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Journal of Virology, January 2002, p. 220-231, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.220-231.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

Benyue Zhang,1,2 Dan F. Spandau,3 and Ann Roman1,2*

Department of Microbiology and Immunology,1 Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, Indiana 46202-5120,2 Departments of Dermatology and Biochemistry and Molecular Biology3

Received 27 June 2001/ Accepted 26 September 2001


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ABSTRACT
 
The human papillomavirus type 16 (HPV16) E5 protein associates with the epidermal growth factor receptor (EGFR) and enhances the activation of the EGFR after stimulation by EGF in human keratinocytes. Phosphatidylinositol 3-kinase (PI3K) and ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK), two signal molecules downstream of the EGFR, have been recognized as participants in two survival signal pathways in response to stress. The fact that E5 can enhance EGFR activation suggests that E5 might act as a survival factor. To test this hypothesis, the apoptotic response of UV B-irradiated primary keratinocytes infected with either control retrovirus, LXSN, or HPV16 2E5-expressing recombinant retrovirus was quantitated. Under the same conditions, LXSN-infected cells showed extensive apoptosis, while E5-expressing cells demonstrated a significant reduction in UV B-irradiation-induced apoptosis. The E5-mediated protection against apoptosis was blocked by wortmannin and PD98059, specific inhibitors of the PI3K and ERK1/2 MAPK pathways, respectively, suggesting that the PI3K and ERK1/2 MAPK pathways are involved in this process. Western blot analysis showed that Akt (also named protein kinase B), which is a downstream effector of PI3K, and ERK1/2 MAPK were activated by EGF. When cells were stimulated by EGF and irradiated by UV B, the levels of phospho-Akt and phospho-ERK1/2 activated by EGF in E5-expressing cells were about twofold greater than those in LXSN-infected cells. Two other UV-activated stress pathways, p38 and JNK, were activated to the same level during UV B irradiation in both LXSN-infected cells and E5-expressing cells, indicating that E5 protein did not affect these two pathways. After UV B irradiation, p53 was activated in both LXSN-infected cells and E5-expressing cells, and cell cycle analysis showed that nearly all cells in both cell populations were growth arrested. These data suggest that unlike HPV16 E6, which blocks apoptosis by inactivation of p53, HPV16 E5 protects cells from apoptosis by enhancing the PI3K-Akt and ERK1/2 MAPK signal pathways.


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INTRODUCTION
 
Human papillomaviruses (HPVs) have been associated with benign epithelial lesions and cancer development (94). Among all of the HPVs, HPV type 6 (HPV6) and HPV11, which are found mainly in benign warts and papillomas, are classified as low-risk types, whereas HPV16 and HPV18 have been found predominantly in malignant lesions (94). The HPV16 genome encodes three oncoproteins, E5, E6, and E7. The major immortalizing and transforming activity of HPV16 resides in the E6 and E7 proteins (38, 44, 59). The high-risk HPV16 E5 protein has been found to be weakly oncogenic and to potentiate the transforming activity of E7 (49, 86). HPV16 E5 is a hydrophobic protein, consisting of 83 amino acids (10, 36), that is found in the endoplasmic reticulum, Golgi apparatus, endosomal compartment, and also the cell membrane (18) and has the potential to form dimers (45). HPV16 E5 associates with the epidermal growth factor receptor (EGFR) (42); increases the ligand-dependent activation of the EGFR in HaCaT cells, an HPV-negative human keratinocyte cell line (23), and in primary human keratinocytes (78); and enhances the EGFR-mediated ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK) activation in mouse NIH 3T3 cells, human HT1080 cells, and monkey COS 1 cells (22, 35). In COS cells, HPV16 E5 binds the 16-kDa subunit (subunit C) of vacuolar proton-ATPase (18) and perturbs its activity, leading to blocking of the acidification of endosomes in human keratinocytes (77). It was reported recently that HPV16 E5 can block endocytic trafficking from early endosomes to late endosomes (83). The alkalization of endosomes or blockage of endocytic trafficking has been suggested to be responsible for the increased recycling of EGFR to the cell surface, resulting in an enhancement of EGFR-mediated biological activity in the presence of EGF (78, 83).

Apoptosis, or programmed cell death, is a physiological cellular response to environmental stress mediated by, for example, growth factor withdrawal, virus infection, genotoxic agents such as chemotherapeutic drugs, ionizing radiation, and nonionizing radiation (40, 54, 79, 82). Recently progress has been made in elucidating the mechanisms of activation of different proapoptotic pathways induced by, for example, Fas ligand, ceramide, and UV B (16, 19, 34, 47, 60, 61). UV B irradiation has been used to induce apoptosis in human keratinocytes (8, 19). Multiple signal pathways have been reported to be involved in the apoptotic response of cells following exposure to UV, including activation of tumor necrosis factor alpha, CD95 (FAS/APO1), MAPK, and a cascade of the caspase (initiative caspases and effective caspases) family of cysteine proteases (2, 32, 50, 65, 72, 80). The p53 tumor suppressor gene has also been shown to play a key role in the cellular response to UV B irradiation (8, 19).

