1 Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel
2 Department of Pathology, Georgetown University Medical School, Washington, DC 2007, USA
Correspondence
Levana Sherman
lsherman{at}post.tau.ac.il
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ABSTRACT |
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INTRODUCTION |
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E6 and E7 contribute to the oncogenic process, at least in part, through their ability to interact with and inactivate key cellular regulatory proteins. E7 associates with tumour suppressor Rb and other cell-cycle regulatory proteins that control cell-cycle progression (Zwerschke & Jansen-Durr, 2000; Munger & Howley, 2002
), whereas the E6 oncoprotein interacts with a variety of cellular proteins involved in different signalling pathways, (for review see Thomas et al., 1999
; Mantovani & Banks, 2001
; Munger & Howley, 2002
), of which the best known is p53. HPV E6 binds to p53 via the E6AP protein, a ubiquitin ligase, and induces p53 degradation through the ubiquitin pathway (Scheffner et al., 1990
; Huibregtse et al., 1991
, 1993
). Other targets of E6 include proteins involved in the regulation of transcription and DNA replication, such as p300/CREB-binding protein (Patel et al., 1999
; Zimmermann et al., 1999
), the transcriptional coactivator ADA3 (Kumar et al., 2002
), interferon regulatory factor-3 (Ronco et al., 1998
), Gps2 (Degenhardt & Silverstein, 2001
), multicopy maintenance protein 7 (hMcm7) (Kuhne & Banks, 1998
; Kukimoto et al., 1998
) and the DNA repair protein 06-methylguanine-DNA methyltransferase (MGMT) (Srivenugopal & Ali-Osman, 2002
); signalling components and enzymes such as PKN (Gao et al., 2000
) and E6 TP1, (Gao et al., 1999
); proteins involved in epithelial organization and differentiation such as paxillin (Tong & Howley, 1997
) and E6BP/ERC-55 (Chen et al., 1995
); proteins involved in cellcell adhesion, polarity and proliferation control that contain a PDZ-binding motif, such as the human homologue of the drosophila large tumour suppressor, hDlg (Lee et al., 1997
; Kiyono et al., 1997
), Scribble (hScrib) (Nakagawa & Huibregtse, 2000
), MAGI-1, 2 and 3 (Glaunsinger et al., 2000
; Thomas et al., 2002
) and Mupp1 (Lee et al., 2000
), and proteins involved in apoptosis such as Bak (Thomas & Banks, 1998
), c-Myc (Gross-Mesilaty et al., 1998
; Veldman et al., 2003
) and tumour necrosis factor receptor (TNF R1) (Filippova et al., 2002
). Many of the E6 interacting proteins (E6AP, E6TP1, hMcm7, Bak, c-Myc, hScrib, hDlg, MAGI 1-3, Mupp1 Gps2 and MGMT) were described as targets for E6 dependent degradation through the ubiquitin-proteasome pathway (for review see Scheffner & Whitaker, 2003
). In addition, high-risk HPV E6 proteins can also bind DNA and activate transcription of several genes including the telomerase subunit, hTERT (Oh et al., 2001
; Veldman et al., 2001
; Gewin & Galloway, 2001
).
However, for most of the E6 binding partners, the consequence of interaction with E6, on the virus life cycle or capacity to transform host cell is not well understood.
Squamous epithelial cells are the natural host cells for HPV infection and in vitro cultured genital human keratinocytes have been used to study the effects of HPV E6 and E7 expression. Previous studies have shown that the viral oncoproteins cooperate to immortalize primary human genital keratinocytes (PHKs) and they inhibit keratinocyte terminal differentiation induced by serum and calcium (Schlegel et al., 1988; Hawley-Nelson et al., 1989
). We showed previously that HPV16 E6 oncoprotein exhibits two separate biological activities in PHKs. E6 protein by itself is capable of inducing colonies of proliferating cells resistant to serum- and calcium-induced differentiation whereas both E6 and E7 are required for immortalization of PHKs (Sherman & Schlegel, 1996
). In further studies, we showed that differentiation of cultured foreskin keratinocytes, triggered by serum and calcium, is a progressive process (23 weeks) associated with morphological and biochemical changes characteristic to keratinocyte terminal differentiation in vivo. At the end of this process cell-death with features of apoptosis is observed. Human keratinocyte terminal differentiation was accompanied with time-related changes in the expression of cellular proteins involved in the control pathways of apoptosis including downregulation of Bcl-2 and p53, and upregulation of Bax that coincided with the appearance of morphological signs of apoptosis. E6 expression significantly reduced cell stratification and apoptosis that correlated with prolonged expression of Bcl-2, reduced elevation of Bax and a complete loss of p53 (Alfandari et al., 1999
).
