Human Papillomavirus E7 Oncoprotein Dysregulates Steroid Receptor Coactivator 1 Localization and Function
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《病菌学杂志》
The Channing Laboratory, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
High-risk human papillomaviruses (HPVs) are present in virtually all cervical carcinomas. However, the majority of women infected with high-risk HPVs do not develop cervical cancer. Therefore, cofactors must contribute to the development and progression of cervical cancer. Although numerous studies have implicated steroid hormones as cofactors in the initiation and progression of cervical neoplasia, the molecular mechanisms by which they contribute to cervical carcinogenesis are currently unknown. These observations led us to investigate a newly discovered association of the high-risk HPV type 16 (HPV16) E7 oncoprotein with steroid receptor coactivator 1 (SRC-1), an essential component of steroid hormone signaling. HPV16 E7 has been previously reported to interact with p300 and p300/CBP-associated factor (PCAF), members of some SRC-1 transcriptional complexes. We demonstrate here that HPV16 E7 associates in vivo and in vitro with SRC-1 independently of p300 and PCAF. Luciferase reporter constructs under the control of either the interleukin-8 promoter or a promoter containing multimerized synthetic estrogen response elements were used to determine the effect of high- and low-risk HPV E7 expression on SRC-1-mediated transcription. In addition, histone acetyltransferase (HAT) assays were performed to determine the effect of HPV E7 on SRC-1-associated HAT activity. These experiments reveal that HPV16 E7 expression down-regulates SRC-1-mediated transcription and SRC-1-associated HAT activity. SRC-1 localization experiments show that SRC-1 is relocalized to the cytoplasm in the presence of high- and low-risk HPV E7 proteins. Our data suggest that HPV E7 proteins dysregulate hormone-dependent gene expression by association with and relocalization of SRC-1. Dysregulation of SRC-1 localization and function by HPV E7 may provide insight into the molecular mechanisms by which steroid hormones act as cofactors in the induction and progression of cervical neoplasia.
INTRODUCTION
Human papillomaviruses (HPVs) are responsible for benign and malignant epithelial lesions such as common warts, genital warts, and cervical cancer. HPVs are icosahedral viruses that contain circular 8-kb double-stranded DNA genomes and are strictly epitheliotropic (reviewed in reference 44). Over 200 different HPVs have been characterized and are classified as mucosal or cutaneous. The majority of HPVs are low risk and usually result only in benign warts, while infections with high-risk types are associated with epithelial lesions that have a propensity for malignant progression in the oral cavity, the anogenital tract, and, most notably, the cervix (9, 14, 23). High-risk HPVs are present in nearly all cervical carcinomas, the second most common malignancy in women worldwide (reviewed in reference 45). HPV type 16 (HPV16) is by far the most prevalent of the high-risk types, followed by HPV types 18, 31, and others (reviewed in reference 46). Although virtually all cervical carcinomas contain high-risk HPVs, only a small fraction of women infected with high-risk HPVs will develop cervical cancer. Therefore, in addition to HPV infection, cofactors must contribute to the development and progression of cervical cancer.
Numerous epidemiological studies implicate steroid hormones as cofactors in the initiation and progression of cervical neoplasia (reviewed in reference 31). A large multinational WHO collaborative study has demonstrated an increased risk of cervical cancer with steroid hormone use (2). Increased duration of oral contraceptive use was also reported to increase the relative risk of adenocarcinoma in situ of the cervix and contribute to the pathogenesis of these tumors in women (27). In another study controlled for social, sexual, and cervical screening variables, the incidence of precursor and invasive lesions was highest among long-term oral contraceptive users (39). A recent report evaluated the role of steroid hormones in association with HPV positivity in cervical carcinogenesis; the risk of cervical carcinoma was increased up to fourfold in HPV-positive women with histories of long-term use of oral contraceptives (32).
Experimental evidence has also suggested a link between steroid hormones, HPV, and cervical cancer. Patterns of steroid hormone receptor expression are reported to be altered in HPV-associated lesions (12, 30). Additionally, HPV gene expression can be transcriptionally modulated by steroid hormones through response elements located in the upstream regulatory region of the viral genome (36, 40); estrogen has been shown to stimulate transcription of HPV16 in SiHa cervical carcinoma cells (29), and treatment with progesterone was shown to alter growth and viral gene expression in HPV16-positive CaSki cervical cancer cells and HPV16-containing Hep2 cells (43). However, the strongest experimental evidence that steroid hormones act as cofactors in cervical cancer has resulted from studies using a transgenic mouse model where HPV16 is expressed under the K14 promoter. Arbeit et al. showed that none of the K14-HPV16 transgenic mice spontaneously developed cervical cancer (irrespective of pregnancy number), whereas 100% of HPV16 transgenic mice developed cervical cancer upon long-term exposure to low doses of estrogen (3). Interestingly, in this model, the HPV16 oncogenes were expressed under the control of a keratin promoter, implying that synergy between steroid hormones and HPV in cervical cancer cannot be explained solely by an increase in viral gene expression. Despite the evidence implicating steroid hormones as cofactors in cervical carcinogenesis, the molecular pathways that link steroid hormones and HPV in the initiation and/or progression of cervical cancer remain undefined.
A possible candidate linking steroid hormones and HPV in cervical cancer is steroid receptor coactivator 1 (SRC-1), a component of steroid hormone signaling. We report that the HPV16 E7 oncoprotein associates with SRC-1, a member of the p160 steroid receptor coactivator family required for full transcriptional activity of the steroid receptor superfamily (35). Regulation of hormone-dependent gene expression by SRCs has been an area of intense study and has proved to be quite complex. SRCs, including SRC-1, modulate target gene expression by interaction with ligand-bound nuclear receptors, recruitment of histone acetyltransferases (HATs) and methylases, subsequent chromatin remodeling, and assembly of transcription factors (reviewed in reference 41). Selective recruitment of SRCs by different nuclear receptors can form specific, unique coactivator complexes to confer specificity to transcriptional regulation (8, 25). Additionally, functionally distinct SRC-1 isoforms likely play different roles in estrogen receptor-mediated transcription (21).
There is evidence to suggest that viral proteins can dysregulate SRC-1 transcriptional complexes, which have been shown to include the HATs p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF). SRC-1 is a very weak HAT, and SRC-1-associated HAT activity is derived largely by recruitment of other HATs, such as p300 and PCAF (reviewed in reference 41). Adenovirus E1A, structurally and functionally similar to HPV E7, was shown to bind CBP and prevent assembly of an SRC-1 coactivation complex, a critical step in hormone-dependent gene expression (42). More recently, p300, CBP, and PCAF were shown to be targets for the E1A proteins of all six adenovirus subgroups, demonstrating the importance of these interactions (38). Interestingly, HPV E7s from both high- and low-risk HPV types have been previously reported to interact with p300 and PCAF (4, 5, 19). However, our results suggest that HPV E7 associates directly with SRC-1, independently of p300 and PCAF, and dysregulates the function of SRC-1 transcriptional complexes. We also provide evidence that HPV E7 retains or relocalizes SRC-1 to the cytoplasm, removing SRC-1 from its transcriptional targets.
With the present study, we have begun to explore the molecular mechanisms by which steroid hormones act as cofactors with HPV in cervical carcinogenesis. In this report, we provide evidence for the dysregulation of SRC-1 localization and function by the HPV E7 oncoprotein.