EGF can protect epithelial cells against Fas-induced apoptosis through an EGF-EGFR-phosphatidylinositol 3-kinase (PI3K)-Akt pathway (34). In response to EGF binding, the EGFR undergoes dimerization and autophosphorylation which activates the receptor tyrosine kinase. PI3K and ERK1/2 MAPK are signaling molecules on two major downstream pathways initiated by the activation of EGFR (11). Akt (also named protein kinase B [PKB]), which is the downstream effector of PI3K, and ERK1/2 have been well recognized as two major survival factors against apoptosis (3, 7, 31, 32, 33, 47, 63, 69, 91). Under stress conditions, PI3K and its putative effector Akt/PKB is down regulated (56, 92, 93), and cells normally show an inactivation of ERK1/2 with activation of JNK and p38 (5).

Since HPV16 E5 can enhance EGFR activation, experiments were designed to test whether HPV16 E5 can block UV B-irradiation-induced apoptosis. The results indicate that HPV16 E5, in the presence of EGF, can act as a stress-resistant factor which keeps the Akt and ERK1/2 levels elevated in UV B-irradiated human keratinocytes. Therefore, E5-expressing cells are protected from UV B-irradiation-induced apoptosis.


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MATERIALS AND METHODS
 
Cell culture. Human foreskin keratinocytes (HFKs) were isolated from neonatal foreskins (67). The foreskin was digested with trypsin-EDTA, and keratinocytes released from the tissue were plated in E medium in the presence of mitomycin C-treated 3T3/J2 fibroblasts (67). At 60% confluency, cells were subcultured in E medium on the feeder cells. At about 80% confluency, the cells were frozen. The frozen cells were subsequently thawed in complete serum-free medium (K-SFM; GIBCO/BRL) supplemented with human recombinant EGF (GIBCO/BRL) and bovine pituitary extract (GIBCO/BRL). PA317 cell lines producing either control virus, LXSN, or HPV16 E5-expressing virus [L(16E5)SN] were kindly provided by Denise Galloway and were grown in Dulbecco modified Eagle medium (GIBCO/BRL) with 10% fetal bovine serum (HyClone).

Retrovirus infection. Third-passage HFKs were grown to about 40% confluency in a 10-cm-diameter dish and infected with 5 ml of the recombinant retrovirus or parental virus (106 virus particles/ml) in the presence of 8 µg of Polybrene per ml. After 6 h the cells were fed with complete K-SFM and kept at 37°C with 5% CO2 for 48 h. The cells were then transferred to four 10-cm-diameter dishes and selected with G418 (200 µg/ml) for 3 to 4 days. Selected cells were expanded and used for experiments, generally when at 80% confluency.

Northern blotting. Total RNAs from uninfected cells and cells infected with either LXSN or L(16E5)SN were extracted using TRI REAGENT (Molecular Research Center, Inc.). Ten micrograms of the RNA was separated on a 7% formaldehyde-1% agarose gel. After electrophoresis, the RNA was transferred to nitrocellulose and probed with [{alpha}-32P]dATP-labeled HPV16 E5 DNA.

UV B irradiation. UV B irradiation of keratinocyte cultures was accomplished using a Philips F20T12/UV B source (wavelength, 270 to 390 nm). The intensity of the radiation from the UV B source was measured prior to each experiment using an IL-1700 radiometer and a SED240 UV B detector (International Light, Newburyport, Mass.). Cells irradiated with UV B were then incubated in the complete K-SFM, except as noted below, at 37°C with 5% CO2 for the indicated times.

TUNEL assay. Nick end labeling of fragmented DNA was performed using the terminal deoxynucleotidyl transferase-mediated fluorescein isothiocyanate (FITC)-dUTP nick end labeling (TUNEL) assay (Cell Death Detection Kit, Fluorescein; Boehringer Mannheim Biochemicals). Infected HFKs (105) were grown on 35-mm-diameter glass-bottomed dishes (MatTek Corporation) coated with type 1 collagen (Sigma). Sixteen hours after being exposed to 0 and 400 J of UV B per m2, cells were air dried, fixed with a freshly prepared paraformaldehyde solution (4% in phosphate-buffered saline [PBS], pH 7.4) for 60 min at room temperature, rinsed once with PBS, and incubated in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate in PBS) for 2 min on ice. The cells were then rinsed twice with PBS and air dried, and 50 µl of TUNEL reaction mixture was added to the cells. The cells were then incubated for 60 min in a humidified chamber at 37°C in the dark, rinsed three times with PBS, and then incubated in 20 µM 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) (Sigma) in PBS. After a further rinse with PBS, the cells were analyzed with a Zeiss epifluorescence microscope equipped with narrow-band-pass DAPI and fluorescein filters. FITC-positive cells are apoptotic cells, and DAPI, which stained all nuclei, was used to see the morphological change of the nucleus and count the total cell number. The percentage of apoptotic cells was calculated by scoring five randomly chosen fields.

