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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Hepatitis C virus (HCV) perturbs the host's lipid metabolism and often results in hepatic steatosis. In nonalcoholic fatty liver disease, the intrahepatic down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a critical mechanism leading to steatosis and its progression toward fibrosis and hepatocellular carcinoma. However, whether an HCV infection triggers the formation of large lipid droplets through PTEN-dependent mechanisms is unknown. We assessed PTEN expression in the livers of patients infected with HCV genotype 1 or 3 with or without steatosis. The role of PTEN in the HCV-induced biogenesis of lipid droplets was further investigated in vitro with hepatoma cells transduced with the HCV core protein of genotype 1b or 3a. Our data indicate that PTEN expression was down-regulated at the posttranscriptional level in steatotic patients infected with genotype 3a. Similarly, the in vitro expression of the HCV genotype 3a core protein (but not 1b), typically leading to the appearance of large lipid droplets, down-regulated PTEN expression by a mechanism involving a microRNA-dependent blockade of PTEN messenger RNA translation. PTEN down-regulation promoted in turn a reduction of insulin receptor substrate 1 (IRS1) expression. Interestingly, either PTEN or IRS1 overexpression prevented the development of large lipid droplets, and this indicates that the down-regulation of both PTEN and IRS1 is required to affect the biogenesis of lipid droplets. However, IRS1 knockdown per se did not alter the morphology of lipid droplets, and this suggests that other PTEN-dependent mechanisms are involved in this process. Conclusion: The down-regulation of PTEN and IRS1 is a critical event leading to the HCV genotype 3a–induced formation of large lipid droplets in hepatocytes. (HEPATOLOGY 2011;)

Metabolic syndrome and hepatitis C virus (HCV) infection are major causes of progressive liver disease.1 Interestingly, these two conditions share some clinical and histological features, such as insulin resistance (IR), hepatic steatosis, inflammation, fibrosis, and cirrhosis.2 In addition, hepatocellular carcinoma (HCC) is a potential end-stage complication of both disorders.3

Abnormal signaling through the phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/Akt pathway is involved in the pathogenesis of liver manifestations associated with metabolic syndrome, that is, nonalcoholic fatty liver disease (NAFLD) and HCC.4 The deregulated expression/activity of PTEN, a potent regulator of PI3K signaling in hepatocytes, importantly contributes to the occurrence of NAFLD and HCC.5 Indeed, liver-specific PTEN knockout mice spontaneously develop NAFLD and HCC.6, 7 In agreement with these studies, PTEN expression is down-regulated in steatotic livers of obese human subjects and in fatty livers of rodent models.8 We have further demonstrated that fatty acids cause hepatic steatosis, nonalcoholic steatohepatitis, and aberrant cell proliferation through the down-regulation of PTEN expression.8-10 Finally, PTEN is a well-established tumor suppressor that is frequently mutated/deleted in human cancers, including HCC.11, 12

Thus, it is tempting to speculate that the expression or function of PTEN may also be altered in HCV infections and may contribute to the development of steatosis in patients with chronic hepatitis C. HCV induces steatosis, especially in individuals infected with HCV genotype 3,13 and this phenomenon has been reproduced experimentally.14 The link between HCV and IR is also well established, although the association of IR with specific HCV genotypes is less clear.15 Discrepancies among studies may be partially explained by the poor reproducibility of the assays generally used to measure IR in clinical practice.16 Thus, it is unclear whether HCV genotypes exert a differential impact on glucose metabolism and, therefore, whether some correlations exist with HCV-induced steatosis. Understanding the mechanisms of metabolic alterations induced by HCV is important because of the potential impact on the management of patients.

In this study, we provide evidence that PTEN expression is down-regulated in the livers of patients with chronic hepatitis C who are infected with HCV genotype 3 (but not HCV genotype 1). Using an in vitro model, we then demonstrate that the core protein of HCV genotype 3a down-regulates PTEN expression by altering PTEN messenger RNA (mRNA) translation and thereby induces the formation of large lipid droplets. We finally show that in hepatocytes expressing the core 3a protein, the appearance of large lipid droplets induced by PTEN down-regulation is mediated by the reduced expression of insulin receptor substrate 1 (IRS1); we thus suggest a molecular link between HCV-induced steatosis and IR in genotype 3a infections.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Reagents, Antibodies, Plasmids, Primers, and Small Interfering RNAs (siRNAs).