MATERIALS AND METHODS
Cell lines. HeLa (cervical carcinoma, HPV18-positive) cells stably expressing C-terminally Flag- and hemagglutinin (HA)-tagged HPV16 E7 (CE7) have been previously described (20). RKO (colorectal carcinoma) and U2OS (osteosarcoma) cell lines were maintained in McCoy's medium supplemented with 10% calf serum and 1% penicillin (Pen)-streptomycin (Strep). RKO cells stably expressing the pcDNA3 control vector or HPV16 E7 (22) and U2OS cells stably expressing pcDNA3 or C-terminal Flag and CE7, derived by PCR cloning of the CE7 fragment from POZ-CE7 (20) into the pCMV BamNeo vector, were maintained in McCoy's medium supplemented with 10% calf serum, 1% Pen-Strep, and 0.5 mg/ml G418. C33A (cervical carcinoma, HPV-negative) and CaSki (cervical carcinoma, HPV16-positive) cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% calf serum and 1% Pen-Strep. Normal human foreskin keratinocytes (HFKs) were obtained from neonatal foreskins and maintained in keratinocyte-SFM plus supplements (Gibco), 1% Pen-Strep, 0.1% gentamicin, and 0.2% amphotericin B (Fungizone). Briefly, human foreskins (from two to seven individuals per set of foreskins) were obtained and cut into four or five pieces each. Each piece was floated (epidermis side up) in a dispase solution (250 mg/10 ml phosphate-buffered saline [PBS]) overnight at 4°C. The epidermis was separated from the dermis, cut into small pieces, and exposed to trypsin at 37°C for 10 min. After trypsinization, the keratinocytes were pelleted and plated. Trypsinization and plating were sequentially performed three times on each set of foreskins.
CoIPs, Western immunoblot analysis, and cell fractionation. Cells at 80 to 90% confluence (four to six 15-cm dishes) were scraped in PBS, pelleted, and resuspended in 3 volumes of 0.3B lysis/immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 8.0, 0.3 M KCl, 5 mM MgCl2, 10% glycerol, 0.1% Tween 20, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride). Lysates were rotated for 30 min at 4°C after one freeze-thaw cycle and then cleared by centrifugation (30 min, 16,000 x g). Two to 5 μg of each antibody (-SRC-1, Upstate clone 1135; -E7, Zymed C-8; -FLAG agarose, Sigma M-2; -p300, Upstate clone RW128) was coupled to 50 μl protein G (Promega) slurry (equilibrated with 0.3B buffer) by gently rotating at 4°C for 45 min. Coupled antibody was added to 10 to 20 mg cleared lysate, and the mixture was gently rotated at 4°C for 4 h. Coimmunoprecipitations (coIPs) were washed with 0.3B lysis/IP buffer three times and resuspended in loading buffer. Samples were resolved on a 10 to 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subsequently Western blotted. -PCAF, Santa Cruz E-8, and the above-mentioned antibodies were used for detection.
Nuclear and cytoplasmic fractions of cells were obtained using a nondetergent method on ice. Cells were pelleted, washed in PBS, washed in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride), and then incubated in 4 volumes of hypotonic buffer for 10 min. Cellular membranes were disrupted with 10 strokes of a mortar and tight pestle. Intact nuclei were pelleted at 1,500 x g for 5 min at 4°C. Nuclei were then lysed in 0.3B buffer with one freeze-thaw cycle. In order to visualize p300 by Western analysis in nuclear and cytoplasmic fractions, 25 mM N-ethylmaleimide was added to the lysis and hypotonic buffers. CoIPs were performed as described above on nuclear and cytoplasmic fractions.
In vitro transcription/translation and GST pull-downs. SRC-1 was in vitro transcribed and translated per kit instructions (TNT T7-coupled wheat germ extract system; Promega) using a Transcend nonradioactive translation detection system (Promega). In this system, biotinylated lysine residues are incorporated into nascent proteins during translation, allowing for nonradioactive proteins synthesized in vitro to be visualized by binding streptavidin-horseradish peroxidase. Glutathione S-transferase (GST) fusion proteins were made and purified per kit instructions (MagneGST protein purification system; Promega). Pull-down assays were performed using conditions the same as those described above for coIPs.
Transfections and luciferase reporter assays. Cells were cotransfected using either TransFast (Promega) or Fugene (Roche) per the manufacturer instructions. Luciferase reporters (500 ng) under the control of the interleukin-8 (IL-8) promoter (provided by A. Bonni) or of three estrogen response element (ERE) repeats (3x EREs) (Panomics) were cotransfected with pcDNA3 (vector only) or with 100 ng of plasmids expressing HPV16 CE7, HPV18 E7, HPV45 E7, HPV1 E7, HPV6b E7, and 40 ng of the control Renilla luciferase plasmid (see Fig. 3 and 4). One hundred nanograms of SRC-1 (provided by M. Brown and B. O'Malley) was also used for cotransfection (see Fig. 4). Forty-eight to 72 h posttransfection, luciferase activity was determined using a Dual Luciferase reporter assay system (Promega). One hundred fifty nanograms of pcDNA3 or plasmids expressing HPV16 E7, HPV1 E7, and HPV6b E7 was transfected into RKO cells using Fugene (Roche), and cells were subsequently used for immunofluorescence (see Fig. 6).
HAT assay. CoIPs were performed as described above using an -SRC-1 antibody (clone 1135; Upstate). Beads containing SRC-1 and its associated proteins were washed three times in 0.3B buffer and resuspended in a HAT activity buffer from a nonradioactive HAT assay kit (Upstate). Relative HAT activity was determined for each sample according to kit guidelines.
Immunofluorescence. Each cell line was grown to 60 to 70% confluence on glass coverslips and fixed in 4% paraformaldehyde for 10 min. Cells were rinsed with PBS and then permeabilized for 10 min with PBS-0.1% Triton X-100. After two PBS washes, cells were blocked with 5% bovine serum albumin. Staining for SRC-1 was performed using -SRC-1 antibodies (Santa Cruz C-20 and Upstate clone 1135) at 4°C for 8 h. Cells were washed two times with PBS and then incubated with secondary fluorescein isothiocyanate-conjugated -mouse (1:1,000) or -rabbit (1:2,000) antibodies (Jackson Labs) for at 37°C for 1 h. Cells were rinsed three times with PBS and counterstained with Hoechst. Coverslips were mounted and analyzed.
RESULTS
HPV16 E7 associates with SRC-1, p300, and PCAF. SRC-1 was initially identified as a potential HPV16 E7-interacting protein by tandem affinity purification (TAP) assay. A C-terminally double-tagged HPV16 E7 (CE7) was expressed in HeLa cells, sequential HA and FLAG coIPs were performed, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and E7-interacting bands were sequenced by mass spectrometry (20). Due to the evidence suggesting that steroid hormones act as cofactors with HPV in the initiation and progression of cervical neoplasia, we sought to verify and examine the interaction between E7 and SRC-1, an essential component of steroid hormone signaling.
Cell lysates from RKO (colorectal carcinoma) and U2OS (osteosarcoma) cells expressing HPV16 E7 or HPV16 CE7 were used to perform coIPs. Both tagged (CE7) and untagged (E7) exogenously expressed HPV16 E7 proteins were found to associate with endogenous SRC-1 (Fig. 1A, left panel). Lysates prepared from C33A (HPV-negative) and CaSki (HPV16-positive) human cervical carcinoma lines were used in coIPs to determine whether endogenous levels of HPV16 E7 could interact with SRC-1. Endogenous E7 and SRC-1 do in fact associate in the CaSki cervical cancer cell line, as detected by both -SRC-1 and -E7 coIPs (Fig. 1A, right panel).
Previous studies have reported that HPV16 E7 can associate with p300 and PCAF (4, 5, 19). To determine whether E7 could interact with these proteins, we performed coIPs under conditions the same as those used for the detection of the SRC-1 interaction. These experiments clearly showed that endogenous levels of both p300 and PCAF were found to associate with E7 (Fig. 1B). These results demonstrate that HPV16 E7 coprecipitates SRC-1, p300, and PCAF. Since SRC-1 has been shown to recruit p300 and PCAF and since E7 has been detected in association with each of the three members of the SRC-1-containing transcriptional complex, E7's interaction with members of this complex may serve to dysregulate/subvert SRC-1 function.