Sub-G1 assay. Sixteen hours after UV B irradiation, cells were harvested and fixed in cold 70% ethanol overnight at 4°C. The cells were then washed with 5 ml of PBS and resuspended in 1 ml of PBS containing 50 µg of propidium iodide (PI) (Sigma) per ml and 100 µg of RNase A (Qiagen) per ml. Fluorescence-activated cell sorter (FACS) analysis was performed with a Becton Dickinson FACScan. In some cases the cells were pretreated with 50 µM PD98059 (New England Biolabs) or 1 µM wortmannin (Sigma) for 1 h before irradiation or kept in medium devoid of EGF after irradiation.

Cell cycle analysis. Cells were grown to 80% confluency in 10-cm-diameter dishes and then irradiated with UV B (400 J/m2) and incubated in 37°C with 5% CO2 for 15 h. The cells were then labeled with 10 µM bromodeoxyuridine (BrdU) for 1 h and washed with PBS, harvested, and fixed with cold 70% ethanol. The cells were then incubated in 2 N HCl with 0.5% Triton X-100 to denature the DNA and permeabilize the membranes, neutralized with 0.1 M Na2B4O7, and counted. Cell concentrations were adjusted to 106 cells/100 µl of blocking buffer (1% bovine serum albumin, 0.5% Tween 20 in PBS), and 10 µl of FITC-conjugated anti-BrdU antibody (DAKO) was added per 106 cells. After 30 min, cells were washed with PBS, incubated for 30 min with PI (Sigma) (5 µg/ml in PBS), and analyzed by flow cytometry using the dual-color system.

Western blotting. Cell lysates were made using cell lysis buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA). Protease, phosphatase, and protein kinase inhibitors were added before use: 1 mM phenylmethylsulfonyl fluoride, 40 mM ß-glycerophosphate, 125 µM Na3VO4, 50 mM NaF, 2 µg of leupeptin per ml, 2 µg of aprotinin per ml, 2 µg of pepstatin per ml, and 1 mM dithiothreitol. Protein was quantitated using the Bio-Rad protein assay. Forty micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), the nitrocellulose membrane was incubated with primary antibody in 5% bovine serum albumin in TBST overnight at 4°C, except for antibodies to p53 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), for which the incubation was in 5% nonfat milk in TBST for 1 h at room temperature. The membrane was then washed three times with TBST and incubated with secondary antibody in 5% milk in TBST. After being washed with TBST five times, the membrane was developed with ECL (Amersham Pharmacia). Antibodies to phospho-EGFR (Tyr845), phospho-Akt (Ser473), total Akt, phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-p38 (Thr180/Tyr182), total p38, phospho-JNK1/2 (Thr183/Tyr185), and total JNK1/2 were from New England BioLabs. Antibodies to total EGFR and p53 (DO-1) were from Santa Cruz. Antibody to GAPDH was from Chemikon.


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RESULTS
 
HPV16 E5 protects cells from UV B-irradiation-induced apoptosis. Primary HFKs were infected with either LXSN (control retrovirus) or L(16E5)SN (the recombinant retrovirus encoding HPV16 E5). Retrovirus infection was analyzed by Northern blot hybridization to detect HPV16 E5 transcripts (Fig. 1). The uninfected cells and LXSN-infected cells did not show any E5-specific message, while L(16E5)SN-infected cells expressed an E5-containing mRNA of about 3 kb, the predicted size for a transcript initiated in the retroviral long terminal repeat.



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  		FIG. 1. Northern blot analysis indicates that the E5 gene is expressed. Total cellular RNA (10 µg) from uninfected, LXSN-infected, and L(16E5)SN-infected cells was separated on a 1% formaldehyde gel. (A) The gel was stained with ethidium bromide, showing relatively even loading of the RNA and the positions of 28S RNA and 18S RNA. (B) The Northern blot was hybridized with a radiolabeled HPV16 E5 DNA probe.

