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Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: Involvement of invasive potential

TAE-WOOK CHUNG^*, YOUNG-CHOON LEE and CHEORL-HO KIM^*,1

^* National Research Laboratories for Glycobiology, Ministry of Science and Technolgoy, and Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyungju, South Korea; and
Faculty of Biotechnology, Dong-A University, Busan, South Korea

¹Correspondence: National Research Laboratories for Glycobiology and Department of Biochemistry and Molecular Biology, College of Oriental medicine, Dongguk University, Sukjang-Dong 707, Kyungju City, Kyungbuk 780-714, Korea. E-mail: chkimbio{at}dongguk.ac.kr

SPECIFIC AIMS

The X protein (HBx) of hepatitis B virus (HBV) has been shown to be essential for the development of hepatocellular carcinoma (HCC). We recently found that HBx causes the progression of liver cancer through down-expression of PTEN, known as a tumor suppressor gene (Chung et al., Cancer Res., 2003). The prognosis for HCC depends mainly on the clinicopathological characteristics regarding invasion and metastasis. The expression of matrix metalloproteinase-9 (MMP-9) has been implicated for an important role in HCC invasion and metastasis. Unfortunately, there is no direct evidence that HBx-induced MMP-9 expression can induce HCC invasion and metastasis, although many reports have been published. The aim of the present study was to determine whether 1) HBx activates the mitogen-activated protein kinase (MAP kinase) and phosphatidylinositol 3-kinase (PI-3K) signal cascade, which is essential for activation of transcription factor AP-1 and nuclear factor-B (NF-B), 2) HBx stimulates the activities of extracellular signal-regulated kinase 1/2 (ERK 1/2) as well as the PI-3K/AKT, 3) the expression of MMP-9 in HBx-transfected cells is enhanced by activation of AP-1 and NF-B transcriptional activity through activation of ERK and PI-3K-AKT/PKB pathways, and 4) enhanced expression of MMP-9 by activation of these signals is eventually associated with the invasive potential of cells.

PRINCIPAL FINDINGS

1. HBx stimulates activation of ERK and PI-3K/AKT/PKB signal pathways for enhanced MMP-9 expression related to invasion and metastasis of liver cancer cells
MMP-9 is specifically related to cancer invasion and metastasis; therefore, ERK and PI-3K activities were assessed by measuring their degrees of regulatory phosphorylation using phosphospecific antibodies. ERK1/2 phosphorylation was markedly elevated in HBx-transfected cells (Fig. 1 A). An ERK-specific inhibitor, U0126, blocked HBx-induced ERK1/2 activation. The HBx promoted activation of AKT, a downstream target of PI-3K (Fig. 1B ), and AKT activation was ablated by the PI-3K inhibitor wortmannin. To examine directly whether activation of ERKs by HBx regulates MMP-9 expression, cells were stimulated in the presence of an ERK inhibitor. Cells pretreated with U0126 showed that HBx-induced MMP-9 expression and ERK1/2 activation were both inhibited (Fig. 1C ). To determine whether PI-3K participates in the HBx-mediated induction of MMP-9 secretion, we also used wortmannin. MMP-9 expression and AKT phosphorylation were dramatically decreased by wortmannin in HBx-transfected cells (Fig. 1D ). To investigate whether p38 MAPK activation modulates expression of MMP-9 in HBx-transfected cells, we used SB203580, a specific p38 MAPK inhibitor. HBx-induced MMP-9 was not affected by SB203505 (Fig. 1C, D ).

View larger version (48K): [in this window] [in a new window]   Figure 1. Activation of ERKs and PI-3K on MMP-9 expression by HBx and their inhibition by U0126 and wortmannin. Chang-pEGFP-X cells were cotreated in serum-free DMEM with PMA and ERK or PI-3K inhibitors in a time-dependent manner (0–60 min). For activity of ERKs (A) and AKT (B), cell lysates were analyzed by Western blot analysis. To determine ERK (C) and PI-3K (D) activity on MMP-9 expression, cultures of each cell were incubated for 24 h in serum-free DMEM with or without PMA, U0126, SB203580, or wortmannin. The conditioned medium was collected, then analyzed by gelatin zymography, and cell lysates were analyzed by Western blot analysis.

