Induction of C/EBP?-Thr266 Phosphorylation by HCV Infection is Indispensable for the Zone-3 Phenotype and Proliferation in Cultured Human Hepatocytes

doi:10.15406/jhvrv.2014.01.00021

Journal of

eISSN: 2373-6453

Human Virology & Retrovirology

Short Communication Volume 1 Issue 4

Induction of C/EBP?-Thr266 Phosphorylation by HCV Infection is Indispensable for the Zone-3 Phenotype and Proliferation in Cultured Human Hepatocytes

Martina Buck,^1,2,3

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Mario Chojkier^1,2,3

¹Veterans Affairs San Diego Healthcare System, USA
²Department of Medicine, University of California, USA
³Biomedical Sciences Program, University of California, USA

Correspondence: Martina Buck, Veterans Affairs San Diego Healthcare System, Department of Medicine, Biomedical Sciences Program, University of California, San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161; USA, Tel 858-552-8585, Fax 858-552-4366

Received: August 12, 2014 | Published: September 25, 2014

Citation: John Martin W, Stoneburner J (2014) Induction of C/EBPb-Thr266 Phosphorylation by HCV Infection is Indispensable for the Zone-3 Phenotype and Proliferation in Cultured Human Hepatocytes. J Hum Virol Retrovirol 1(4): 00021. DOI: 10.15406/jhvrv.2014.01.00021

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Abstract

Liver acinar zonation allows for diverse functions to be performed on demand by specialized hepatocytes. Therefore, understanding how hepatitis C virus (HCV) infection may modulate the acinar zone phenotypes should provide insights into the pathophysiology of HCV infection. We assessed the effects HCV infection in the highly-differentiated primary human hepatocyte culture that we have developed for the efficient replication of intact HCV virions.

We found that HCV genotype 1 infection stimulated the zone-3 phenotype after 72 hr since the cultured hepatocytes expressed GS, β-Catenin and HIF-1α , markers of zone-3 hepatocytes. HCV infection also induced phosphorylated-C/EBPβ-Thr266 and proliferation in these cultured hepatocytes. A peptide designed to selectively inhibit phosphorylation of C/EBPβ-Thr266 blocked this phosphorylation, the zone-3 phenotype and hepatocyte proliferation induced by HCV infection in these cultured human hepatocytes. In addition, we found that in normal, uninfected human hepatocytes, unphosphorylated-C/EBPβ-Thr266 was associated with protein von Hippel Lindau (pVHL) and Axin (inhibitors of HIF-1α and β-catenin activation, respectively). These associations were blocked by the HCV infection since phosphorylated C/EBPβ-Thr266 was not associated with either pVHL or Axin. The inhibition of C/EBPβ-Thr266 phosphorylation normalized the association of unphosphorylatable C/EBPβ-Thr266 with pVHL and Axin, which is expected to inhibit the activation of HIF-1α and β−catenin. Collectively, these results strongly suggest that C/EBPβ-Thr266 phosphorylation is indispensable for the activation of GS, HIF-1α and β-catenin, and the induction of zone-3 phenotype in HCV-infected human hepatocytes.

Keywords: Human hepatocytes, Hepatitis C, Liver zone-3, C/EBPβ, Glutamine synthetase, HIF-1, β-catenin

Abbreviations

C/EBP, CCAAT/Enhancer Binding Protein; HIF, Hypoxia Inducible Factor; GS, Glutamine Synthetase; HCC, Hepatocellular Carcinoma; pVHL, protein von Hippel Lindau; HCV, Hepatitis C Virus

Introduction

The hepatocytes in different liver acinar zones are under different Ο₂ tension and perform different metabolic activities.¹Hepatocytes in acinar zone-1 are under normoxic conditions, which stimulate aerobic (mitochondrial oxidative) metabolism. By contrast hepatocytes in acinar zone-3 are exposed to hypoxia, which induces survival by hypoxic/‘anaerobic’ (non-mitochondrial) metabolism. Zone-3 hepatocytes are characterized by the expression of Glutamine Synthetase (GS), β-Catenin and Hypoxia-Inducible Factor (HIF)-1.^2-5 Expression of the GS gene in zone-3 hepatocytes is stimulated by C/EBPβ.⁶