Association of HPV16 E7 with SRC-1 does not require p300 or PCAF. In light of the results that E7 could associate with SRC-1, p300, and PCAF, we wanted to determine whether p300 or PCAF was required for E7's association with SRC-1. After examination of p300 and PCAF levels in a number of cell lines, we found that the RKO cell line contained nearly undetectable levels of p300 (Fig. 2A, lanes 1 and 2), while the CaSki cell line contained reduced levels of PCAF (Fig. 2A, lane 5). Because these cell lines represent "natural knock-downs" of p300 and PCAF, we used them to perform SRC-1 coIPs to determine association with E7. Our results show that SRC-1 efficiently coprecipitates E7 in RKO cell lysates (Fig. 1A, left panel) in the absence of detectable association with p300 (Fig. 2A, lane 7). Similarly, SRC-1 coprecipitates E7 in CaSki cells (Fig. 1A, right panel) in the absence of detectable association with PCAF (Fig. 2A, lane 10). These results suggest that the association of E7 with SRC-1 does not critically depend on p300 or PCAF association.
To further explore the association between HPV16 E7 and SRC-1, we performed in vitro binding experiments using purified GST, GST-HPV16 E7, or various GST-HPV16 E7 mutants and SRC-1 produced in wheat germ extract (Promega) by in vitro transcription/translation. By producing SRC-1 with wheat germ extract, we reduced the presence of proteins with significant mammalian homology that may be required for SRC-1/E7 binding. SRC-1 does associate with wild-type E7, as well as with all E7 mutants tested, in these in vitro binding experiments (Fig. 2B). These results indicate that multiple HPV16 E7 domains may be necessary for association with SRC-1. The interaction detected between SRC-1 and E7 in vitro further supports the likelihood that the interaction is independent of p300 and PCAF. Taken together, these results demonstrate that HPV16 E7 associates with SRC-1 and the SRC-1 transcriptional complex members p300 and PCAF; however, the association of E7 with SRC-1 does not appear to require p300 or PCAF and may be direct.
HPV E7 dysregulates SRC-1-mediated transcription. Since SRC-1 is a component of steroid hormone signaling that regulates hormone-dependent gene expression, and given our results that E7 can associate with SRC-1, we sought to determine whether expression of HPV E7 could affect SRC-1-mediated transcription. SRC-1 is known to mediate signaling through EREs, and the IL-8 promoter has been previously shown to be modulated by expression of the HPV16 E7 oncoprotein (19). Luciferase reporters under control of either the IL-8 promoter or an artificial promoter containing 3x EREs were cotransfected with a Renilla control vector into the C33A and CaSki human cervical cancer cell lines to determine the effects of endogenously expressed HPV16 E7 on SRC-1-mediated transcription. Dual-luciferase activity was determined in triplicate for each cell line and reporter construct, and relative light unit values were normalized to that for the Renilla control vector. Luciferase activity for each was expressed as a percentage of the value for the control (C33A cells that do not contain HPV16) (Fig. 3). SRC-1-mediated transcription was dramatically decreased in the HPV16-containing CaSki cell line for both the 3x ERE (Fig. 3A) and the IL-8 (Fig. 3B) reporters. To determine whether the effect of HPV16 on SRC-1-mediated transcription is due to E7 expression, both reporters were also transfected along with the vector only or with HPV16 E7 plasmids into RKO, C33A, and HFKs. Expression of HPV16 E7 alone did likewise significantly decrease SRC-1-mediated transcription in each cell line tested for both the 3x ERE (Fig. 3A) and IL-8 (Fig. 3B) reporters. SRC-1-mediated transcription determined using the 3x ERE reporter in HFKs was a notable exception to these results (data not shown). This was an anticipated result and is likely due to the hormonal receptor differences in HFKs.
We also sought to functionally map the E7-interacting domain by using the above-described reporter assay with the IL-8 reporter and plasmids expressing HPV16 E7 mutants in C33A cells. The effects of E7 on SRC-1-mediated transcription did not map to a specific site on E7, as all the mutants tested decreased SRC-1-mediated transcription similarly to wild-type E7 (Fig. 3C). However, these reporter assay results obtained using mutants of E7 do agree with our in vitro binding data (Fig. 2B). All E7 mutants tested were able to bind SRC-1 and decrease SRC-1-mediated transcription. Since these mutants are inclusive of the domains required for E7's interaction with p300 and PCAF (4, 5, 19), these results provide further support to the assertion that the interaction between E7 and SRC-1 and the subsequent effects of E7 on SRC-1's transcriptional activities are independent of p300 and PCAF.
Using this assay as a biological readout, we wanted to determine the effects of high- and low-risk HPV E7s on SRC-1-mediated transcription. The assay was performed as described above by use of the IL-8 reporter in the C33A cell line. Expression of both high-risk (HPV types 16, 18, and 45) and low-risk (HPV types 1 and 6b) E7s decreased SRC-1-mediated transcription (Fig. 4). Ensuring that E7's effect on transcription was indeed SRC-1 mediated, transcriptional repression induced by E7 expression was relieved upon SRC-1 overexpression (Fig. 4). These results illustrate that expression of both high- and low-risk HPV E7s down-regulates SRC-1-mediated transcription. Moreover, these results demonstrate that SRC-1 is the rate-limiting factor for E7's effect on SRC-1-mediated transcription.
HPV16 E7 expression decreases SRC-1-associated HAT activity. SRC-1 recruits HATs in order to modify chromatin and exert transcriptional control. Given our results demonstrating that HPV E7 can dysregulate SRC-1-mediated transcription, we wanted to determine if SRC-1-associated HAT activity was affected by expression of HPV E7. To examine this, we performed SRC-1 coIPs with cells expressing HPV16 E7 and with those not expressing HPV16 E7. We then used the immunoprecipitates in a nonradioactive HAT assay. SRC-1-associated HAT activity was measured and expressed as a percentage of the value for the control (cells not expressing HPV16 E7) (Fig. 5). In CaSki cells expressing endogenous HPV16 E7 and upon expression of HPV16 E7 alone (C33A and RKO cells), SRC-1-associated HAT activity was reduced by more than 50% (Fig. 5). The results shown are from experiments using whole-cell lysates, although experiments done with nuclear extracts yielded similar results (data not shown). Down-regulation of SRC-1-mediated transcription by HPV E7 is therefore likely a result of a decreased HAT activity. These results provide further support to the observation that SRC-1-mediated transcription is dysregulated by expression of HPV E7 and suggest that HPV E7 dysregulates SRC-1-mediated gene expression by interfering with the activity of SRC-1 transcriptional complexes.
HPV16 E7 associates with SRC-1 in the cytoplasm. Although all known functions of SRC-1 are thought to take place in the nuclear compartment, a recent study using live-cell imaging demonstrated that SRC-1 can shuttle between the nucleus and the cytoplasm (1). Additionally, our analysis of fractionations from many cell lines expressing HPV16 E7 reveals that E7 does exist in the cytoplasmic compartment (Fig. 6D and data not shown), and this has also been reported by others (11). Finally, since the SRC-1/E7 interaction was initially identified in the cytoplasmic fraction by our TAP assay data, we sought to conclusively determine the localization of the SRC-1/E7 interaction. Cytoplasmic and nuclear fractions were obtained from HeLa cells expressing HPV16 CE7, and coIPs were performed on both fractions using an -FLAG antibody. Association between SRC-1 and E7 was detected in the cytoplasmic fraction of cells (Fig. 6A) but not in the nuclear fraction (data not shown).
HPV E7 expression relocalizes SRC-1 to the cytoplasm. Our results demonstrating that association between HPV16 E7 and SRC-1 occurs in the cytoplasmic fraction of cells were initially puzzling, especially given the experimental data demonstrating that E7 expression affects the nuclear activities of SRC-1 (Fig. 3, 4, and 5). These results led us to question whether E7 expression could affect SRC-1 subcellular localization. To address this question, we used immunofluorescence to visualize SRC-1 localization in HPV-negative (C33A) cells, and we compared this localization to that seen for cells containing endogenous HPV16 (CaSki and SiHa) and HPV18 (HeLa). Localization of SRC-1 is primarily nuclear without HPV expression; however, in cells expressing high-risk HPVs, SRC-1 is dramatically, but not completely, relocalized to the cytoplasm of cells (Fig. 6B, top panel).
Comparison of RKO cells expressing vector only or HPV16 E7 revealed that E7 alone relocalizes SRC-1, demonstrating that this relocalization effect is not due to expression of other viral proteins (Fig. 6C, top panel). The low-risk HPV1 and HPV6b E7 proteins also exhibit the ability to relocalize SRC-1 to the cytoplasm (Fig. 6C, top panel).