All reagents, antibodies plasmids, primers, and siRNAs used in this study are described in the Supporting Information.

Lentiviral Vectors.

Lentivectors expressing PTEN short hairpin RNAs (shRNAs) or the core proteins of genotypes 1b_109B (HM53611) and 3a_452 (DQ437509) have been described elsewhere.8, 17 The construction of lentivectors expressing PTEN is described in the Supporting Information.

Cell Cultures, Lentiviral Transductions, and Transfections.

Human Huh-7 and HepG2 cells were cultured in Dulbecco's modified Eagle's medium/10% fetal bovine serum with penicillin/streptomycin. Lentiviral transductions were performed as previously described.8, 17 For the overexpression or down-regulation of IRS1, Huh-7 cells were transiently transfected with Mammalian Gateway® expression vector pCMV·SPORT6 encoding human IRS1 or IRS1 siRNAs with Lipofectamine.

Luciferase Assays.

The 3′-untranslated region (3′-UTR) of PTEN cloned downstream of luciferase complementary DNA [i.e., the plasmid encoding the luciferase gene coupled to the 3′-UTR end of the PTEN gene (pLuc-PTEN-3′-UTR)] was purchased from Signosis. Luciferase assays were performed as previously described.10

Immunoblot Analyses.

Cells were lysed in RIPA buffer containing phosphatase and protease inhibitors. Polyubiquitinated proteins were isolated with a ubiquitin enrichment kit from Thermo Scientific. Equal amounts of proteins were resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (5%-20% gradient), blotted to nitrocellulose membranes, and detected with enhanced chemiluminescence. Quantifications were performed with ChemiDoc XRS from Bio-Rad.

Human Samples.

Liver biopsy samples from 21 patients with histologically confirmed chronic hepatitis C (11 with HCV genotype 1 and 10 with HCV genotype 3) and surgically resected liver specimens from healthy living donors were examined. Demographic data, including age, sex, weight, and height, were collected at the time of liver biopsy. HCV RNA was quantified by real-time polymerase chain reaction (RT-PCR) and was expressed as international units per milliliter. HCV genotyping was performed with a second-generation reverse hybridization line probe assay (INNO-LiPA HCV II). Studies were performed in accordance with the ethical standards of the Declaration of Helsinki.

Histology and Immunohistochemistry.

Liver biopsy samples were formalin-fixed, paraffin-embedded, and processed for histological staining. Steatosis, activity, and fibrosis (METAVIR scoring system) were scored by an experienced pathologist.18 Steatosis was graded as follows: (0) <2% (none), (1) 2% to 30% (mild), (2) 31% to 60% (moderate), and (3) >60% (severe). An immunohistochemical analysis of PTEN and IRS1 expression was performed as previously described.8 Staining was scored by two independent observers as follows: (−) negative staining, (+) weakly positive staining, (++) moderately positive staining, and (+++) strongly positive staining.

RT-PCR.

Total RNA was extracted with the RNeasy mini kit. Complementary DNA was synthesized from 100 ng of RNA with SuperScript II reverse transcriptase and random hexanucleotides. RT-PCR and quantifications were performed as described.19

Confocal Microscopy.

Cells were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 before incubation with primary and Alexa-conjugated secondary antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole, and neutral lipids were stained with Oil Red O (ORO) as previously described.17, 19 Images were acquired with a confocal microscope (LSM510 Meta, Zeiss) and were analyzed with Metamorph software (Molecular Devices, Sunnyvale, CA).

Statistical Analysis.

The results were expressed as means and standard deviations (or standard errors) of three independent experiments. The results were analyzed with the Student t test. P < 0.001, P < 0.01, and P < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

PTEN Is Down-Regulated Posttranscriptionally in the Livers of HCV Genotype 3a–Infected Patients.

