Department of Microbiology, College of Natural Sciences, Pusan National University, Pusan 609-735, Korea1
Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea2
Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Pusan 614-052, Korea3
Author for correspondence: Kyung Lib Jang. Fax +82 51 514 1778. e-mail kljang{at}pusan.ac.kr
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Abstract |
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Introduction |
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HCV core protein promotes cell proliferation through several mechanisms, for example by upregulation of the cyclin E expression level (Cho et al., 2001a ), modulation of the Rb pathway through pRb downregulation and E2F-1 upregulation (Cho et al., 2001b
), or activation of the MAP kinase pathway (Tsuchihara et al., 1999
; Hayashi et al., 2000
). HCV core protein also represses transcription of the universal cyclin-dependent kinase (CDK) inhibitor p21 gene, as demonstrated by in vitro transient expression assays using murine fibroblasts (NIH 3T3), human hepatocellular carcinoma (HepG2) and human cervical carcinoma (HeLa) cells (Ray et al., 1998
; Jung et al., 2001
; Yoshida et al., 2001
). Considering the anti-proliferative function of p21, the effect might play an important role during HCV-mediated hepatocellular carcinogenesis. The effect might result from the inhibition of a major p21 upstream regulator, p53, either by proteinprotein interactions or transcriptional repression. Otherwise, HCV core protein may repress the p21 gene through a p53-independent pathway. In this study we performed a detailed mutational analysis of the p21 promoter to investigate the mechanism by which HCV core protein represses transcription of the p21 gene. Furthermore, we tested whether the repression effect actually stimulates cell cycle progression in Core-expressing cells.
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Methods |
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Transfection and luciferase assay.
NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum. Cells were seeded at 2x105 cells per 60 mm diameter plate and transfected the next day with a calcium phosphateDNA precipitate containing 3 µg each of target and effector plasmid DNAs as previously described (Gorman et al., 1982 ). To control for variation in transfection efficiency, 2 µg of plasmid pCH110 (Pharmacia) containing the E. coli lacZ gene under control of the SV40 promoter was cotransfected. After 48 h, the level of expression from the target gene (luciferase activity) was analysed and values obtained were normalized to the
-galactosidase activity measured in the corresponding cell extracts. Each experiment was repeated at least three times.
Semi-quantitative RTPCR and Western blotting analysis.
Total RNA was prepared from cells 48 h after transfection by the guanidinium isothiocyanate procedure (Chomczynski & Sacchi, 1987 ). For RTPCR, 3 µg of RNA was reverse transcribed with the corresponding antisense primer. One-quarter of the reverse transcribed RNA was amplified with Taq polymerase (95 °C, 5 min; 30 cycles of 95 °C for 1 min, 56 °C for 1 min, 72 °C for 30 s; final elongation step 72 °C, 5 min) using the appropriate primers. The primer pairs for p21 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were described previously (Ahn et al., 2001
). For the detection of HCV core transcripts, sense primer 5' TCC GGA TCC CTG TCA TCT TCT GTC CCT 3' and antisense primer 5' TCG CTT AGT GGA TCC TGG GGG CAG 3' were used.
For Western blotting analysis, cells were lysed in buffer (50 mM TrisHCl, pH 8, 150 mM NaCl, 0·1% SDS, 1% NP-40) supplemented with protease inhibitors. Ten µg of cell extracts was separated by SDSPAGE and transferred onto a nitrocellulose membrane (Hybond PVDF; Amersham). Western blotting was performed with either anti-p53 monoclonal antibody, anti-p21 rabbit polyclonal IgG or anti-actin monoclonal IgG (all from Santa Cruz), and subsequently detected by chemiluminescent ECL (Amersham) as recommended by the manufacturer.
Generation of stable cell lines and determination of cell growth rate.
NIH 3T3 cells transfected with HCV Core-expressing plasmid were selected and amplified to obtain stable cell lines as described previously (Kwun et al., 2001 ). The expression level of transfected genes was checked by either RTPCR or Western blotting analysis. For the determination of cell growth rate, 5x104 cells were plated in six-well plates (Nunc) and the total number of cells in each well was counted after incubation under the appropriate conditions.