Although our TAP data revealed interaction of E7 and SRC-1 in the cytoplasmic compartment, the TAP analysis provided no evidence of association with p300 or PCAF in the cytoplasm. To determine whether HPV E7 also relocalizes these known members of the SRC-1 transcriptional complex to the cytoplasm, we performed cellular fractionation of RKO cells expressing HPV16 E7. Western analysis was performed on both the nuclear and cytoplasmic fractions. p300 and PCAF were detected only in the nuclear fractions of cells expressing E7, although the majority of E7 detected was in the cytoplasmic fraction (Fig. 6D). Thus, our data demonstrate that E7 does not relocalize p300 and PCAF to the cytoplasm; rather, E7 specifically targets and relocates SRC-1. These results demonstrate that in the presence of high- or low-risk HPV E7, SRC-1 is relocalized to the cytoplasm. These results complement the finding that both high- and low-risk HPV E7s can dysregulate SRC-1-mediated transcription (Fig. 3 and 4). Collectively, our findings suggest a model whereby HPV E7 dysregulates SRC-1 function by relocalization to the cytoplasm, thereby decreasing the levels of transcriptionally active, nuclear SRC-1 (Fig. 7) independently of p300 and PCAF.
DISCUSSION
The studies reported here were initiated to explore the molecular mechanism(s) by which steroid hormones may contribute to HPV-associated cervical neoplasia. These studies yielded the following important observations. (i) HPV16 E7 associates with a component of steroid hormone signaling, SRC-1 (Fig. 1). (ii) E7's association with SRC-1 is not strictly dependent on the SRC-1 complex members p300 and PCAF (Fig. 2). (iii) Expression of high- and low-risk HPV E7 proteins dysregulates SRC-1-mediated transcription (Fig. 3 and 4). (iv) SRC-1-associated HAT activity decreases upon HPV16 E7 expression (Fig. 5). (v) High- and low-risk HPV E7s relocalize SRC-1 to the cytoplasm (Fig. 6). Overall, these data support a model whereby either HPV E7 associates with SRC-1 and relocalizes it to the cytoplasm, or HPV E7 associates with a cytoplasmic pool of SRC-1 and inhibits its nuclear translocation. This association with HPV E7, which likely occurs independently of p300 and PCAF, leads to SRC-1 retention in the cytoplasm, resulting in displacement of SRC-1 from its transcriptional targets. Consequently, SRC-1 nuclear transcriptional activity is dysregulated by HPV E7 (Fig. 7).
Expression of the high-risk HPV E6/E7 oncogenes is necessary and sufficient to immortalize primary human keratinocytes (17, 33), and continued expression of the E6 and E7 oncoproteins is essential to maintain a transformed phenotype in cervical cancer cells (15). HPV E7s from both high- and low-risk HPV types have been shown to interact with p300 (5) and PCAF (19). Association of E7 with PCAF was found to be necessary for down-regulation of a target promoter (19) and was also shown to reduce HAT activity of PCAF in vitro (4). Additional reports have demonstrated that the ability of E7 to associate with histone-modifying enzymes contributes to promotion of cell growth (6), deregulates transcriptional silencing (24), extends the life span of keratinocytes, and contributes to stable maintenance of viral genomes through association with histone deacetylases (26). Together, these studies establish that HPV E7 subverts the normal activity of chromatin-remodeling transcriptional complexes. We discovered that HPV16 E7 associates with SRC-1 (Fig. 1), a component of chromatin-remodeling transcriptional complexes, independently of p300 and PCAF (Fig. 2). Our results demonstrate that E7 expression induces significant relocalization of SRC-1 to the cytoplasm (Fig. 6B and C). Conversely, the localization of PCAF and p300 does not change upon E7 expression (Fig. 6D). Therefore, it is likely that the composition of the SRC-1 transcriptional complex is disrupted upon binding of E7 to SRC-1. These findings suggest that dysregulation of the SRC-1 transcriptional complex results from the direct association between E7 and SRC-1.
Perhaps one of the most exciting observations in this report is the relocalization of SRC-1 by HPV E7 to the cytoplasm (Fig. 6B and C). Although steroid hormone receptors are mainly localized in the nucleus, a recent live-cell imaging study using fluorescently tagged SRC-1 has demonstrated that SRC-1 is imported into the nucleus after synthesis in the cytoplasm and then exported back into the cytoplasm, possibly for degradation (1). That study also suggests nuclear export may be dependent on the interaction of SRC-1 with other proteins, which could regulate steroid hormone signaling. Similarly, association of HPV E7 with SRC-1 initially in the nucleus may result in such nuclear export. Putative nuclear export signals, which are not exposed in the nuclear conformation of SRC-1, may become exposed upon conformational changes induced by interaction with other proteins, such as the E7 oncoprotein. The mechanism by which HPV E7 relocalizes SRC-1 to the cytoplasm is the focus of ongoing studies. Interestingly, nuclear accumulation of E2F4 in response to HPV E7 expression has been reported (37). These observations suggest that the HPV E7 oncoprotein may alter the normal subcellular localization of host proteins as a general mechanism to promote oncogenesis.
Association of HPV16 E7 and SRC-1 was detected only in the cytoplasmic compartment of cells (Fig. 6A). However, we cannot rule out the possibility that E7 may also play a role in SRC-1-mediated transcription by interaction with SRC-1 in the nucleus. Given that we typically observe lower levels of E7 in the nucleus than in the cytoplasm and that E7 expression relocalizes SRC-1 to the cytoplasm, an association between E7 and SRC-1 in the nucleus might simply be below the level of detection. Nevertheless, the effects of E7 expression on SRC-1-associated HAT activity (Fig. 5) and SRC-1-mediated transcription (Fig. 3 and 4) are likely due to the relocalization of SRC-1 to the cytoplasm (Fig. 6B and C). In addition to reducing the nuclear activities of SRC-1 by removing it from its nuclear targets, E7 may serve to disrupt an as-yet-undiscovered cytoplasmic role of SRC-1. The observation that SRC-1 is not completely relocalized to the cytoplasm in E7-expressing cells is also very interesting. SRC-1's regulation of gene expression is complex, and several differentially spliced SRC-1 transcripts have been reported to function differently under various conditions (7, 10, 16, 18, 28). It is possible that only a specific isoform of SRC-1 is relocalized upon E7 expression, resulting in a highly specific mechanism that dysregulates SRC-1 function.
We did not detect an interaction between low-risk HPV E7s and SRC-1 by coimmunoprecipitation (data not shown). This does not rule out the possibility that low-risk HPV E7 proteins may associate with SRC-1; however, the efficiency of the interaction may be below the level of detection in our assays. Other cellular proteins that efficiently associate with high-risk HPV E7, most notably pRb, have been demonstrated to bind low-risk HPV E7 proteins at a decreased efficiency. The findings that both high- and low-risk HPV E7 proteins dysregulate SRC-1 localization (Fig. 6) and function (Fig. 4) demonstrate that this particular function of E7 may be conserved and suggest that dysregulation of SRC-1 localization and function by E7 may be of general importance for the biology of HPVs. This hypothesis is currently under investigation in our laboratory.
Because of the synergy between HPV and steroid hormones in cervical cancer, down-regulation of SRC-1 function by HPV E7 may seem counterintuitive initially. Hormone-dependent gene expression, however, is a complex, exquisitely regulated process. In a recent report demonstrating the complexity of SRC-mediated gene regulation, SRC family members and corepressors (histone deacetylases, silencing mediator of retinoid and thyroid receptors [SMRT] and NcoR) were recently shown to be recruited simultaneously to the same promoter; the final regulation results from a dynamic balance between coactivators and corepressors (13). Additionally, SRC-1 has been reported to act not only as a coactivator of steroid hormone-dependent gene expression but also as a coregulator of nonsteroid receptors (34). Similarly, results reported here indicate that E7 expression interferes with SRC-1-mediated transcription for hormonally regulated (Fig. 3A) and non-hormonally regulated (Fig. 3B and 4) reporters. It is therefore plausible that relocalization of SRC-1 by HPV E7 may disrupt the balance of coactivators and corepressors on promoters, leading to dysregulation of SRC-1-mediated transcription. It will be important in the future to determine target genes that are affected by this dysregulation.