Proteins of the Bcl-2 family are important regulators of apoptosis and common targets of viral oncoproteins (for review see Reed, 1998; Tsujimoto & Shimizu, 2000
; Thomson, 2001
). In squamous epithelium, the expression and topographical distribution of these proteins were shown to be related to stages of differentiation. Bcl-2, a suppressor protein of apoptosis, is expressed exclusively in the undifferentiated basal layer of the epithelium (Hockenbery et al., 1991
; Polakowska et al., 1994
; Jordan et al., 1996
), while expression of Bax, a pro-apoptotic protein, is increased in the suprabasal layers (Jordan et al., 1996
; Maruoka et al., 1997
; Delehedde et al., 1999
). The changes observed in Bcl-2 and Bax expression during serumcalcium differentiation of E6 expressing PHKs (Alfandari et al., 1999
) could be a reflection of E6 inhibition of terminal differentiation or the direct effect of E6.
In the present study, the effect of HPV16 E6 on Bax expression and stability was investigated. Bax, a multidomain pro-apoptotic member of the Bcl-2 family, functions in mitochondrial membrane permeabilization and the release of cytochrome c, major events in apoptosis induction (Esposti & Dive, 2003; Scorrano & Korsmeyer, 2003
). Bax was described previously as a target of the adenovirus E1B19K (Han et al., 1998
), and the EBV Bcl-2 homologue, BALF1, (Marshall et al., 1999
).
Results of the present study demonstrate that HPV16 E6 expression reduces both Bax mRNA levels and protein stability in human keratinocytes, and stimulates the degradation of Bax protein in stable and transiently expressing cells. E6 enhancement of Bax degradation was exhibited in Saos-2 cells that lack p53. Using annexin-V binding as a marker for apoptosis, we demonstrate that E6 abrogates Bax-induced apoptosis in transiently transfected cells. Finally, we defined the carboxy-terminal region of E6, spanning aa 120132, as being essential for E6 function in degradation of Bax and inhibition of Bax-induced apoptosis.
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METHODS |
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Cell culture, transfection and retroviral infection.
PHKs cultured from neonatal foreskins were grown in keratinocyte serum-free medium (K-SFM) supplemented with 5 ng epidermal growth factor ml1 and 50 µg bovine pituitary extract (Gibco-BRL) ml1.
Infection of PHKs with retroviral vectors carrying the E6 gene, and selection in G418 (Geneticin), were carried out as described previously (Alfandari et al., 1999). For induction of differentiation, monolayers formed after growth in K-SFM were switched to Dulbecco's modified Eagle medium (DMEM) containing high-calcium and serum supplemented with 1 µg hydrocortisone ml1 (Schlegel et al., 1988
) and then maintained in this medium for 13 weeks. Human 293, 293T and Saos-2 cells were maintained in DMEM supplemented with 10 % fetal calf serum (FCS). Cells were transfected by a modified calcium phosphate procedure as described previously (Sherman & Schlegel, 1996
).
Protein degradation assays in vitro and in vivo.
In vitro degradation assays with 35S-cysteine-methionine-labelled E6 protein translated in wheat germ extract (WGE) and p53 or Bax proteins translated in reticulocyte cell lysate (RTL) were performed at 25 °C as described previously (Sherman et al., 1997, 2002
). For the in vivo assays, cells were co-transfected in duplicates with 2 µg haemagglutinin (HA)-Bax expression plasmid, together with 515 µg E6 or mutant E6 plasmid, or vector pJS55 and 2 µg GFP expression plasmid that was used as a control for transfection efficiency. Cells were harvested in RIPA buffer after 48 h and the remaining Bax and GFP levels were determined by immunoblot analysis as described previously (Sherman et al., 2002
).
Immunoblotting analysis of protein abundance.