PTEN expression was investigated in liver specimens from healthy patients (n = 3) and in biopsy samples from patients who were infected with HCV genotype 1 (n = 11) or 3 (n = 10) and had a body mass index < 24 kg/m2 (to prevent the bias of steatosis originating from metabolic defects). Patients infected with HCV genotype 3 (but not genotype 1) showed extensive steatosis (Table 1). Because PTEN expression can undergo complex posttranscriptional regulation,5, 11 we assessed the PTEN protein level by immunohistochemistry. PTEN was homogeneously expressed in the cytoplasm and nuclei of all hepatocytes in uninfected control individuals (Fig. 1A and Table 1). Almost all HCV genotype 1–infected cases were also highly positive for PTEN, with slight variations among individuals (Fig. 1B and Table 1). However, in HCV genotype 3–infected patients, PTEN expression was significantly decreased in areas with fatty infiltration (Fig. 1C and Table 1), whereas in the absence of steatosis (n = 3), PTEN expression was comparable to that of healthy patients and genotype 1–infected patients. In contrast to what was previously observed in the steatotic livers of HCV-negative obese patients,8 the intrahepatic PTEN mRNA levels of HCV genotype 3–infected patients were comparable to those of patients with genotype 1 (Fig. 1D). These data indicate that PTEN expression is reduced in hepatocytes from patients infected with HCV genotype 3 (but not HCV genotype 1) through posttranscriptional mechanisms, and this PTEN reduction correlates with the accumulation of lipid droplets in these cells.

Table 1. Clinical, Histological, and Immunohistochemical Characteristics of HCV-Infected Patients and Uninfected Control Patients
 Age (Years)/SexBody Mass Index (kg/m2)Serum HCV RNA (IU/mL)HCV GenotypeHistology ScoresPTEN ExpressionIRS1 Expression
ActivityFibrosisSteatosis
HCV genotype 1 (n = 11)31/F19332,0001000++++
42/F19.8356,0001110+++++
24/F20.5200,0001110+++
36/F21.3250,0001000++++
44/M22.3ND1130++++
42/M221,100,0001000+++
45/M21823,0001130+++++
41/M24884,0001230+++++
31/F24.5862,0001100++ND
34/M223,200,0001120++++
49/M2310,000,0001110++ND
        Steatotic AreasNonsteatotic Areas 
  • For each liver specimen, the antibody-stained sections were carefully examined by two independent observers, and the staining was scored as follows: (−) negative staining, (+) weakly positive staining. (++) moderately positive staining, and (+++) strongly positive staining. The PTEN staining in uninfected human samples, the PTEN staining in patients infected with HCV genotype 1 (the patients displayed no steatosis), and the PTEN staining in nonsteatotic patients infected with genotype 3 were comparable in intensity. When steatosis was present in HCV genotype 3a–infected patients, the PTEN expression was quantified in both steatotic and nonsteatotic areas.

  • Abbreviations: F, female; M, male; ND, not determined.

  • *

    Nonsteatotic areas were absent.

HCV genotype 3 (n = 10)37/F221,300,0003002+++++
44/F21234,0003122+/−++
28/M22.5200,0003112+++++
36/M22.87,600,0003112+/−+++
54/F21.5ND3233+/−+++
45/M22.9ND3123+/−*+
49/MND784,0003123+++++
49/F231,630,0003130++++
397F21.3111,0003000++++
 30/M21318,0003000+++
Uninfected controls (n = 3)86/MND000++++++
32/FND000++++++
 36/MND000++++++
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Figure 1. Posttranscriptional down-regulation of PTEN in HCV genotype 3a–infected patients. Representative PTEN immunohistochemistry (immunoperoxidase staining) of (A) uninfected patients (n = 3), (B) HCV genotype 1–infected patients (n = 11), and (C) steatotic HCV genotype 3–infected patients (n = 10). (D) PTEN mRNA expression in liver biopsy samples of HCV genotype 1–infected patients and HCV genotype 3–infected patients. Means and standard deviations were derived from eight liver biopsy samples from different patients infected with HCV genotype 1 or 3.

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The Core Protein of HCV Genotype 3a Triggers PTEN Down-Regulation in Hepatocytes Through a 3′-UTR–Dependent Blockade of PTEN mRNA Translation.