To determine whether expression of HPV16 E5 can influence UV B-irradiation-induced apoptosis of HFKs, LXSN-infected and L(16E5)SN-infected keratinocytes were analyzed following nuclear staining with 20 µM DAPI and TUNEL. Cell morphology (phase), nuclear morphology (nuclear staining), and end labeling of DNA breaks (TUNEL) were documented microscopically from the same field (Fig. 2). In the absence of UV B (0 J/m2), LXSN-infected cells and L(16E5)SN-infected cells exhibited little if any evidence of cellular and nuclear morphology changes or TUNEL positivity (Fig. 2)A. Sixteen hours after irradiation with 400 J of UV B per m2, LXSN-infected cells demonstrated cellular morphology changes indicative of apoptosis (e.g., membrane blebbing), DNA condensation, and fragmentation in the nucleus and TUNEL positivity. In E5-expressing cells, the nucleus looked relatively intact and there were few TUNEL-positive cells (Fig. 2B). Five random images were chosen from each set of cells to quantitate the percentage of apoptotic cells, using the TUNEL image to count apoptotic cells and the DAPI image to count total cell number. Three different foreskin keratinocyte populations were analyzed for their responses to UV B irradiation and the effect of HPV16 E5 expression. The results showed that there was a reduction of apoptosis in all three of the HFK populations. The average reduction of apoptosis in E5-expressing cells was 45% ± 5%. The result from a representative HFK population is shown in Fig. 2C, where the percentages of cells undergoing apoptosis were 39% ± 5% in the LXSN-infected cells and 21% ± 3% in the E5-expressing cells.



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  		FIG. 2. TUNEL results show that E5 protects HFKs from UV B-irradiation-induced apoptosis. (A and B) LXSN-infected and L(16E5)SN-infected keratinocytes were grown in complete K-SFM in 35-mm-diameter glass-bottomed dishes and irradiated with 0 (A) and 400 (B) J of UV B per m2, and apoptosis assays were done 16 h later. Images for phase (cellular morphology), DAPI staining (nuclear morphology), and TUNEL labeling were obtained from the same field under the fluorescence microscope. (C) The results of a representative experiment are shown as the average percentage of apoptotic cells ± standard deviation from five randomly chosen fields.

The sub-G1 assay was conducted as a second assay for apoptosis. The apoptotic cells will show a lower level of PI staining, which appears as a peak before the G1 peak, i.e., the sub-G1 peak, using flow cytometry analysis. The percentage of apoptotic cells was calculated as a ratio between the events in the sub-G1 peak and the total events. Consistent with the results shown in Fig. 2, in the absence of UV B irradiation (0 J/m2), both LXSN-infected and L(16E5)SN-infected cells showed a very small sub-G1 peak (less than 2%), suggesting that little apoptosis was occurring in these cells (Fig. 3). When cells were irradiated with 400 J of UV B per m2, a large sub-G1 peak was seen in LXSN-infected cells but not in E5-expressing cells. Quantitation showed that the average percentage of apoptotic cells was 29% in LXSN-infected cells and 9% in E5-expressing cells at 16 h following UV B irradiation (Fig. 4A). These results again suggest that HPV16 E5 protects HFKs from UV B-irradiation-induced apoptosis. The difference in the percentages of apoptotic cells determined by TUNEL and by the sub-G1 assay is probably due to the fact that the TUNEL assay is more sensitive, since it detects DNA breaks in both low-molecular-weight DNA and high-molecular-weight DNA while the sub-G1 assay detects only the loss of low-molecular-weight DNA.



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  		FIG. 3. Sub-G1 assay shows that E5 protects HFKs from UV B-irradiation-induced apoptosis. LXSN-infected and L(16E5)SN-infected keratinocytes were grown in complete K-SFM and irradiated with 0 and 400 J of UV B per m2, and 16 h later the cells were fixed with 70% ethanol at 4°C overnight. Cells were then washed with 5 ml of PBS and resuspended in 1 ml of PBS containing 50 µg of PI per ml and 100 µg of RNase A per ml. FACS analysis was performed with a Becton Dickinson FACScan to evaluate the sub-G1 ratio. The results of a representative experiment are shown.



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  		FIG. 4. Protection against UV B-irradiation-induced apoptosis by HPV16 E5 is EGF dependent and can be blocked by wortmannin and PD98059. Histograms show the percentages of LXSN-infected and L(16E5)SN-infected cells in the sub-G1 peak under various conditions. (A) HFKs were grown and maintained in complete K-SFM after UV B irradiation (0 and 400 J/m2). (B) HFKs were irradiated with 0 and 400 J of UV B per m2 and then incubated in K-SFM without supplementation with EGF. (C) HFKs were pretreated with 1 µM wortmannin (WO) for 1 h and then irradiated with 0 and 400 J of UV B per m2. (D) HFKs were pretreated with 50 µM PD98059 (PD) for 1 h and then irradiated with 0 and 400 J of UV B per m2. Sixteen hours after UV B irradiation, the cells were prepared and analyzed by FACS as described for Fig. 3. The results represent the average ± standard deviation from three experiments.