Preparation of cell lysates in modified RIPA buffer, electrophoresis on polyacrylamide gels and transfer to nitrocellulose membranes were described previously (Alfandari et al., 1999). Filters were cut into strips and reacted with the specific antibodies. Primary antibodies used were as follows: the Bax rabbit polyclonal antibody (SC-493; Santa Cruz Biotechnology) and p53 DO-1 monoclonal antibody (mAb) (SC-126; Santa Cruz Biotechnology), the anti-GFP mAb mixture (Cat. No. 1814460; Boehringer Mannheim), the human involucrin mAb (19018; Sigma), the human actin mAb (1378996; Boehringer Mannheim). Proteins were visualized by enhanced chemiluminesence (ECL) (Amersham), using peroxidase-conjugated anti-mouse IgG (115-035-003; Jackson Immuno Research Laboratories) or protein A-peroxidase (NA9120; Amersham) according to the manufacturer's instructions. Protein amounts were determined by densitometric scanning (Dinco and Rhenium Biological Imaging System BIS 202). Non-saturated exposures of ECL films were scanned and analysed by using the TINA software.
Co-immunoprecipitation.
35S-labelled Bax or p53 translated in vitro in RTL were mixed with 35S-labelled E6A protein translated in WGE. Proteins were suspended in 300 µl lysis buffer containing 1 % NP-40, 100 mM Tris/HC1 (pH-8·0) and 100 mM NaCl (LSAB) or in 300 µl cell lysates prepared from 293T or PHKs using the same buffer. After 2 h incubation with rotary shaking at 4 °C, samples were precleaned once with protein A Sepharose and then incubated with anti-Bax or p53 antibody and protein A Sepharose for an additional 2 h with continued rotation. Beads were then collected, washed four times with LSAB and subjected to SDS-12·5 % PAGE. Proteins were detected by autoradiography.
RNA isolation and Northern blot analysis.
Total RNA was isolated from the keratinocyte cultures by using the RNADNA, protein reagent, TriReagent (Molecular Research Center), according to the manufacturer's protocol. RNA was separated on 1·2 % formaldehyde agarose gel, transferred to Hybond-N-filter (Amersham Pharmacia Biotech) and detected after hybridization to a 32P-labelled random-primed DNA probe. The Bax insert released by EcoRI digest from pSFFV-Bax was purified by gel extraction, and randomly labelled with [-32P]dCTP by using the Megaprime DNA labelling kit (Amersham Pharmacia Biotech). Molecular hybridization probes also included a cDNA for human involucrin (0·65 kbp) inserted into pBR322 (Eckert & Green, 1986
) and human glyceraldehyde phosphate dehydrogenase. Hybridization was carried out as described previously (Sherman et al., 1988
).
Protein stability assay.
Cells were incubated with 80 µg cycloheximide ml1 for the time periods indicated. Cell extracts were obtained by lysis in a modified RIPA buffer. Proteins (100 µg per lane) were analysed by SDS-12·5 % PAGE electrophoresis and immunoblotting.
Detection of apoptosis by annexin-V labelling.
293T cells were transfected with the HA-Bax expression plasmid together with E6A, vector or Bcl-2 plasmid. After 24 h, transfected cells were trypsinized, washed once in DMEM/10 % FCS and once in PBS and then stained with annexin V-Alexa (Boehringer Mannheim) according to the manufacturer's instructions.
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RESULTS |
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DISCUSSION |
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In the present study, we show that HPV16 E6 reduces Bax mRNA expression and protein stability in differentiating human keratinocytes. Bax was shown to be transcriptionally regulated by p53 (Miyashita et al., 1994; Miyashita & Reed, 1995
), as well as by c-Myc (Mitchell et al., 2000
), both targets of E6 dependent degradation (Scheffner et al., 1990
; Gross-Mesilaty et al., 1998
). The reduction in Bax mRNA levels could be due to E6-induced degradation of these transcriptional activators. Data from this study and previous data (Alfandari et al., 1999
) indicated marked reduction in p53 protein levels in E6 expressing PHKs. Levels of c-Myc were not evaluated in the present study, however, in a recent study carried out with E6 retrovirus-infected PHKs a reduction in Myc protein levels was not detected (Veldman et al., 2003
), thus questioning the role of Myc in the E6 effect on Bax transcription. In addition to the effect on Bax mRNA, data described herein indicate that E6 stimulates the degradation of Bax in vivo. Enhancement of Bax turnover was demonstrated in half-life determination assays, carried out in PHKs that were switched to differentiation medium and in co-expression assays, in transiently transfected cells, using Bax expression plasmid that directs HA-Bax transcription from a heterologous promoter (SFFV). Stimulation of Bax degradation was demonstrated in 293T cells as well as Saos-2 cells that lack p53, thereby showing that this activity is not associated with E6 effect on p53. Previous studies (Thomas & Banks, 1999
) failed to detect E6 dependent degradation of Bax, in vivo, in transient expression assays that clearly showed the degradation of Bak. The reason for this apparent discrepancy from our study is not clear. One possible explanation is that levels of expression of the E6 protein were different, reflecting differences in vectors used and cells employed.