We report here the association between an HPV oncoprotein and a component of steroid hormone signaling. Our data demonstrate that the HPV E7 oncoprotein dysregulates SRC-1 localization and function. Since SRC-1 is a regulator of hormone-dependent gene expression, dysregulation of SRC-1 by the HPV E7 oncoprotein likely dysregulates hormone-dependent gene expression, possibly contributing to the process of cervical carcinogenesis.
ACKNOWLEDGMENTS
We thank Myles Brown (Dana Farber Cancer Institute, Boston, MA) and Bert O'Malley (Baylor College of Medicine, Houston, TX) for kindly providing the SRC-1 expression vectors and Azad Bonni (Pathology Department, Harvard Medical School, Boston, MA) for providing the IL-8 luciferase reporter construct.
This work was supported by National Institutes of Health grants F32CA112978-01A1 (A.B.), T32-CAO9031-28 (A.B.), and R01CA66980 (K.M.).
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ABSTRACT
High-risk human papillomaviruses (HPVs) are present in virtually all cervical carcinomas. However, the majority of women infected with high-risk HPVs do not develop cervical cancer. Therefore, cofactors must contribute to the development and progression of cervical cancer. Although numerous studies have implicated steroid hormones as cofactors in the initiation and progression of cervical neoplasia, the molecular mechanisms by which they contribute to cervical carcinogenesis are currently unknown. These observations led us to investigate a newly discovered association of the high-risk HPV type 16 (HPV16) E7 oncoprotein with steroid receptor coactivator 1 (SRC-1), an essential component of steroid hormone signaling. HPV16 E7 has been previously reported to interact with p300 and p300/CBP-associated factor (PCAF), members of some SRC-1 transcriptional complexes. We demonstrate here that HPV16 E7 associates in vivo and in vitro with SRC-1 independently of p300 and PCAF. Luciferase reporter constructs under the control of either the interleukin-8 promoter or a promoter containing multimerized synthetic estrogen response elements were used to determine the effect of high- and low-risk HPV E7 expression on SRC-1-mediated transcription. In addition, histone acetyltransferase (HAT) assays were performed to determine the effect of HPV E7 on SRC-1-associated HAT activity. These experiments reveal that HPV16 E7 expression down-regulates SRC-1-mediated transcription and SRC-1-associated HAT activity. SRC-1 localization experiments show that SRC-1 is relocalized to the cytoplasm in the presence of high- and low-risk HPV E7 proteins. Our data suggest that HPV E7 proteins dysregulate hormone-dependent gene expression by association with and relocalization of SRC-1. Dysregulation of SRC-1 localization and function by HPV E7 may provide insight into the molecular mechanisms by which steroid hormones act as cofactors in the induction and progression of cervical neoplasia.
INTRODUCTION
Human papillomaviruses (HPVs) are responsible for benign and malignant epithelial lesions such as common warts, genital warts, and cervical cancer. HPVs are icosahedral viruses that contain circular 8-kb double-stranded DNA genomes and are strictly epitheliotropic (reviewed in reference 44). Over 200 different HPVs have been characterized and are classified as mucosal or cutaneous. The majority of HPVs are low risk and usually result only in benign warts, while infections with high-risk types are associated with epithelial lesions that have a propensity for malignant progression in the oral cavity, the anogenital tract, and, most notably, the cervix (9, 14, 23). High-risk HPVs are present in nearly all cervical carcinomas, the second most common malignancy in women worldwide (reviewed in reference 45). HPV type 16 (HPV16) is by far the most prevalent of the high-risk types, followed by HPV types 18, 31, and others (reviewed in reference 46). Although virtually all cervical carcinomas contain high-risk HPVs, only a small fraction of women infected with high-risk HPVs will develop cervical cancer. Therefore, in addition to HPV infection, cofactors must contribute to the development and progression of cervical cancer.
Numerous epidemiological studies implicate steroid hormones as cofactors in the initiation and progression of cervical neoplasia (reviewed in reference 31). A large multinational WHO collaborative study has demonstrated an increased risk of cervical cancer with steroid hormone use (2). Increased duration of oral contraceptive use was also reported to increase the relative risk of adenocarcinoma in situ of the cervix and contribute to the pathogenesis of these tumors in women (27). In another study controlled for social, sexual, and cervical screening variables, the incidence of precursor and invasive lesions was highest among long-term oral contraceptive users (39). A recent report evaluated the role of steroid hormones in association with HPV positivity in cervical carcinogenesis; the risk of cervical carcinoma was increased up to fourfold in HPV-positive women with histories of long-term use of oral contraceptives (32).
Experimental evidence has also suggested a link between steroid hormones, HPV, and cervical cancer. Patterns of steroid hormone receptor expression are reported to be altered in HPV-associated lesions (12, 30). Additionally, HPV gene expression can be transcriptionally modulated by steroid hormones through response elements located in the upstream regulatory region of the viral genome (36, 40); estrogen has been shown to stimulate transcription of HPV16 in SiHa cervical carcinoma cells (29), and treatment with progesterone was shown to alter growth and viral gene expression in HPV16-positive CaSki cervical cancer cells and HPV16-containing Hep2 cells (43). However, the strongest experimental evidence that steroid hormones act as cofactors in cervical cancer has resulted from studies using a transgenic mouse model where HPV16 is expressed under the K14 promoter. Arbeit et al. showed that none of the K14-HPV16 transgenic mice spontaneously developed cervical cancer (irrespective of pregnancy number), whereas 100% of HPV16 transgenic mice developed cervical cancer upon long-term exposure to low doses of estrogen (3). Interestingly, in this model, the HPV16 oncogenes were expressed under the control of a keratin promoter, implying that synergy between steroid hormones and HPV in cervical cancer cannot be explained solely by an increase in viral gene expression. Despite the evidence implicating steroid hormones as cofactors in cervical carcinogenesis, the molecular pathways that link steroid hormones and HPV in the initiation and/or progression of cervical cancer remain undefined.
A possible candidate linking steroid hormones and HPV in cervical cancer is steroid receptor coactivator 1 (SRC-1), a component of steroid hormone signaling. We report that the HPV16 E7 oncoprotein associates with SRC-1, a member of the p160 steroid receptor coactivator family required for full transcriptional activity of the steroid receptor superfamily (35). Regulation of hormone-dependent gene expression by SRCs has been an area of intense study and has proved to be quite complex. SRCs, including SRC-1, modulate target gene expression by interaction with ligand-bound nuclear receptors, recruitment of histone acetyltransferases (HATs) and methylases, subsequent chromatin remodeling, and assembly of transcription factors (reviewed in reference 41). Selective recruitment of SRCs by different nuclear receptors can form specific, unique coactivator complexes to confer specificity to transcriptional regulation (8, 25). Additionally, functionally distinct SRC-1 isoforms likely play different roles in estrogen receptor-mediated transcription (21).
There is evidence to suggest that viral proteins can dysregulate SRC-1 transcriptional complexes, which have been shown to include the HATs p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF). SRC-1 is a very weak HAT, and SRC-1-associated HAT activity is derived largely by recruitment of other HATs, such as p300 and PCAF (reviewed in reference 41). Adenovirus E1A, structurally and functionally similar to HPV E7, was shown to bind CBP and prevent assembly of an SRC-1 coactivation complex, a critical step in hormone-dependent gene expression (42). More recently, p300, CBP, and PCAF were shown to be targets for the E1A proteins of all six adenovirus subgroups, demonstrating the importance of these interactions (38). Interestingly, HPV E7s from both high- and low-risk HPV types have been previously reported to interact with p300 and PCAF (4, 5, 19). However, our results suggest that HPV E7 associates directly with SRC-1, independently of p300 and PCAF, and dysregulates the function of SRC-1 transcriptional complexes. We also provide evidence that HPV E7 retains or relocalizes SRC-1 to the cytoplasm, removing SRC-1 from its transcriptional targets.