Protection against UV B-irradiation-induced apoptosis by HPV16 E5 is EGF dependent and can be blocked by wortmannin and PD98059. After being irradiated with 400 J of UV B per m2, both the LXSN-infected and L(16E5)SN-infected cells were incubated with K-SFM devoid of EGF. After 16 h, the sub-G1 assay was done to evaluate the percentage of apoptotic cells. The results show that without EGF, HPV16 E5 no longer protects keratinocytes from UV B-induced apoptosis (Fig. 4B).

Specific inhibitors of the PI3K-Akt and MEK1/2-ERK1/2 MAPK pathways were used to determine whether E5-mediated protection from UV B-irradiation-induced apoptosis required the activation of either PI3K or ERK1/2 MAPK. Wortmannin specifically inhibits PI3K activation, and PD98059 specifically inhibits ERK1/2 MAPK activation (1, 90). The LXSN-infected and L(16E5)SN-infected cells were pretreated with 1 µM wortmannin or 50 µM PD98059 (47) for 1 h and then irradiated with 400 J of UV B per m2. Sixteen hours later, the sub-G1 assay was performed to evaluate the percentage of apoptotic cells following pretreatment with 1 µM wortmannin or 50 µM of PD98059. Both LXSN-infected and L(16E5)SN-infected cells showed extensive apoptosis after UV B irradiation (Fig. 4C and D). These results suggest that the protection generated by HPV16 E5 involves both the PI3K and the ERK1/2 MAPK pathways.

The antiapoptotic signaling pathways downstream of the EGFR are enhanced by HPV16 E5 after UV B irradiation. HPV16 E5 has been reported to increase the activation of the EGFR in primary and immortalized human keratinocytes (23, 78) and to enhance the activation of ERK1/2 MAPK in several permanent cell lines (22, 35). To determine whether retrovirally expressed HPV16 E5 enhanced activation of EGFR in primary foreskin keratinocytes, cells were starved for EGF for 24 h and then stimulated with 5 ng of EGF per ml for 0, 15, 30, or 120 min. Cell lysates were subjected to Western analysis with antibodies to phospho-EGFR, total EGFR, and GAPDH (as a loading control). The results show that the EGFR was phosphorylated rapidly after EGF stimulation in both LXSN-infected and L(16E5)SN-infected cells but that HPV16 E5 strongly enhanced the activation of EGFR in primary human keratinocytes (Fig. 5).



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  		FIG. 5. HPV16 E5 enhances the activation of EGFR in HFKs stimulated by EGF. HFKs were starved for EGF for 24 h and then stimulated with 5 ng of EGF per ml for 0, 15, 30, and 120 min. Cell lysates containing 40 µg of protein were separated by SDS-PAGE on a 10% gel, transferred to nitrocellulose, and analyzed with antibodies to phospho-EGFR (Tyr845), total EGFR, and GAPDH (as a loading control).

To analyze downstream signals of EGFR, LXSN-infected and L(16E5)SN-infected cells were starved for EGF for 24 h, either stimulated or not with 5 ng of EGF per ml for 1 min, and then irradiated with 0 and 400 J of UV B per m2. In parallel, some starved cells were pretreated with 1 µM wortmannin or 50 µM PD98059 for 1 h before being stimulated with EGF and UV B irradiation. All cells were lysed 15 min after UV B irradiation. Western blot analysis was used to determine the levels of phospho-Akt, total Akt, phospho-ERK1/2, and total ERK1/2 (Fig. 6). Densitometric analysis of the Western blot results was done to determine the relative phospho-Akt and phospho-ERK1/2 levels. The phospho-Akt value was normalized to total Akt, and the value for EGF-starved, unirradiated LXSN-infected cells was set to 1.0. In the absence of UV B irradiation, Akt was activated approximately twofold by EGF in LXSN-infected and L(16E5)SN-infected cells. When LXSN-infected and L(16E5)SN-infected cells were starved for EGF and irradiated with UV B, the relative values of phospho-Akt of both cells types were down regulated to similar levels. However, when cells were stimulated with EGF and irradiated with UV B, only L(16E5)SN-infected cells showed the approximately twofold increase in phospho-Akt level activated by EGF. Pretreatment of the EGF-stimulated and UV B-irradiated cells with 1 µM wortmannin, the specific inhibitor of PI3K, nearly completely blocked the activation of Akt, while pretreatment of such cells with PD98059 had no effect on Akt activation. Like the cells without pretreatment, only the PD98059-pretreated, L(16E5)SN-infected cells maintained an elevated level of phospho-Akt (Fig. 6A).



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  		FIG. 6. HPV16 E5 enhances the activation of Akt and ERK1/2 in HFKs after UV B irradiation in the presence of EGF. LXSN-infected and L(16E5)SN-infected cells were grown in K-SFM without EGF for 24 h, either stimulated or not with 5 ng of EGF per ml for 1 min, and then irradiated with 0 and 400 J of UV B per m2. In parallel, some cells were pretreated with 1 µM wortmannin (WO) or 50 µM PD98059 (PD) for 1 h. All cells were lysed 15 min after UV B irradiation. Cell lysates containing 40 µg of protein were separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and analyzed with antibodies to phospho-Akt (p-Akt) and total Akt (A) and phospho-ERK1/2 and total ERK1/2 (B).