2. HBx-mediated MMP-9 expression depends on intact NF-B and AP-1 binding sites within the MMP-9 promoter region
To further confirm that HBx is directly involved in the AP-1 and NF-B-mediated transcriptional activation of MMP-9, we examined whether HBx is correlated to the binding of AP-1 and NF-B to wild-type oligonucleotides that contain the sequence for the AP-1 and NF-B binding sites from the MMP-9 promoter by EMSA. We confirmed that nuclear lysates isolated from Chang-pEGFP-X cells induced an electromobility shift only when [-³²P]-labeled AP-1-1, AP-1-2, and NF-B wild-type oligonucleotides were introduced (Fig. 2 ). Formation of an electrophoretically retarded complex was inhibited when an unlabeled wild-type oligonucleotide was introduced. Nuclear lysates isolated from Chang, Chang-EGFP, and Chang-pEGFP-X cells did not induce an electromobility shift when both [-³²P]-labeled and unlabeled AP-1-1, AP-1-2, and NF-B mutant-type oligonucleotides were introduced. The HBx-mediated MMP-9 expression depended on intact NF-B and AP-1 binding sites within the MMP-9 promoter region.

View larger version (42K): [in this window] [in a new window]   Figure 2. Enhanced activity of AP-1 and NF-B-mediated MMP-9 promoter by HBx. Nuclear extract was isolated from each cell incubated with [-32P] ATP-labeled oligonucleotides corresponding to the wild-type AP-1-1, AP-1-2, and NF-B binding site of the MMP-9 promoter. Competition was performed using unlabeled wild-type oligo or a mutant oligo. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5 x TBE buffer at 250 V for 20 min. The gel was dried and subjected to autoradiography.

CONCLUSIONS AND SIGNIFICANCE

HBx protein and MMP-9 enzyme activity have been reported to relate to the development of hepatocellular carcinoma (HCC) and HCC invasion/metastasis, respectively, but there was no result on direct relationship between HBx and HCC invasion/metastasis. Our present study demonstrates for the first time that the HBx protein stimulates activities of the PI-3K-AKT/PKB as well as ERK 1/2, but not p38 MAPK. We have demonstrated that HBx enhanced expression of MMP-9 by dual transcriptional up-regulations of AP-1 and NF-B transcriptional activity through activation of ERKs and PI-3K/AKT pathways. For metastatic potential of the HBx, these transcriptional regulations were directly associated with the invasive potential of cells. Our data show that HBx induces the expression of MMP-9 by ERK and PI-3K signal pathways and that ERK and PI-3K inhibitors both inhibit HBx-induced MMP-9 production. The invasiveness of HBx-transfected cells is increased in an MMP-9-dependent manner, and both U0126 and wortmannin significantly inhibited the invasiveness of HBx-transfected cells. As illustrated by Fig. 3 , HBx induces PI-3K/AKT and ERK pathways, but not the p38 MAPK pathway. Stimulation of these signal pathways by HBx leads to activation of NF-B and the AP-1 transcription factor. Translocation of NF-B and AP-1 activated by HBx into the nucleus results in an increase of MMP-9 expression. Therefore, these findings extend the current knowledge of HBx function during hepatitis B viral hepatocarcinogenesis and invasive metastasis, and point out a novel mechanism whereby HBx plays a role in the induction of MMP-9; this in turn plays an important role in cancer invasion and metastasis in liver cells and the increment of HBx-transfected cell’s invasion by the up-regulation of MMP-9 via activation of ERK and PI-3K signal pathways.

View larger version (55K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating molecular mechanism of HBX-induced MMP-9 expression. HBx induces PI-3K/AKT and ERK pathways but not the p38 MAPK pathway. Stimulation of these signal pathways by HBx leads to the activation of NF-B and AP-1 transcription factor. Translocation of NF-B and AP-1 activated by HBx into the nucleus results in an increase of MMP-9 expression. Therefore, HBx regulates the transcriptional activation of MMP-9, which plays an important role in invasion and metastasis of tumor via PI-3K/AKT and ERK pathways.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1429fje; doi: 10.1096/fj.03-1429fje

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