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Hepatitis B virus X protein induces angiogenesis by stabilizing hypoxia-inducible factor-1¹

EUN-JOUNG MOON^*,, CHUL-HO JEONG^*, JOO-WON JEONG^*, KWANG ROK KIM^*, DAE-YEUL YU, SEISHI MURAKAMI, CHUL WOO KIM^|| and KYU-WON KIM^*,2

^* Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea,
Department of Molecular Biology, Pusan National University, Busan 609-735, Korea,
Korea Research Institute of Bioscience and Biotechnology, Daejon 305-333, Korea,
Department of Molecular Biology, Division of Molecular Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920, Japan,
^|| Cancer Research Center, Department of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea

²Correspondence: College of Pharmacy, Seoul National University, Seoul 151-742, Korea. E-mail: qwonkim{at}plaza.snu.ac.kr

SPECIFIC AIMS

Hepatocellular carcinoma (HCC) is a highly vascularized solid tumor. One of the major risk factors for HCC is the infection of hepatitis B virus (HBV). Although HBx, the protein encoded by the X gene of the HBV genome, has been implicated in HBV-mediated hepatocarcinogenesis, the relationship between HBx and the hypervascularity of HCC remains unclear. Therefore, the aim of this study is to determine the role of HBx in the enhanced angiogenesis of HCC.

We previously reported that HBx increases the expression of vascular endothelial growth factor (VEGF), suggesting that HBx may act as a mediator of hypoxia-induced angiogenesis. Since HBx is a coactivator of transcription factor, it requires a certain transcription factor to increase the transcription level of VEGF. The expression of VEGF is tightly regulated by hypoxia-inducible factor-1 (HIF-1), a key transcription factor that upregulates a variety of genes involved in angiogenesis under hypoxia. HIF-1 is a heterodimer consisting of HIF-1 and HIF-1ß subunits. HIF-1 is targeted for von Hippel-Lindau protein (pVHL)-mediated destruction by the cellular oxygen concentration, whereas HIF-1ß is constitutively expressed, therefore, the transcriptional activity of HIF-1 is under the control of HIF-1. On the basis of these backgrounds, we focused on the regulatory mechanism of HBx on the stability of HIF-1 in HBx-mediated angiogenesis.

PRINCIPAL FINDINGS

1. HBx increases the protein level of HIF-1 through direct interaction
We investigated whether HBx induces HIF-1 in human embryonic kidney (HEK) 293 cell lysates transfected with a pCEP4-HBx vector and exposed to hypoxic (1% O₂) or normoxic (21% O₂) condition for 24 h. We observed that HBx increases the protein level but not the mRNA level of HIF-1 under both normoxic and hypoxic conditions.

To examine the possibility that HBx interacts directly with the HIF-1 protein, we performed GST-pull down assay. Full-length HIF-1 bound to GST-HBx but not to GST alone. Among the fragments of HIF-1, the bHLH/PAS (amino acids 1-400) region of HIF-1 interacted with HBx. We confirmed that HBx interacts with HIF-1 under both normoxic and hypoxic conditions by coimmunoprecipitation analysis in vivo.

2. HBx inhibits the interaction of pVHL with HIF-1 and blocks ubiquitin-dependent degradation
HIF-1 is rapidly degraded by the pVHL-mediated ubiquitin-proteasome pathway under normoxic condition. To investigate the mechanism by which HBx stabilizes HIF-1 protein, we first examined whether HBx affects the binding of pVHL to HIF-1. A coimmunoprecipitation assay showed that HBx inhibits the binding of pVHL to HIF-1 (Fig. 1 A). However, HBx did not interact with pVHL in a GST pull-down analysis. Next, we investigated whether HBx modulates the ubiquitin-proteasome degradation of HIF-1. HIF-1 was strongly detected in cells transfected with HIF-1 (Fig. 1B , lane 2). However, the expression of HIF-1 was decreased in cells cotransfected with HIF-1 and FLAG-tagged ubiquitin vectors (Fig. 1B , lane 3). After treatment with proteasome inhibitor MG132, the stability of HIF-1 was restored (Fig. 1B , lane 4). Similar to MG132 treatment, HBx also increased HIF-1 stability (Fig. 1B , lane 5), suggesting that HBx blocks the ubiquitin-proteasome degradation pathway of HIF-1.

View larger version (28K): [in this window] [in a new window]   Figure 1. HBx decreases the interaction of pVHL with HIF-1 and blocks ubiquitination-dependent degradation. A) HEK 293 cells were cotransfected with expression vectors encoding GFP-HIF-1 and pCEP4-HBx, as indicated. Aliquots of anti-HIF-1 immunoprecipitates (IP) and whole cell lysates were analyzed by immunoblot assays with antibodies against pVHL and HIF-1. Total RNA was isolated from these cells and mRNA expression for HBx and ß-actin were analyzed by RT-PCR. B) HEK 293 cells were cotransfected with pCEP4-HBx, FLAG-ubiquitin, and pBOS-HIF-1 vectors, as indicated. The transfected cells were treated with 30 µM MG132 for 3 h or left untreated prior to lysis. The blot was analyzed with an antibody against HIF-1.

3. HBx increases the transcriptional activity of HIF-1 and angiogenesis
In addition to the induction of HIF-1, we examined whether HBx activates the transcriptional activity of HIF-1 through an HRE-dependent reporter gene assay. In cells cotransfected with the pCEP4-HBx vector, the reporter gene expression was increased 2- to threefold compared with mock-transfected cells under both normoxic and hypoxic conditions (Fig. 2 A). Moreover, the expression of VEGF was increased in both HBx-expressing and hypoxia-treated cell lysates by immunoblot analysis (Fig. 2B ).