The activation of ERK1/2 was similarly quantitated. The value of phospho-ERK1/2 was normalized to total ERK1/2, and the value for EGF-starved, unirradiated LXSN-infected cells was set to 1.0. ERK1/2 was activated approximately 10-fold by EGF in both unirradiated LXSN-infected and L(16E5)SN-infected cells. When cells were starved for EGF and irradiated with UV B, both LXSN-infected and L(16E5)SN-infected cells showed a reduction in phospho-ERK1/2 compared to the unirradiated cells. When cells were stimulated with EGF and irradiated with UV B, the L(16E5)SN-infected cells showed a ninefold increase of the phospho-ERK1/2 level, similar to that seen in the absence of UV B irradiation. In contrast, the increase seen in the LXSN-infected cells was only about fourfold. PD98059, the specific ERK1/2 MAPK inhibitor, nearly completely blocked the activation of ERK1/2, while wortmannin had no effect on ERK1/2 activation. The relative phospho-ERK1/2 levels seen in wortmannin-pretreated cells were similar to those in cells without pretreatment (Fig. 6B). These results show that in the presence of EGF, HPV16 E5 can enhance both Akt and ERK1/2 MAPK activation after UV B irradiation.

HPV16 E5 does not affect the activation of p38 and JNK induced by UV B irradiation. p38 and JNK are two stress-induced MAPKs that are induced by UV B irradiation and can provide an apoptotic signal to cells (15, 43, 48, 84). To determine whether HPV16 E5 might protect cells from apoptosis by inhibiting the activation of either of these kinases, Western blot analysis was conducted on cell lysates from cells treated as described above. No phospho-p38 or phospho-JNK could be seen in cells that were not irradiated with UV B. Both p38 and JNK, as expected, were activated by UV B irradiation (Fig. 7). Phospho-p38 and phospho-JNK increased to similar levels in both LXSN-infected and L(16E5)SN-infected cells, whether or not the cells were stimulated with EGF. These results suggest that HPV16 E5 does not affect p38 and JNK activation.



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  		FIG. 7. p38 and JNK are activated by UV B in both LXSN-infected and L(16E5)SN-infected cells. LXSN-infected and L(16E5)SN-infected cells were starved for EGF for 24 h, either stimulated or not with 5 ng of EGF per ml for 1 min, and then irradiated with 0 and 400 J of UV B per m2. In parallel, some cells were pretreated with 1 µM wortmannin (WO) or 50 µM PD98059 (PD) for 1 h. All cells were lysed 15 min after UV B irradiation. Cell lysates containing 40 µg of protein were separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and analyzed with antibodies to phospho-p38 (p-p38) and total p38 or to phospho-JNK1/2 and total JNK1/2.

HPV16 E5 does not affect the steady-state level of p53 after UV B irradiation. One of the main cellular responses to UV B irradiation is the activation of the gene for the tumor suppressor p53 (12, 37). Once the p53 gene is activated, the p53 protein level is increased and the conformation is modified to be more stable (20, 41, 58). p53 then migrates to the nucleus and interacts with the DNA replication machinery (20) and regulates the transcription of genes involved in both cell cycle arrest (52) and apoptosis (74). Transfection with the E6 or E6/E7 genes of HPV16 in human diploid fibroblasts leads to inactivation of p53 function and significantly increases radioresistance (85). To determine whether the E5 protein of HPV16 can also affect p53 activation, cell lysates were made from LXSN-infected and L(16E5)SN-infected keratinocytes at 0, 2, 5, 15, and 24 h after irradiation with 400 J of UV B per m2 and subjected to Western blot analysis with antibodies to p53 (DO-1). The results show that the p53 level increased gradually after 5 h and reached similar levels at 24 h in LXSN-infected and L(16E5)SN-infected cells (Fig. 8). These results suggest that HPV16 E5 does not affect p53 expression and stability after UV B irradiation.



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  		FIG. 8. p53 is up regulated in both LXSN-infected and L(16E5)SN-infected HFKs after UV B irradiation. LXSN-infected and L(16E5)SN-infected HFKs were grown in complete K-SFM and then irradiated with 400 J of UV B per m2, washed with cold PBS, and lysed at 0, 2, 5, 15, and 24 h after UV B irradiation. Cell lysates containing 40 µg of protein were separated on an SDS-10% polyacrylamide gel, transferred to nitrocellulose, and analyzed with antibodies to p53 (DO-1) and GAPDH (as a loading control).