With the present study, we have begun to explore the molecular mechanisms by which steroid hormones act as cofactors with HPV in cervical carcinogenesis. In this report, we provide evidence for the dysregulation of SRC-1 localization and function by the HPV E7 oncoprotein.
MATERIALS AND METHODS
Cell lines. HeLa (cervical carcinoma, HPV18-positive) cells stably expressing C-terminally Flag- and hemagglutinin (HA)-tagged HPV16 E7 (CE7) have been previously described (20). RKO (colorectal carcinoma) and U2OS (osteosarcoma) cell lines were maintained in McCoy's medium supplemented with 10% calf serum and 1% penicillin (Pen)-streptomycin (Strep). RKO cells stably expressing the pcDNA3 control vector or HPV16 E7 (22) and U2OS cells stably expressing pcDNA3 or C-terminal Flag and CE7, derived by PCR cloning of the CE7 fragment from POZ-CE7 (20) into the pCMV BamNeo vector, were maintained in McCoy's medium supplemented with 10% calf serum, 1% Pen-Strep, and 0.5 mg/ml G418. C33A (cervical carcinoma, HPV-negative) and CaSki (cervical carcinoma, HPV16-positive) cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% calf serum and 1% Pen-Strep. Normal human foreskin keratinocytes (HFKs) were obtained from neonatal foreskins and maintained in keratinocyte-SFM plus supplements (Gibco), 1% Pen-Strep, 0.1% gentamicin, and 0.2% amphotericin B (Fungizone). Briefly, human foreskins (from two to seven individuals per set of foreskins) were obtained and cut into four or five pieces each. Each piece was floated (epidermis side up) in a dispase solution (250 mg/10 ml phosphate-buffered saline [PBS]) overnight at 4°C. The epidermis was separated from the dermis, cut into small pieces, and exposed to trypsin at 37°C for 10 min. After trypsinization, the keratinocytes were pelleted and plated. Trypsinization and plating were sequentially performed three times on each set of foreskins.
CoIPs, Western immunoblot analysis, and cell fractionation. Cells at 80 to 90% confluence (four to six 15-cm dishes) were scraped in PBS, pelleted, and resuspended in 3 volumes of 0.3B lysis/immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 8.0, 0.3 M KCl, 5 mM MgCl2, 10% glycerol, 0.1% Tween 20, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride). Lysates were rotated for 30 min at 4°C after one freeze-thaw cycle and then cleared by centrifugation (30 min, 16,000 x g). Two to 5 μg of each antibody (-SRC-1, Upstate clone 1135; -E7, Zymed C-8; -FLAG agarose, Sigma M-2; -p300, Upstate clone RW128) was coupled to 50 μl protein G (Promega) slurry (equilibrated with 0.3B buffer) by gently rotating at 4°C for 45 min. Coupled antibody was added to 10 to 20 mg cleared lysate, and the mixture was gently rotated at 4°C for 4 h. Coimmunoprecipitations (coIPs) were washed with 0.3B lysis/IP buffer three times and resuspended in loading buffer. Samples were resolved on a 10 to 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subsequently Western blotted. -PCAF, Santa Cruz E-8, and the above-mentioned antibodies were used for detection.
Nuclear and cytoplasmic fractions of cells were obtained using a nondetergent method on ice. Cells were pelleted, washed in PBS, washed in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride), and then incubated in 4 volumes of hypotonic buffer for 10 min. Cellular membranes were disrupted with 10 strokes of a mortar and tight pestle. Intact nuclei were pelleted at 1,500 x g for 5 min at 4°C. Nuclei were then lysed in 0.3B buffer with one freeze-thaw cycle. In order to visualize p300 by Western analysis in nuclear and cytoplasmic fractions, 25 mM N-ethylmaleimide was added to the lysis and hypotonic buffers. CoIPs were performed as described above on nuclear and cytoplasmic fractions.
In vitro transcription/translation and GST pull-downs. SRC-1 was in vitro transcribed and translated per kit instructions (TNT T7-coupled wheat germ extract system; Promega) using a Transcend nonradioactive translation detection system (Promega). In this system, biotinylated lysine residues are incorporated into nascent proteins during translation, allowing for nonradioactive proteins synthesized in vitro to be visualized by binding streptavidin-horseradish peroxidase. Glutathione S-transferase (GST) fusion proteins were made and purified per kit instructions (MagneGST protein purification system; Promega). Pull-down assays were performed using conditions the same as those described above for coIPs.
Transfections and luciferase reporter assays. Cells were cotransfected using either TransFast (Promega) or Fugene (Roche) per the manufacturer instructions. Luciferase reporters (500 ng) under the control of the interleukin-8 (IL-8) promoter (provided by A. Bonni) or of three estrogen response element (ERE) repeats (3x EREs) (Panomics) were cotransfected with pcDNA3 (vector only) or with 100 ng of plasmids expressing HPV16 CE7, HPV18 E7, HPV45 E7, HPV1 E7, HPV6b E7, and 40 ng of the control Renilla luciferase plasmid (see Fig. 3 and 4). One hundred nanograms of SRC-1 (provided by M. Brown and B. O'Malley) was also used for cotransfection (see Fig. 4). Forty-eight to 72 h posttransfection, luciferase activity was determined using a Dual Luciferase reporter assay system (Promega). One hundred fifty nanograms of pcDNA3 or plasmids expressing HPV16 E7, HPV1 E7, and HPV6b E7 was transfected into RKO cells using Fugene (Roche), and cells were subsequently used for immunofluorescence (see Fig. 6).
HAT assay. CoIPs were performed as described above using an -SRC-1 antibody (clone 1135; Upstate). Beads containing SRC-1 and its associated proteins were washed three times in 0.3B buffer and resuspended in a HAT activity buffer from a nonradioactive HAT assay kit (Upstate). Relative HAT activity was determined for each sample according to kit guidelines.
Immunofluorescence. Each cell line was grown to 60 to 70% confluence on glass coverslips and fixed in 4% paraformaldehyde for 10 min. Cells were rinsed with PBS and then permeabilized for 10 min with PBS-0.1% Triton X-100. After two PBS washes, cells were blocked with 5% bovine serum albumin. Staining for SRC-1 was performed using -SRC-1 antibodies (Santa Cruz C-20 and Upstate clone 1135) at 4°C for 8 h. Cells were washed two times with PBS and then incubated with secondary fluorescein isothiocyanate-conjugated -mouse (1:1,000) or -rabbit (1:2,000) antibodies (Jackson Labs) for at 37°C for 1 h. Cells were rinsed three times with PBS and counterstained with Hoechst. Coverslips were mounted and analyzed.
RESULTS
HPV16 E7 associates with SRC-1, p300, and PCAF. SRC-1 was initially identified as a potential HPV16 E7-interacting protein by tandem affinity purification (TAP) assay. A C-terminally double-tagged HPV16 E7 (CE7) was expressed in HeLa cells, sequential HA and FLAG coIPs were performed, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and E7-interacting bands were sequenced by mass spectrometry (20). Due to the evidence suggesting that steroid hormones act as cofactors with HPV in the initiation and progression of cervical neoplasia, we sought to verify and examine the interaction between E7 and SRC-1, an essential component of steroid hormone signaling.
Cell lysates from RKO (colorectal carcinoma) and U2OS (osteosarcoma) cells expressing HPV16 E7 or HPV16 CE7 were used to perform coIPs. Both tagged (CE7) and untagged (E7) exogenously expressed HPV16 E7 proteins were found to associate with endogenous SRC-1 (Fig. 1A, left panel). Lysates prepared from C33A (HPV-negative) and CaSki (HPV16-positive) human cervical carcinoma lines were used in coIPs to determine whether endogenous levels of HPV16 E7 could interact with SRC-1. Endogenous E7 and SRC-1 do in fact associate in the CaSki cervical cancer cell line, as detected by both -SRC-1 and -E7 coIPs (Fig. 1A, right panel).