The mechanism of Bax accelerated degradation by E6 remains to be established. The ubiquitin-proteasome system is involved in the degradation of several of the E6 targets (reviewed by Scheffner & Whitaker, 2003). Binding of E6 to the ubiquitin ligase E6AP, mediates ubiquitination and subsequent degradation of several of the E6 targets. It is not known whether E6AP plays a role in Bax degradation. However, the presence of E6AP, which was supplemented in the reticulocyte lysates, was not sufficient for inducing Bax degradation in vitro, in mixing experiments that were carried out at 25 or 30 °C. Other targets of HPV E6 proteins such as Bak (Thomas & Banks, 1999
), Gps2 (Degenhardt & Silverstein, 2001
) and ADA3 (Kumar et al., 2002
) were also shown to be targeted for degradation by E6 in vivo and not in vitro, although all these proteins were demonstrated to form a complex with E6. The mechanisms that control Bax stability have not been elucidated. Some reports indicated the accumulation of Bax in the presence of proteasome inhibitors, implicating the ubiquitin proteasome system in the control of Bax stability (Chang et al., 1998
; Li & Dou, 2000
). Other studies, however, failed to show this effect (Dewson et al., 2003
). Additional data, point to the possibility that Bax has different rates of proteasome dependent protein degradation in different cell types (Marshansky et al., 2001
). Other studies provided evidence on the involvement of a calcium activated calpain in Bax cleavage during apoptosis (Wood et al., 1998
; Gao & Dou, 2000
).
Data from our recent experiments with the proteasome inhibitor MG 132 and stably transduced normal human keratinocytes, failed to reveal the accumulation of Bax either in the vector or E6 cells upon treatment for 4 h with 1040 µM of the inhibitor (data not shown). Further studies are needed to reveal the molecular pathways involved in the control of Bax stability in the presence and absence of E6, particularly during serumcalcium differentiation of PHKs.
Our attempts to detect direct interaction between E6 and Bax, have not been successful, consistent with a previous report (Thomas & Banks, 1999). Possible explanations for the inability to detect complex formation are that E6 binding to Bax is conformation dependent and proteins synthesized in vitro lack this conformation or that cellular components that are unstable or destroyed in the procedure of cell extraction are required for binding. Binding of the Adeno 19K protein to Bax was shown to occur upon apoptotic trigger that induces conformational change in Bax and exposure of the amino-terminal domain (Perez & White, 2000
). Another possibility is that Bax degradation induced by E6 is indirect and mediated through a yet unknown, cellular component that controls Bax stability. E6 could possibly increase the transcription or stability of this putative protein. Using a series of E6 carboxy-terminal deletion mutants, we defined the region spanning aa 132120 as being essential for Bax degradation activity. M132 that lacks 19 aa from the carboxy terminus, was still able to induce Bax degradation, while M120 that lacks 32 aa was completely defective in this function. M132 was shown previously to be completely inactive in p53 degradation (Sherman et al., 1997
), thus further supporting the p53 independent activity of E6 on Bax protein downregulation.
The mutational analyses carried out in the present study showed correlation between the ability of E6 to induce Bax degradation and ability of E6 to reduce Bax-induced apoptosis, suggesting a potential function for E6 in circumventing cellular signals for apoptosis. This apparently is not the only activity of E6 that is required for the formation of differentiation resistant colonies in PHKs (Schlegel et al., 1988; Sherman & Schlegel, 1996
). M132 that showed significant ability to degrade Bax and low, yet consistent activity in protection from Bax-induced apoptosis, was completely defective in its ability to induce differentiation resistant colonies in PHKs. These data suggest that other cellular regulators are involved that contribute to the colony formation activity.
In conclusion, we identified the human pro-apoptotic protein, Bax, as a proteolytic effector of the HPV16 E6 oncoprotein and demonstrated that E6 abrogates Bax function in apoptosis. This provides additional strategy for HPV to prevent cell death in differentiated keratinocytes thereby creating the cellular environment in which HPV late gene expression and assembly can occur.
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ACKNOWLEDGEMENTS |
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Received 19 July 2004;
accepted 9 November 2004.
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