We have shown that TGFα induces phosphorylation of mouse C/EBPβ on Thr217 as well as hepatocyte proliferation.⁷ The C/EBPβ-Thr217 phosphoacceptor is highly conserved through evolution.^7,8We reported that expression of a catalytically inactive mutant RSK, which behaves as a dominant negative, blocks hepatocyte proliferation induced by TGFα, indicating that RSK activity is important for this effect.⁷

Phosphorylation of C/EBPβ on Thr217 is required for the stimulation of hepatocyte proliferation by TGFα since hepatocytes expressing a C/EBPβ-Ala217 mutant, lacking the critical phosphoacceptor, are refractory to the stimulation of hepatocyte proliferation by TGFα. Also, expression of the phosphorylation-mimic C/EBPβ−Glu217 transgene was sufficient to induce hepatocyte proliferation in the absence of TGFα. In contrast, expression of the C/EBPβ−Ala217 transgene blocked hepatocyte proliferation in the presence of TGFα.⁷ Thus, C/EBPβ PhosphoThr217 plays an active role in inducing hepatocyte proliferation after treatment with TGFα.

In this study we asked whether Hepatitis C Virus infection in cultured human hepatocytes stimulates C/EBPβ-Thr266 phosphorylation (the human homologue phosphoacceptor) and whether it is required for hepatocyte proliferation and a zone-3 phenotype.

Conclusion

In this study, we assessed the effects HCV infection in the highly-differentiated primary human hepatocyte culture that we have developed for the efficient replication of intact HCV virions. We used primary human hepatocytes (Invitro technologies; Baltimore, MD) cultured on a collagen matrix and infected on day-3 after plating the cells, with serum from a patient with a chronic HCV genotype 1 infection as described previously.⁹The matrix was rat-tail collagen( BD Biosciences); the collagen matrix was prepared within 24 hr of hepatocyte plating , at a concentration of 50 mg/ml or greater; the culture plates were coated with polylysine; the suspended hepatocytes were allowed to attach in 20% fetal calf serum for not more than 18 hr; the hepatocyte-specific media was given for at least 24 hr prior to the HCV infection; the hepatocytes were >85% confluent until the time of infection; and hepatocyte media was changed every 72 hr.⁹

This normal human hepatocyte culture system is permissible to the infection with, and physiologically significant amplification of, naturally occurring HCV.⁹

We found that HCV genotype 1 infection after 72 hr (viral load ∼ 7 x10⁶ IU/uL) stimulated a zone-3 phenotype since the cultured hepatocytes expressed HIF-1α and GS.^2,5HCV infection also induced phosphorylated-C/EBPβ-Thr266 (the human homologous phosphoacceptor^7,8,10) in these cultured hepatocytes. Control uninfected, normal quiescent cultured human hepatocytes did not express phosphorylated-C/EBPβ-Thr266, HIF-1α or GS (Figure 1).

Figure 1 Phosphorylation of human C/EBPβ-Thr266 induced by HCV infection is indispensable for the zone-3 phenotype. Primary human hepatocytes infected with HCV (viral load ~ 7 x106 IU/uL after 72) also express phosphorylation of C/EBPβ-Thr266 and a zone-3 phenotype (HIF-1α; GS). Blocking phosphorylation of C/EBPβ-Thr266 with the dominant negative peptide (100 nM for 24 hr) prevents expression of the zone-3 phenotype. Nuclear stain and the merge images are shown.

We found that HCV infection did not affect the expression of unphosphorylated C/EBPβ-Thr266 compared to control uninfected hepatocytes but stimulated hepatocyte proliferation as determined by the expression of ki67. Hepatocyte proliferation occurred in zone-3 phenotypic hepatocytes, judging by the expression of β-Catenin, another marker of zone-3^3,4 (Figure 2).