Both LXSN-infected and L(16E5)SN-infected cells are growth arrested after UV B irradiation. Cells undergo growth arrest and DNA repair or apoptosis after UV B irradiation (19). Cell cycle analysis was done to determine whether HPV16 E5 affects the cell cycle arrest after UV B irradiation. LXSN-infected and L(16E5)SN-infected keratinocytes were grown in complete K-SFM, irradiated with 0 or 400 J of UV B per m2, and 15 h later labeled with BrdU for 1 h. Unirradiated LXSN-infected and L(16E5)SN-infected cells showed 16 and 18% of cells in S phase, respectively (Fig. 9). When irradiated with UV B, both cell populations were growth arrested nearly completely (Fig. 9). This growth arrest is observed for at least 1 week (data not shown). These results suggest that HPV16 E5 does not affect the cell cycle arrest after UV B irradiation.



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  		FIG. 9. Both LXSN-infected and L(16E5)SN-infected cells are growth arrested after UV B irradiation. LXSN-infected and L(16E5)SN-infected HFKs were grown in complete K-SFM, irradiated with UV B (0 and 400 J/m2), and, 15 h later, labeled with BrdU for 1 h and stained with FITC-conjugated anti-BrdU antibody and PI. The percentage of cells in S phase was determined by flow cytometry using the dual-color system.


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DISCUSSION
 
The HPV16 E5 gene encodes a hydrophobic protein located in the membrane of the Golgi apparatus and endoplasmic reticulum as well as in the plasma membrane (18). HPV16 E5 has been reported to associate with the EGFR (42), to increase the activation of the EGFR in an immortalized keratinocyte cell line (HaCaT) (23), and to enhance the EGFR-mediated ERK1/2 MAPK activation in several permanent cell lines (22, 35). Consistent with a previous report (78), we show here that HPV16 E5 also enhances EGFR activation in primary HFKs after stimulation by EGF (Fig. 5).

The EGFR is a 170-kDa transmembrane tyrosine kinase receptor, encoded by the cellular proto-oncogene c-ErbB-1. PI3K and ERK1/2 MAPK are two major signaling effectors downstream of the EGFR (11). Akt is a serine/threonine protein kinase downstream of PI3K. Akt has been shown to play an important role in the inhibition of apoptosis (3, 31, 47, 69, 91) and cell growth (87). The mechanisms underlying how Akt exerts its antiapoptotic effect in cells have attracted much attention. Targets of Akt related to apoptosis include BAD (25, 28), human caspase-9 (14), forkhead transcriptional factors (6, 9, 46, 66), NF-{kappa}B (64, 68), glycogen synthase kinase 3b (21), and CREB (30). ERK1/2 has also been shown to have significant survival functions in some cell types (7, 32, 33, 63). The mechanism by which ERK1/2 can function to protect cells from apoptosis is possibly by phosphorylating BAD and activating Bcl-XL and Bcl-2 (39, 71).

Given the mechanism of action of EGFR and the available information on E5, we developed a model for the mechanism of E5-mediated protection from UV B irradiation-induced apoptosis (Fig. 10). The model suggests that E5 enhances the ability to activate the EGFR and thereby its downstream signals, PI3K-Akt and ERK1/2 MAPK. This, in turn, provides protection from apoptosis. To test this hypothesis, we used an established model system, UV B irradiation, to induce apoptosis in primary HFKs. TUNEL assays, nuclear staining, and sub-G1 assays showed that HPV16 E5 could protect cells from UV B-induced apoptosis (Fig. 2 and 3). In addition, we showed that the ability of HPV16 E5 to protect cells from UV B-induced apoptosis was EGF dependent and could be blocked by wortmannin or PD98059, specific inhibitors of PI3K and ERK1/2 MAPK, respectively (Fig. 4). These data suggest that HPV16 E5-mediated protection involves both PI3K and ERK1/2 MAPK pathways.



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  		FIG. 10. Model for the mechanism by which HPV16 E5 protects HFKs from UV B-irradiation-induced apoptosis. The model is described in the text. The gray bars represent HPV16 E5.

We have examined levels of phospho-Akt and phospho-ERK1/2, activated kinases that provide the two main survival signals in the cells after UV B irradiation. Even though E5 can enhance EGFR activation in unirradiated primary foreskin keratinocytes (Fig. 5), in keratinocytes that were not irradiated by UV B, the LXSN-infected and L(16E5)SN-infected keratinocytes showed similar increases of Akt and ERK1/2 activation after stimulation of EGF. Consistent with our model (Fig. 10), we observed that after UV B irradiation E5-expressing cells, like the unirradiated cells, respond to EGF stimulation with an increase in phospho-Akt and phospho-ERK1/2. In contrast, LXSN-infected cells no longer show this up regulation. The survival of the cells depends on the balance between the survival signal pathways (mediated by PI3K-Akt and ERK1/2 MAPK) and the stress signal pathways (mediated by p38 and JNK) (13). Our results show that levels of activated p38 and JNK were up regulated by UV B irradiation and that HPV16 E5 did not affect p38 and JNK activation. Thus, the enhancement of Akt and ERK1/2 activation stimulated by EGF after UV B irradiation in E5-expressing cells appears to be responsible for protection from UV B-induced apoptosis.