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Results and Discussion |
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More direct evidence that the Sp1 binding site is not responsible for the loss of Core responsiveness was obtained with 93-S mut#2.3. This construct contains a mutation of bases -76 and -77 from CT to GG, and thus maintains the consensus Sp1 binding site, but showed a significantly reduced ability to be activated by TGF- (Datto et al., 1995a
). Actually, the Core-responsive element in the p21 promoter defined above exactly overlapped the TGF-
responsive element (T
RE), which is known to mediate transcriptional activation of the p21 gene by TGF-
(Datto et al., 1995a
). The promoter activity in 93-S mut#2.3 was not repressed by Core at all (Fig. 1b
), suggesting that destruction of the T
RE in both 93-S mut#2 and 93-S mut#2.3 is responsible for the loss of Core responsiveness. Moreover, pGL2 T+I 4xT
RE, which contains four copies of the TGF-
responsive element inserted 5' of the TATA box (Datto et al., 1995a
), was responsive to core protein in a dose-dependent manner (Fig. 2a
), suggesting that the T
RE is sufficient for the effect of Core. Therefore, from the above studies we concluded that the T
RE between -83 and -74 of the p21 promoter is the Core-responsive element. The decrease in repression observed with other 93-S mutants was probably due to their low basal activities resulting from the loss of Sp1 sites.
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The Core-responsive region defined in this study also exactly overlaps the main butyrate responsive element between -87 and -72, which mediates the effect of butyrate on the p21 promoter (Nakano et al., 1997 ). Butyrate arrests cell growth by activating p21 promoter through specific Sp1 sites in a p53-independent fashion. In contrast to the effect of core protein on the TGF-
pathway, Core does not repress the effect of butyrate (to induce p21 transcription) at all (Fig. 2a
, c
), suggesting that core protein does not act through a butyrate pathway. This result is consistent with the mutational analysis of p21 promoter because the Sp1 sites on the p21 promoter were not related to the effect of core protein.
Next, we tried to confirm that inhibition of the TGF- pathway by core protein, as demonstrated with the T
RE-luciferase construct, is responsible for the repression of p21 promoter by core protein. As expected, in the presence of either TGF-
or butyrate, the core protein inhibited only the effect of TGF-
on the p21 promoter (Fig. 2c
). We therefore concluded that Core represses p21 transcription through inhibition of a TGF-
pathway.
Repression of endogenous p21 gene expression by HCV Core
To elucidate whether expression of the endogenous p21 gene is actually repressed by HCV core protein at the transcriptional level, we measured the level of endogenous p21 RNA. According to a semi-quantitative RTPCR analysis, p21 was repressed by expression of the core protein (Fig. 3a, lanes 1 and 2). The endogenous p21 protein level was also decreased by HCV Core (Fig. 3b
, lanes 1 and 2). In addition, the core protein could clearly repress the expression of endogenous p21 gene elevated by TGF-
(Fig. 3
, lanes 3 and 4) but not by butyrate (lanes 5 and 6), which is consistent with the results of reporter assay. The expression level of p53 was not significantly affected by core protein under the conditions used in this study.
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This study may provide a clue to elucidate the transcription mechanism of p21. The p21 gene is regulated by a rapidly growing list of physiological and pathological factors. In particular, the region between -83 and -74 in the p21 promoter has been shown to be required for induction of p21 by several factors such as TGF- (Datto et al., 1995a
), Ca2+ (Prowse et al., 1997
), butyrate (Nakano et al., 1997
) and lovastatin (Lee et al., 1998
). Among these, TGF-
and butyrate inhibit cell proliferation and induce G1 cell cycle arrest in various cell types (Alexandrow & Moses, 1995
; Datto et al., 1995b
; Nakano et al., 1997
; Reynisdottir et al., 1995
). Sp1 and Sp3 bind to this region in TGF-
- and butyrate-treated cells (Li et al., 1998
; Matsukawa et al., 1997
; Moustakas & Kardassis, 1998
), suggesting that stabilization of Sp1 binding mediates induction of the p21 promoter by TGF-
and butyrate. However, it is still questionable because the pattern of Sp1 binding in TGF-
- and butyrate-treated cells was not much different from that in untreated cells (Pardali et al., 2000
; Nakano et al., 1997
). Furthermore, the two pathways showed different responses to HCV core protein for expression of p21 gene, as demonstrated in this study. This study may provide a good model system to study the TGF-
pathway because it specifically responds to Core protein. Another important point to be elucidated is how Core protein inhibits the TGF-
pathway. Core protein may inhibit the function of intracellular effectors of TGF-
such as Smad3 and Smad4, either directly or indirectly by proteinprotein interactions, or may augment the activity of inhibitory signalling molecules such as Smad6 and Smad7. To provide an answer to this question, more detailed studies on the mechanism by which Core regulates the TGF-
pathway should be carried out.
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Acknowledgments |
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References |
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Received 28 January 2002;
accepted 23 April 2002.