Figure 2 Phosphorylation of human C/EBP-Thr266 induced by HCV infection is indispensable for the expression of -Catenin and hepatocyte proliferation. Experiments were performed as described in Figure 1. Normal or HCV-infected primary human hepatocytes express unphosphorylated C/EBPβ-Thr266. HCV-infected primary human hepatocytes express -Catenin and a marked increase in zone-3 hepatocyte proliferation (ki67 + hepatocytes). Blocking phosphorylation of C/EBPβ-Thr266 with the dominant negative peptide (100 nM for 24 hr in 72-hr HCV-infected human hepatocytes) prevented expression of the zone-3 -Catenin and hepatocyte proliferation. Nuclear stain and merge images are shown.

To test whether phosphorylated-C/EBPβ-Thr266 is required for zone-3 phenotype and hepatocyte proliferation in HCV infection, we treated 72 hr HCV-infected hepatocytes cultures with a peptide designed to selectively inhibit phosphorylation of C/EBPβ-Thr266.⁸ The peptide blocked this phosphorylation (Figure 1) but did not affect the expression of unphosphorylated C/EBPβ-Thr266 (Figure 2). The peptide also blocked the zone-3 phenotype (expression of HIF-1, GS and β-Catenin as well as hepatocyte proliferation induced by HCV infection (Figures 1 and 2).

In addition, we found that in normal, uninfected human hepatocytes, unphosphorylated-C/EBPβ-Thr266 was associated with protein von Hippel Lindau (pVHL) and Axin (inhibitors of HIF-1α and β-catenin activation, respectively)^11,12 (Figure 3).

Figure 3 Unphosphorylated-C/EBPβ-Thr217 is physically associated with pVHL and Axin. Experiments were performed as described in Figure 1. C/EBPβ was immunoprecipitated from the 72-hr HCV-infected human hepatocyte’s cell lysates. Unphosphorylated-C/EBPβ-Thr217 was physically associated with pVHL and Axin in uninfected normal primary human hepatocytes (Left lane) and in HCV-infected cultures treated with the inhibitory peptide (Right lane). In HCV-infected human hepatocytes not treated with the peptide, phospho-C/EBPβ-Thr266 is not associated with either pVHL or Axin (Center lane).

These associations were blocked by the HCV infection since phosphorylated C/EBPβ-Thr266, which stimulates HIF-1α and β-catenin activation and expansion of zone-3 phenotype (and the resultant increase in hepatocyte proliferation), was not associated with either pVHL or Axin. The inhibition of C/EBPβ-Thr266 phosphorylation normalized the association of unphosphorylatable C/EBPβ-Thr266 with pVHL and Axin (Figure 3), which is expected to inhibit the activation of HIF-1α and β−catenin (Figure 1 and 2). Further, the interaction between β-catenin and HIF-1 promotes cellular adaptation to hypoxia.¹³

Collectively, these results strongly suggest that C/EBPβ-Thr266 phosphorylation is indispensable for the activation of GS, HIF-1 and catenin, and the induction of zone-3 phenotype in HCV-infected human hepatocytes. These novel findings support the activation of catenin and HIF-1 by phospho-C/EBPβ-Thr217 in zone-3 and their inhibition by the peptide blocking the zone-3 phenotype.

We hypothesize that the induction of hepatocyte proliferation confers a survival advantage to the HCV infection. The HCV-induced hepatocyte proliferation is mediated in cultured human hepatocytes by C/EBPβ-Thr266 phosphorylation and the adoption of a zone-3 hepatocyte phenotype.

Acknowledgments

We thank Daniela Traykova and Caitlin Stalling for their technical support. This study was supported by following grants: RC1 Challenge-DK 087031 (to MB and MC); MERIT R37-DK-46071 (to MC); R01-DK-084139 (to MB), and the Department of Veterans Affairs: Merit Review Award (to MC).