Previous studies have reported that HPV16 E7 can associate with p300 and PCAF (4, 5, 19). To determine whether E7 could interact with these proteins, we performed coIPs under conditions the same as those used for the detection of the SRC-1 interaction. These experiments clearly showed that endogenous levels of both p300 and PCAF were found to associate with E7 (Fig. 1B). These results demonstrate that HPV16 E7 coprecipitates SRC-1, p300, and PCAF. Since SRC-1 has been shown to recruit p300 and PCAF and since E7 has been detected in association with each of the three members of the SRC-1-containing transcriptional complex, E7's interaction with members of this complex may serve to dysregulate/subvert SRC-1 function.
Association of HPV16 E7 with SRC-1 does not require p300 or PCAF. In light of the results that E7 could associate with SRC-1, p300, and PCAF, we wanted to determine whether p300 or PCAF was required for E7's association with SRC-1. After examination of p300 and PCAF levels in a number of cell lines, we found that the RKO cell line contained nearly undetectable levels of p300 (Fig. 2A, lanes 1 and 2), while the CaSki cell line contained reduced levels of PCAF (Fig. 2A, lane 5). Because these cell lines represent "natural knock-downs" of p300 and PCAF, we used them to perform SRC-1 coIPs to determine association with E7. Our results show that SRC-1 efficiently coprecipitates E7 in RKO cell lysates (Fig. 1A, left panel) in the absence of detectable association with p300 (Fig. 2A, lane 7). Similarly, SRC-1 coprecipitates E7 in CaSki cells (Fig. 1A, right panel) in the absence of detectable association with PCAF (Fig. 2A, lane 10). These results suggest that the association of E7 with SRC-1 does not critically depend on p300 or PCAF association.
To further explore the association between HPV16 E7 and SRC-1, we performed in vitro binding experiments using purified GST, GST-HPV16 E7, or various GST-HPV16 E7 mutants and SRC-1 produced in wheat germ extract (Promega) by in vitro transcription/translation. By producing SRC-1 with wheat germ extract, we reduced the presence of proteins with significant mammalian homology that may be required for SRC-1/E7 binding. SRC-1 does associate with wild-type E7, as well as with all E7 mutants tested, in these in vitro binding experiments (Fig. 2B). These results indicate that multiple HPV16 E7 domains may be necessary for association with SRC-1. The interaction detected between SRC-1 and E7 in vitro further supports the likelihood that the interaction is independent of p300 and PCAF. Taken together, these results demonstrate that HPV16 E7 associates with SRC-1 and the SRC-1 transcriptional complex members p300 and PCAF; however, the association of E7 with SRC-1 does not appear to require p300 or PCAF and may be direct.
HPV E7 dysregulates SRC-1-mediated transcription. Since SRC-1 is a component of steroid hormone signaling that regulates hormone-dependent gene expression, and given our results that E7 can associate with SRC-1, we sought to determine whether expression of HPV E7 could affect SRC-1-mediated transcription. SRC-1 is known to mediate signaling through EREs, and the IL-8 promoter has been previously shown to be modulated by expression of the HPV16 E7 oncoprotein (19). Luciferase reporters under control of either the IL-8 promoter or an artificial promoter containing 3x EREs were cotransfected with a Renilla control vector into the C33A and CaSki human cervical cancer cell lines to determine the effects of endogenously expressed HPV16 E7 on SRC-1-mediated transcription. Dual-luciferase activity was determined in triplicate for each cell line and reporter construct, and relative light unit values were normalized to that for the Renilla control vector. Luciferase activity for each was expressed as a percentage of the value for the control (C33A cells that do not contain HPV16) (Fig. 3). SRC-1-mediated transcription was dramatically decreased in the HPV16-containing CaSki cell line for both the 3x ERE (Fig. 3A) and the IL-8 (Fig. 3B) reporters. To determine whether the effect of HPV16 on SRC-1-mediated transcription is due to E7 expression, both reporters were also transfected along with the vector only or with HPV16 E7 plasmids into RKO, C33A, and HFKs. Expression of HPV16 E7 alone did likewise significantly decrease SRC-1-mediated transcription in each cell line tested for both the 3x ERE (Fig. 3A) and IL-8 (Fig. 3B) reporters. SRC-1-mediated transcription determined using the 3x ERE reporter in HFKs was a notable exception to these results (data not shown). This was an anticipated result and is likely due to the hormonal receptor differences in HFKs.
We also sought to functionally map the E7-interacting domain by using the above-described reporter assay with the IL-8 reporter and plasmids expressing HPV16 E7 mutants in C33A cells. The effects of E7 on SRC-1-mediated transcription did not map to a specific site on E7, as all the mutants tested decreased SRC-1-mediated transcription similarly to wild-type E7 (Fig. 3C). However, these reporter assay results obtained using mutants of E7 do agree with our in vitro binding data (Fig. 2B). All E7 mutants tested were able to bind SRC-1 and decrease SRC-1-mediated transcription. Since these mutants are inclusive of the domains required for E7's interaction with p300 and PCAF (4, 5, 19), these results provide further support to the assertion that the interaction between E7 and SRC-1 and the subsequent effects of E7 on SRC-1's transcriptional activities are independent of p300 and PCAF.
Using this assay as a biological readout, we wanted to determine the effects of high- and low-risk HPV E7s on SRC-1-mediated transcription. The assay was performed as described above by use of the IL-8 reporter in the C33A cell line. Expression of both high-risk (HPV types 16, 18, and 45) and low-risk (HPV types 1 and 6b) E7s decreased SRC-1-mediated transcription (Fig. 4). Ensuring that E7's effect on transcription was indeed SRC-1 mediated, transcriptional repression induced by E7 expression was relieved upon SRC-1 overexpression (Fig. 4). These results illustrate that expression of both high- and low-risk HPV E7s down-regulates SRC-1-mediated transcription. Moreover, these results demonstrate that SRC-1 is the rate-limiting factor for E7's effect on SRC-1-mediated transcription.
HPV16 E7 expression decreases SRC-1-associated HAT activity. SRC-1 recruits HATs in order to modify chromatin and exert transcriptional control. Given our results demonstrating that HPV E7 can dysregulate SRC-1-mediated transcription, we wanted to determine if SRC-1-associated HAT activity was affected by expression of HPV E7. To examine this, we performed SRC-1 coIPs with cells expressing HPV16 E7 and with those not expressing HPV16 E7. We then used the immunoprecipitates in a nonradioactive HAT assay. SRC-1-associated HAT activity was measured and expressed as a percentage of the value for the control (cells not expressing HPV16 E7) (Fig. 5). In CaSki cells expressing endogenous HPV16 E7 and upon expression of HPV16 E7 alone (C33A and RKO cells), SRC-1-associated HAT activity was reduced by more than 50% (Fig. 5). The results shown are from experiments using whole-cell lysates, although experiments done with nuclear extracts yielded similar results (data not shown). Down-regulation of SRC-1-mediated transcription by HPV E7 is therefore likely a result of a decreased HAT activity. These results provide further support to the observation that SRC-1-mediated transcription is dysregulated by expression of HPV E7 and suggest that HPV E7 dysregulates SRC-1-mediated gene expression by interfering with the activity of SRC-1 transcriptional complexes.
HPV16 E7 associates with SRC-1 in the cytoplasm. Although all known functions of SRC-1 are thought to take place in the nuclear compartment, a recent study using live-cell imaging demonstrated that SRC-1 can shuttle between the nucleus and the cytoplasm (1). Additionally, our analysis of fractionations from many cell lines expressing HPV16 E7 reveals that E7 does exist in the cytoplasmic compartment (Fig. 6D and data not shown), and this has also been reported by others (11). Finally, since the SRC-1/E7 interaction was initially identified in the cytoplasmic fraction by our TAP assay data, we sought to conclusively determine the localization of the SRC-1/E7 interaction. Cytoplasmic and nuclear fractions were obtained from HeLa cells expressing HPV16 CE7, and coIPs were performed on both fractions using an -FLAG antibody. Association between SRC-1 and E7 was detected in the cytoplasmic fraction of cells (Fig. 6A) but not in the nuclear fraction (data not shown).