The HCV core protein is sufficient to induce the formation of large lipid droplets in hepatocyte cell lines.14 In the absence of HCV, steatosis can be induced by the down-regulation of PTEN.8 Thus, we investigated whether the core protein of HCV genotype 3a could reduce PTEN expression in hepatocytes. Huh-7 and HepG2 cells were transduced with lentivectors encoding the core protein of HCV genotype 1b or 3a. PTEN protein and mRNA expression levels were then measured. The core protein of HCV genotype 3a (but not 1b) induced a 50% decrease in the PTEN protein expression level in Huh-7 and HepG2 cells (Fig. 2A,B and Supporting Information Fig. 1). However, in agreement with data for liver specimens from HCV genotype 3–infected patients (Fig. 1), the core 3a protein did not significantly affect PTEN mRNA levels in these cells (Fig. 2C). We then investigated the potential posttranscriptional mechanisms by which core 3a could induce PTEN down-regulation in hepatocytes. HCV core 3a likely modulates PTEN expression indirectly because no physical interactions of core 3a and PTEN were detected with coimmunoprecipitation experiments (data not shown). PTEN down-regulation in core 3a–expressing cells was also unrelated to increased ubiquitination and proteosomal degradation, modifications of the redox status, and increased phosphorylation of PTEN, which can affect protein stability and expression (Supporting Information Fig. 2).11 In contrast, HCV core 3a induced a 3′-UTR–dependent blockade of PTEN mRNA translation. Indeed, luciferase expression in Huh-7 cells transfected with pLuc-PTEN-3′-UTR decreased by 44% ± 15% in cells expressing HCV core 3a (but not 1b), and this was consistent with the extent of PTEN down-regulation in the same cells (Fig. 2D). These data suggest that the core 3a protein triggers a 3′-UTR–mediated blockade of PTEN mRNA translation because the PTEN mRNA levels in HCV core 3a–expressing cells were comparable to the levels in controls.

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Figure 2. The HCV core 3a protein down-regulates PTEN expression in Huh-7 cells via a 3′-UTR blockade of PTEN mRNA translation. (A) Representative immunoblots of PTEN and β-actin expression and quantification of the PTEN protein levels in Huh-7 cells transduced with the HCV core 1b protein, the HCV core 3a protein, or GFP as a control. (B) Quantification of the PTEN mRNA levels. (C) pLuc-PTEN-3′-UTR luciferase activity in cells expressing or not expressing the HCV core 3a and 1b proteins. The results are the means and standard errors of three independent experiments. **P < 0.01. Abbreviation: GFP, green fluorescent protein; Empty, cells transduced with an empty lentivirus; UT, untransduced cells.

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The HCV Core 3a Protein Causes the Accumulation of Large Lipid Droplets by Down-Regulating PTEN Expression.

To test whether PTEN down-regulation could be sufficient to account for the HCV genotype 3a core protein–induced accumulation of large lipid droplets, we stained neutral lipids within cytoplasmic lipid droplets with ORO in cells transduced with the HCV core 3a protein and overexpressing or not overexpressing PTEN (Fig. 3A). The size of the lipid droplets was then quantified (Fig. 3B). As expected, the expression of the genotype 3a core protein induced the appearance of large lipid droplets, with the viral protein typically localized at their surface (Fig. 3Ae-h). Although PTEN overexpression did not affect the localization of the core 3a protein, the development of large lipid droplets was completely inhibited (Fig. 3Ai-l). Overexpression of PTEN per se did not alter the lipid droplet morphology (Fig. 3Am-p). Finally, in agreement with the working hypothesis that core 3a may alter the biogenesis of lipid droplets by down-regulating PTEN, the cellular depletion of PTEN by shRNAs also triggered the formation of large lipid droplets (Fig. 3Aq-t).

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Figure 3. PTEN-dependent accumulation of large lipid droplets in Huh-7 cells expressing the HCV core 3a protein. (A) Representative single optical confocal immunofluorescence sections of (a,e,i,m,q) ORO, (b,f,j,n,r) GFP, and (c,g,k,o,s) core 3a staining in (a-d) untransduced control cells, (e-h) cells transduced with core 3a, (m-p) cells transduced with PTEN, (i-l) cells transduced with both core 3a and PTEN, and (q-t) cells transduced with shRNAs targeting PTEN. Overlay images are shown in panels d, h, l, p, and t. (B) Quantification of individual lipid droplet sizes in 20 or more cells with Metamorph software. Abbreviation: GFP, green fluorescent protein.