The p53 tumor suppressor gene is activated by UV B irradiation (12, 37), resulting in an increase in the p53 protein level (20, 41, 58). The p53 migrates to the nucleus and interacts with the DNA replication machinery (20) and regulates the transcription of genes involved in both cell cycle arrest (52) and apoptosis (74). Both p53-mediated transcriptional activation and a p53 function that does not require transcriptional activation play roles in apoptosis (51). In keratinocytes, p53 activation is one of the main apoptotic signals in UV B-irradiation-induced apoptosis (19). HPV16 E6 can block apoptosis by binding the p53 protein and targeting it for degradation (70). In contrast, our result shows that E5 does not affect the level of p53 expression (Fig. 8). Preliminary data also indicate that p53 translocates to the nucleus in both LXSN-infected and L(16E5)SN-infected cells following UV B irradiation (data not shown). After irradiation with 400 J of UV B per m2, both control cells and E5-expressing cells were growth arrested (Fig. 9). Thus, E5 does not appear to have an effect on the p53 pathway but does, via the PI3K and ERK1/2 MAPK pathways, alter the induction of apoptosis.

Apoptosis is a physiological mechanism of cell death that occurs during normal development and under certain pathological conditions (40, 54, 79, 82). Apoptosis is generally controlled and plays roles in both embryonic development and tissue homeostasis. Apoptosis also plays a protective role, eliminating cells that might prove harmful if they were to survive, e.g., cells that acquired mutations following irradiation or virally infected cells. During virus infection, host cells undergo apoptosis as a defense mechanism to prevent further spread of the virus in the organism. Some viruses have evolved mechanisms either to block apoptosis, in order to prevent premature death of the host cell and maximize virus progeny from a lytic infection and/or facilitate a persistent infection, or to trigger apoptosis, serving to spread virus progeny to neighboring cells while evading host inflammatory responses (62). Many viral gene products induce apoptosis, including adenovirus E1a (81), human immunodeficiency virus type 1 Tat protein (53), and parvoviruses B19 (57). Some other viral gene products play a role in blocking apoptosis during viral infection, including adenovirus E1B 19- and 55-kDa proteins (17, 26), simian virus 40 large T antigen (27), bovine papillomavirus E5 (62), and polyomavirus middle T antigen (24). Some of the viral proteins block apoptosis by interacting with p53, and some seem to block apoptosis through p53-independent pathways.

In HPVs, at least two proteins can induce apoptosis. The E7 protein of HPV16 sensitizes primary human keratinocytes to apoptosis (76). The E2 protein has also been shown to induce apoptosis (29, 88). The E6 protein of HPVs, in contrast, blocks apoptosis by binding p53 and targeting this tumor suppressor for proteolysis via the ubiquitin pathway (70, 89). Our finding that HPV16 E5 protects primary human keratinocytes from UV B-induced apoptosis suggests that E5 might play a role in the life cycle of HPV16 as an antiapoptotic factor. Blocking of apoptosis by E5 and E6 can inhibit the premature death of host cells and potentiate a maximum infection. The E5 protein is predominantly located in the basal and granular layers, with the highest level found to be coincidental with the onset of virion morphogenesis (55). The most abundant mRNA transcripts in low-grade cervical intraepithelial neoplasia are E5 and E4 (75). The E5 gene is often deleted from the integrated viral genome in cervical cancers (4, 73). Taken together, these findings suggest that expression of E5 may also have an important function in the early neoplastic changes of infected epithelium (45, 75). Thus, it is reasonable to postulate that E5, by activating effectors on survival pathways in primary human keratinocytes, cooperates with E6 and E7, the main oncogenes of HPV16, to promote oncogenesis.


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ACKNOWLEDGMENTS
 
We thank Denise Galloway for generously providing the LXSN-PA317 and L(16E5)SN-PA317 cell lines and Darl Swartz (Department of Anatomy and Cell Biology) for his guidance on all of the immunofluorescence experiments and use of the Zeiss fluorescence microscope in his laboratory. We also thank Harikrishna Nakshatri for his critical review of the manuscript.

This project was supported by grants from the Lilly Center for Women’s Health, the Phi Beta Psi Sorority, and the Walther Cancer Institute.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120. Phone: (317) 274-7275. Fax: (317) 274-4090. Email: aroman{at}iupui.edu. Back


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Journal of Virology, January 2002, p. 220-231, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.220-231.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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