 1 expression (Figs. (Figs.11 and and4).4). Arginase 1 expression may also be up-regulated during chronic liver disease by the appearance of type 2 cytokines, which include interleukin-4 (IL-4) and interleukin-10 (IL-10) (31). Interestingly, type 2 cytokines also tend to promote chronicity, since they are associated with the development of anti-HCV but not with the appearance and persistence of strong cytotoxic T cell responses. It may also be relevant that interferon-γ suppresses arginase 1 expression (31), the latter of which also correlates with the suppression of HCV stimulated cell growth in culture (Fig. 4). Hence, elevated arginase 1 expression may be hepatoprotective during chronic infection by promoting the growth and survival of HCV infected hepatocytes in the presence of inflammatory immune responses that contribute to liver damage but not to the elimination of virus infected cells. However, the finding that HCV stimulates hepatocellular growth in HepG2 and Huh7 cells, both of which are already transformed, suggests that HCV may also promote cell growth during later stages of tumorigenesis.

An important consequence of arginase 1 over-expression in the chronically infected liver (Fig. 3) is that most of the arginine available is likely to be metabolized to polyamines, and that relatively little is available to induce the expression of iNOS. In addition, there is evidence that arginase 1 and iNOS mutually inhibit each other (32), so that under conditions where liver cells are HCV infected, and arginase 1 is over-expressed (Figs. (Figs.11 and and3),3), it is likely that the levels of iNOS and NO would also be depressed. Empirical evidence with HCV in liver cells demonstrates an inverse relationship between arginase 1 and iNOS (Fig. 4A). NO production directly correlates with the levels of iNOS and inversely with the levels of arginase 1 (Fig. 5), suggesting that HCV indirectly suppresses NO production in infected cells. The consequences of altered NO expression may promote hepatocellular growth and tumorigenesis or promote hepatocellular death and tumor regression (29). In light of the empirical data presented in this work (Figs. (Figs.44 and and5),5), it is proposed that in HCV negative cells, a significant amount of arginine is converted to citrulline plus NO, and that under these circumstances, NO would deliver a negative growth regulatory signal. In the context of chronic liver disease, which is characterized partially by severe redox stress, elevated NO may promote cell cycle arrest or cell death (33), especially among uninfected hepatocytes. This may follow from the binding of NO to cytochrome c oxidase in mitochrondria, thereby competing out the binding of the latter to oxygen, resulting in the disruption of the respiratory chain (34). In contrast, among HCV infected cells, most of the available arginine becomes converted to ornithine and polyamines instead, which promote tumorigenesis, and that depressed iNOS and NO production may result in the amelioration of negative growth regulation. At least in vitro, these changes appear to have little impact upon HCV replication/expression (Fig. 2), although it is not clear whether this also occurs in vivo. Hence, the HCV associated up-regulation of arginase 1 may have important implications in the pathogenesis of chronic infection and the development of HCC.

Acknowledgements

This work was supported by Ruijin Hospital and by NIH grants CA104025 and CA111427 to MAF. There are no potential conflicts of interest to report.

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