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IRS1 Down-Regulation Induced by HCV Core 3a Is PTEN-Dependent.

In Huh-7 cells, HCV core 3a causes the accumulation of large lipid droplets and the down-regulation of IRS1, a key factor controlling insulin signaling.20 IRS1 down-regulation has also been observed by immunohistochemistry on paraffin-embedded liver sections of patients infected with HCV genotype 3, and this confirms the relevance of our previous in vitro data (Table 1 and Supporting Information Fig. 3). Because PTEN depletion in hepatocytes also decreases IRS1 levels,8 we hypothesized that core 3a–mediated IRS1 down-regulation might be PTEN-dependent. According to immunoblotting (Fig. 4A-C), HCV core3a–induced IRS1 down-regulation was prevented by PTEN overexpression. PTEN overexpression in control cells did not affect IRS1 protein levels, which were instead significantly reduced by PTEN depletion with shRNAs. IRS1 mRNA levels were up-regulated to the same extent in cells expressing core 3a (concomitantly or not concomitantly with PTEN) and in controls (Fig. 4D); this supports the view that HCV core 3a–mediated PTEN down-regulation accelerates IRS1 protein degradation. These data indicate that PTEN down-regulation in cells expressing HCV core 3a decreases IRS1 levels, probably via posttranscriptional mechanisms.

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Figure 4. Low levels of PTEN expression induced by core 3a triggers the posttranscriptional down-regulation of IRS1. (A) Representative immunoblots of IRS1, PTEN, core 3a, and β-actin expression in Huh-7 cells (1) cotransduced with empty pLVTHM and a lentiviral vector expressing GFP (pLVTHM GFP), (2) cotransduced with empty pLVTHM and a lentiviral vector expressing HCV core 3a (pLVTHM 3a), (3) stably overexpressing PTEN and transduced with a lentiviral vector expressing HCV core 3a (PTEN+3a), (4) stably overexpressing PTEN and transduced with a lentiviral vector expressing GFP (PTEN+GFP), or (5) stably expressing shRNAs targeting PTEN (PTEN shRNA). Quantification of (B) PTEN and (C) IRS1 protein levels. (D) Expression of IRS1 mRNA. The results are means and standard errors of three independent experiments. ***P < 0.001, **P < 0.01, and *P < 0.05. Abbreviation: GFP, green fluorescent protein.

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PTEN-Dependent IRS1 Down-Regulation Contributes to the HCV Core 3a–Induced Formation of Large Lipid Droplets.

IRS1 is an important regulator of lipid metabolism in the liver.21 It is thus possible that IRS1 down-regulation in PTEN-deficient hepatocytes contributes to the increased biogenesis of lipid droplets induced by HCV core 3a. To test this hypothesis, we counteracted IRS1 down-regulation in HCV core 3a–expressing Huh-7 cells through the transient transfection of IRS1 (Fig. 5). When IRS1 was coexpressed with HCV core 3a, the accumulation of large lipid droplets (typically occurring in cells expressing the core 3a protein alone; Fig. 5Ae-h) was significantly reduced (Fig. 5Ai-l). Interestingly, IRS1 overexpression or depletion (>85% inhibition by specific siRNAs; Fig. 6) in Huh-7 cells was not sufficient per se to affect the size of lipid droplet (Figs. 5Am-p and 6). This suggests that IRS1 down-regulation and other mechanisms induced by PTEN depletion are required to trigger the formation of large lipid droplets in cells expressing the HCV core 3a protein.

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Figure 5. IRS1 overexpression prevents the accumulation of large lipid droplets in Huh-7 cells expressing the HCV core 3a protein. (A) Representative single optical confocal immunofluorescence sections of (a,e,i,m) ORO, (b,f,j,n) IRS1, and (c,g,k,o) HCV core 3a staining in (a-d) untransduced control cells, (e-h) cells transduced with HCV core 3a, (i-l) cells transduced with HCV core 3a and overexpressing IRS1, and (m-p) cells transfected with IRS1 alone. Overlay images are shown in panels d, h, l, and p. (B) Quantification of individual lipid droplet sizes in 20 or more cells with Metamorph software.