View larger version (23K): [in this window] [in a new window]   Figure 2. HBx increases the transcriptional activity of HIF-1. A) HEK 293 cells were cotransfected with pBOS-HIF-1, pBOS-ARNT, pCMV-ß-gal, EpoHRE-Luc, and pCEP4-HBx or pCEP4, and exposed to hypoxia (1% O2) for 18 h. Luciferase activity was then measured. Data are means of triplicate measurements from one transfection. B) HEK 293 cells were transfected with pCEP4-HBx or pCEP4 and incubated for 24 h under either normoxic or hypoxic conditions. The blot was analyzed with antibodies against VEGF and -tubulin.

Next, to investigate whether the HBx leads to angiogenesis, we performed in vitro and in vivo angiogenesis assays using the conditioned media (CM) from control cells or HBx-transfected cells. The CM from HBx-transfected cells stimulated the tube formation of HUVECs on a Matrigel matrix as well as angiogenesis on the chorioallantoic membrane (CAM) of chick embryos. However, CM from HBx-transfected cells which preincubated with neutralizing specific VEGF antibody did not stimulate angiogenesis on the chorioallantoic membrane (CAM) of chick embryos. Although it is possible that other mechanisms of HBx-induced VEGF overexpression may be exist independently of HIF-1, these data implicate that HBx stimulates angiogenesis via VEGF signaling pathway driven by HIF-1.

4. Induction of HIF-1, VEGF, and microvessels in the HBx-transgenic mice
It was previously reported that HCC is detected in the livers of the HBx-transgenic mice at the age of 11–18 months. We investigated whether the inductions of HIF-1 and VEGF are involved in HBx-induced angiogenesis using the 12-month-old HBx-transgenic mice. We sectioned the liver tissues near the HCC of the HBx-transgenic mice. In immunohistochemical analysis, HBx was more strongly detected in the dysplastic lesion than in the non-neoplastic region of the HBx-transgenic liver. HIF-1 and VEGF were also strongly detected in the dysplastic lesion of the HBx-transgenic liver. In contrast, HIF-1 and VEGF were rarely detected in the liver tissues of non-transgenic mice. Moreover, capillary-like microvessels stained with PECAM-1 antibody were more frequently and strongly detected in the dysplastic lesion than in the non-neoplastic region of the HBx-transgenic liver.

CONCLUSIONS AND SIGNIFICANCE

The shortage of blood supply due to portal hypertension in liver cirrhosis and rapid proliferation of tumor cells probably develop local hypoxia in the HCC liver tissues. This local hypoxia may function as a strong stimulus for the synthesis of angiogenic factors in HCC. Here, we suggest that HBx is an important factor for the hypoxic-induction of angiogenic factors through HIF-1 in HCC.

Our results demonstrate that HBx increases the protein level of HIF-1 by the direct interaction of HIF-1, which leads to inhibit the interaction between pVHL and HIF-1 and the ubiquitin-dependent degradation of HIF-1 (Fig. 3 ). Under normoxic condition, HIF-1 binds to pVHL, which is the recognition component of an E3 ubiquitin-protein ligase. This binding occurs via the hydroxylation by prolyl hydroxylases of Pro⁵⁶⁴ in the oxygen dependent degradation (ODD) domain of HIF-1. Once bound to pVHL, HIF-1 undergoes ubiquitination and proteasomal degradation. Since HBx binds to the bHLH/PAS domain of HIF-1, while pVHL binds to the ODD domain of HIF-1, HBx may inhibit the interaction between pVHL and HIF-1 but not compete with pVHL for the interaction with HIF-1.

View larger version (29K): [in this window] [in a new window]   Figure 3. Scheme of the proposed role of HBx in the HIF-1-mediated angiogenesis of HCC. HBx interacts and stabilizes HIF-1 through inhibition of the interaction between pVHL and HIF-1 and the ubiquitin-dependent degradation. Subsequently, HBx activates the HIF-1-dependent transcription and leads to angiogenesis. Ub, ubiquitin; HRE, hypoxia-response element.

Moreover, we found that HBx did not bind to pVHL directly and did not diminish the protein level of pVHL. In addition, we determined that the direct interaction of HBx and the bHLH/PAS domain of HIF-1 increases the transcriptional activity of HIF-1. Furthermore, our results showed that HBx stimulates the induction of VEGF via HIF-1 signaling pathway and then increases angiogenesis (Fig. 3) .

Finally, we confirmed our observations using HBx-transgenic mice. The non-neoplastic area of the HBx transgenic liver had an intact lobular architecture without cirrhotic changes and no inflammatory cells in the hepatic parenchyma. The dysplastic lesion showed abnormally large blood vessels, infiltration of blood cells into the vessels, hepatocellular changes, and cell proliferation. In immunohistochemical analysis, we detected the significant inductions of VEGF, HIF-1 and microvessels in the dysplastic lesion of HBx-transgenic mice. These findings suggest that HBx may promote the development of HCC by the overexpression of HIF-1 and the induction of angiogenesis at the early stage of hepatocarcinogenesis. Therefore, HIF-1 may be a good candidate for antiangiogenic therapy to inhibit the growth and the development of HCC by blocking the function of HIF-1 or accelerating the pVHL-mediated degradation pathway of HIF-1.

Collectively, we established that the hypervascularity of HCC might be closely related with the stabilization of HIF-1 mediated by HBx. Furthermore, our data suggest a novel function of HBx as a regulator of hypoxia-induced angiogenesis in HCC.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0153fje;

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This Article Full Text (PDF) All Versions of this Article: 18/2/382 03-0153fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MOON, E.-J. Articles by KIM, K.-W. Search for Related Content PubMed PubMed Citation Articles by MOON, E.-J. Articles by KIM, K.-W.