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Figure 6. IRS1 down-regulation alone does not induce the formation of large lipid droplets in Huh-7 cells. (A) Representative immunoblots and (B) quantification of IRS1 protein levels in cells transfected or not transfected with scrambled siRNAs and IRS1-specific siRNAs. The bars represent the standard errors of three independent experiments. (C) IRS1 mRNA expression in cells transfected or not transfected with scrambled siRNAs and IRS1-specific siRNAs. The bars represent the standard deviations of three independent experiments. (D) Representative single optical confocal immunofluorescence sections of ORO staining and (E) quantification of lipid droplet sizes in cells transfected with scrambled siRNAs and IRS1-specific siRNAs with Metamorph software. The lipid droplet size was quantified in 10 different cells for each condition (>3000 lipid droplets). ***P < 0.001, **P < 0.01, and *P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Steatosis is a histological feature frequently occurring in patients with chronic hepatitis C.22 Although it is mostly associated with metabolic syndrome in the case of non-3 HCV genotypes, it is predominantly due to viral factors in HCV genotype 3a infections.13 However, the molecular mechanisms by which genotype 3a perturbs lipid droplet biogenesis and lipid metabolism remain poorly defined. In this study, we have demonstrated a preponderant role for impaired PTEN expression/activity in mediating the accumulation of large lipid droplets in HCV genotype 3a–infected hepatocytes. HCV genotype 3a–infected patients exhibited a posttranscriptional down-regulation of PTEN in the liver that was associated with the presence of steatosis. In hepatoma cells, the core protein of genotype 3a alone was sufficient to decrease PTEN expression through mechanisms involving a microRNA-dependent blockade of PTEN mRNA translation. We have also demonstrated that IRS1 down-regulation is mediated by a reduction of PTEN expression. Down-regulation of both PTEN and IRS1 was required to accumulate large lipid droplets in cells expressing HCV core 3a (Fig. 7). However, in contrast to PTEN, the depletion of IRS1 was not sufficient per se to induce the formation of large lipid droplets. Together, our data have uncovered a sequence of early molecular events in which the core of HCV genotype 3a affects PTEN and IRS1 expressions, thereby triggering steatosis in infected patients.

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Figure 7. Hypothetical model of the HCV genotype 3a–mediated down-regulation of PTEN and the genesis of large cytoplasmic lipid droplets.

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Liver-specific PTEN knockout mice develop massive steatosis,6, 7 and PTEN down-regulation in hepatocytes has also been observed with NAFLD5; based on these observations, it is likely that a decreased PTEN expression represents one of the primum movens signaling defects promoting steatosis.8 PTEN inactivation by posttranslational phosphorylation in HCV genotype 2–infected cells has been reported to activate sterol regulatory element binding proteins (SREBPs),23 which, together with impaired microsomal triglyceride transfer protein (MTP) activity, may contribute to HCV-associated steatosis.24, 25 These studies suggest that alterations of PTEN expression/activity during an HCV infection may stimulate lipogenesis by modulating MTP and/or SREBP1 activity. Alternatively, the formation of large lipid droplets and the induction of lipogenesis could be distinct events. Indeed, we have previously shown that PTEN depletion in HepG2 cells induces the accumulation of large lipid droplets, but this falls short of altering SREBP/fatty acid synthase (FAS) expression and inducing lipogenesis.8 We have also failed to observe alterations of SREBP/FAS expression or triglyceride biosynthesis in Huh-7 cells transduced with HCV core 3a (data not shown). Thus, it remains to be established whether HCV core 3a–mediated PTEN down-regulation both promotes the formation of large cytoplasmic lipid droplets and stimulates lipogenesis. In this respect, the evidence indicates that a nonstructural viral protein 5A (NS5A) also promotes lipid accumulation.26 The synergistic effects of multiple HCV proteins on the biogenesis of lipid droplets and the lipid metabolism in hepatocytes remain to be evaluated.

We have already shown that the genotype 3a core protein induces IRS1 degradation in hepatocytes.20 As previously reported,8, 27 we have found that IRS1 down-regulation is triggered by low levels of PTEN expression and is crucial for core 3a–induced lipid droplet formation. In agreement with a role for IRS1 in hepatic lipid metabolism,21, 28 we have found IRS1 to be down-regulated in the livers of HCV-infected patients. Furthermore, IRS1 overexpression prevented the formation of large lipid droplets in core 3a–expressing cells. Because IRS1 depletion in cultured cells did not lead to the formation of enlarged lipid droplets, it is likely that, in addition to IRS1 down-regulation, other core 3a–dependent and/or PTEN-dependent mechanisms are required. Notably, it is unlikely that Akt2, which is overactivated in liver-specific PTEN knockout mice and promotes lipogenesis,29 is involved in this process because Akt2 activity was not exacerbated by core 3a expression in our model (data not shown). Further studies are necessary to delineate the precise role of IRS1 versus other effects of core 3a in the generation of large lipid droplets.

Mechanisms regulating PTEN expression have been intensively investigated because of the tumor suppressor activity of PTEN. Posttranscriptional modifications such as phosphorylation, ubiquitination, and redox mechanisms have been shown to control the stability and degradation of the PTEN protein.11 Our data indicate that none of these mechanisms are likely responsible for the core 3a–mediated down-regulation of PTEN. Instead, HCV core 3a expression appears to repress PTEN mRNA translation via PTEN 3′-UTR–dependent mechanisms. Noncoding microRNAs play important roles in protein expression by hybridizing to complementary sites on the 3′-UTR sequences of target mRNAs and thereby inhibiting their translation or triggering their degradation.30 Several microRNAs have been reported to inhibit PTEN expression.11 Interestingly, because the levels of PTEN mRNA are unchanged between control and HCV core 3a–expressing cells, it is likely that core 3a induces the expression of microRNAs, which prevent the translation of this mRNA. Supporting our data, the transcriptome profiling of livers from HCV-infected patients and computational target predictions have suggested that microRNA-mediated posttranscriptional PTEN alterations and the impairment of Akt signaling occur during an HCV infection.31 Notably, miR-21, a microRNA mediating PTEN down-regulation in NAFLD,10 was not increased in HCV core 3a–expressing cells (data not shown); this suggests that other, hitherto unknown PTEN-targeting microRNAs are involved in this process.

Steatosis in patients with chronic hepatitis C is clinically relevant because it influences both the progression of liver disease and the response to antivirals. Whether the clinical impact of steatosis depends on its pathogenesis (i.e., viral versus metabolic) remains a matter of debate.22 In this respect, alterations of PTEN expression/activity induced by HCV not only may lead to a deregulated lipid metabolism and potentially impaired insulin sensitivity but also may contribute to the progression of liver disease toward cirrhosis and HCC. Indeed, PTEN is a well-established tumor suppressor that is frequently mutated/deleted or down-regulated in human cancers, including HCC.11, 12 In addition, liver-specific PTEN knockout mice develop steatohepatitis, fibrosis, and HCC6, 7; this supports a role for PTEN in liver fibrosis and carcinogenesis. Finally, an analysis of cirrhotic and HCC tissues from HCV-infected patients has shown that PTEN is often down-regulated in tumors, and higher PTEN expression levels are a factor predicting prolonged survival.32

Further molecular, clinical, and epidemiological studies are now warranted for determining in greater detail the mechanisms by which an HCV genotype 3a infection alters the function of PTEN in the liver and the role of these PTEN alterations in the pathogenesis of hepatitis C. Furthermore, it remains to be established whether PTEN represents a therapeutic target for preventing the progression of liver disease toward its most ominous complications, cirrhosis and HCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

The authors thank the Genomics Platform of the National Centers of Competence in Research (Geneva, Switzerland) for the RT-PCR analyses and S. Conzelmann, M. Fournier, C. Maeder, and S. Startchik for their invaluable help.

REFERENCES

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_24340_sm_suppinfofig1.doc211KSupporting Information
HEP_24340_sm_suppinfofig2.doc581KSupporting Information
HEP_24340_sm_suppinfofig3.doc194KSupporting Information

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