HPV E7 expression relocalizes SRC-1 to the cytoplasm. Our results demonstrating that association between HPV16 E7 and SRC-1 occurs in the cytoplasmic fraction of cells were initially puzzling, especially given the experimental data demonstrating that E7 expression affects the nuclear activities of SRC-1 (Fig. 3, 4, and 5). These results led us to question whether E7 expression could affect SRC-1 subcellular localization. To address this question, we used immunofluorescence to visualize SRC-1 localization in HPV-negative (C33A) cells, and we compared this localization to that seen for cells containing endogenous HPV16 (CaSki and SiHa) and HPV18 (HeLa). Localization of SRC-1 is primarily nuclear without HPV expression; however, in cells expressing high-risk HPVs, SRC-1 is dramatically, but not completely, relocalized to the cytoplasm of cells (Fig. 6B, top panel).
Comparison of RKO cells expressing vector only or HPV16 E7 revealed that E7 alone relocalizes SRC-1, demonstrating that this relocalization effect is not due to expression of other viral proteins (Fig. 6C, top panel). The low-risk HPV1 and HPV6b E7 proteins also exhibit the ability to relocalize SRC-1 to the cytoplasm (Fig. 6C, top panel).
Although our TAP data revealed interaction of E7 and SRC-1 in the cytoplasmic compartment, the TAP analysis provided no evidence of association with p300 or PCAF in the cytoplasm. To determine whether HPV E7 also relocalizes these known members of the SRC-1 transcriptional complex to the cytoplasm, we performed cellular fractionation of RKO cells expressing HPV16 E7. Western analysis was performed on both the nuclear and cytoplasmic fractions. p300 and PCAF were detected only in the nuclear fractions of cells expressing E7, although the majority of E7 detected was in the cytoplasmic fraction (Fig. 6D). Thus, our data demonstrate that E7 does not relocalize p300 and PCAF to the cytoplasm; rather, E7 specifically targets and relocates SRC-1. These results demonstrate that in the presence of high- or low-risk HPV E7, SRC-1 is relocalized to the cytoplasm. These results complement the finding that both high- and low-risk HPV E7s can dysregulate SRC-1-mediated transcription (Fig. 3 and 4). Collectively, our findings suggest a model whereby HPV E7 dysregulates SRC-1 function by relocalization to the cytoplasm, thereby decreasing the levels of transcriptionally active, nuclear SRC-1 (Fig. 7) independently of p300 and PCAF.
DISCUSSION
The studies reported here were initiated to explore the molecular mechanism(s) by which steroid hormones may contribute to HPV-associated cervical neoplasia. These studies yielded the following important observations. (i) HPV16 E7 associates with a component of steroid hormone signaling, SRC-1 (Fig. 1). (ii) E7's association with SRC-1 is not strictly dependent on the SRC-1 complex members p300 and PCAF (Fig. 2). (iii) Expression of high- and low-risk HPV E7 proteins dysregulates SRC-1-mediated transcription (Fig. 3 and 4). (iv) SRC-1-associated HAT activity decreases upon HPV16 E7 expression (Fig. 5). (v) High- and low-risk HPV E7s relocalize SRC-1 to the cytoplasm (Fig. 6). Overall, these data support a model whereby either HPV E7 associates with SRC-1 and relocalizes it to the cytoplasm, or HPV E7 associates with a cytoplasmic pool of SRC-1 and inhibits its nuclear translocation. This association with HPV E7, which likely occurs independently of p300 and PCAF, leads to SRC-1 retention in the cytoplasm, resulting in displacement of SRC-1 from its transcriptional targets. Consequently, SRC-1 nuclear transcriptional activity is dysregulated by HPV E7 (Fig. 7).
Expression of the high-risk HPV E6/E7 oncogenes is necessary and sufficient to immortalize primary human keratinocytes (17, 33), and continued expression of the E6 and E7 oncoproteins is essential to maintain a transformed phenotype in cervical cancer cells (15). HPV E7s from both high- and low-risk HPV types have been shown to interact with p300 (5) and PCAF (19). Association of E7 with PCAF was found to be necessary for down-regulation of a target promoter (19) and was also shown to reduce HAT activity of PCAF in vitro (4). Additional reports have demonstrated that the ability of E7 to associate with histone-modifying enzymes contributes to promotion of cell growth (6), deregulates transcriptional silencing (24), extends the life span of keratinocytes, and contributes to stable maintenance of viral genomes through association with histone deacetylases (26). Together, these studies establish that HPV E7 subverts the normal activity of chromatin-remodeling transcriptional complexes. We discovered that HPV16 E7 associates with SRC-1 (Fig. 1), a component of chromatin-remodeling transcriptional complexes, independently of p300 and PCAF (Fig. 2). Our results demonstrate that E7 expression induces significant relocalization of SRC-1 to the cytoplasm (Fig. 6B and C). Conversely, the localization of PCAF and p300 does not change upon E7 expression (Fig. 6D). Therefore, it is likely that the composition of the SRC-1 transcriptional complex is disrupted upon binding of E7 to SRC-1. These findings suggest that dysregulation of the SRC-1 transcriptional complex results from the direct association between E7 and SRC-1.
Perhaps one of the most exciting observations in this report is the relocalization of SRC-1 by HPV E7 to the cytoplasm (Fig. 6B and C). Although steroid hormone receptors are mainly localized in the nucleus, a recent live-cell imaging study using fluorescently tagged SRC-1 has demonstrated that SRC-1 is imported into the nucleus after synthesis in the cytoplasm and then exported back into the cytoplasm, possibly for degradation (1). That study also suggests nuclear export may be dependent on the interaction of SRC-1 with other proteins, which could regulate steroid hormone signaling. Similarly, association of HPV E7 with SRC-1 initially in the nucleus may result in such nuclear export. Putative nuclear export signals, which are not exposed in the nuclear conformation of SRC-1, may become exposed upon conformational changes induced by interaction with other proteins, such as the E7 oncoprotein. The mechanism by which HPV E7 relocalizes SRC-1 to the cytoplasm is the focus of ongoing studies. Interestingly, nuclear accumulation of E2F4 in response to HPV E7 expression has been reported (37). These observations suggest that the HPV E7 oncoprotein may alter the normal subcellular localization of host proteins as a general mechanism to promote oncogenesis.
Association of HPV16 E7 and SRC-1 was detected only in the cytoplasmic compartment of cells (Fig. 6A). However, we cannot rule out the possibility that E7 may also play a role in SRC-1-mediated transcription by interaction with SRC-1 in the nucleus. Given that we typically observe lower levels of E7 in the nucleus than in the cytoplasm and that E7 expression relocalizes SRC-1 to the cytoplasm, an association between E7 and SRC-1 in the nucleus might simply be below the level of detection. Nevertheless, the effects of E7 expression on SRC-1-associated HAT activity (Fig. 5) and SRC-1-mediated transcription (Fig. 3 and 4) are likely due to the relocalization of SRC-1 to the cytoplasm (Fig. 6B and C). In addition to reducing the nuclear activities of SRC-1 by removing it from its nuclear targets, E7 may serve to disrupt an as-yet-undiscovered cytoplasmic role of SRC-1. The observation that SRC-1 is not completely relocalized to the cytoplasm in E7-expressing cells is also very interesting. SRC-1's regulation of gene expression is complex, and several differentially spliced SRC-1 transcripts have been reported to function differently under various conditions (7, 10, 16, 18, 28). It is possible that only a specific isoform of SRC-1 is relocalized upon E7 expression, resulting in a highly specific mechanism that dysregulates SRC-1 function.
We did not detect an interaction between low-risk HPV E7s and SRC-1 by coimmunoprecipitation (data not shown). This does not rule out the possibility that low-risk HPV E7 proteins may associate with SRC-1; however, the efficiency of the interaction may be below the level of detection in our assays. Other cellular proteins that efficiently associate with high-risk HPV E7, most notably pRb, have been demonstrated to bind low-risk HPV E7 proteins at a decreased efficiency. The findings that both high- and low-risk HPV E7 proteins dysregulate SRC-1 localization (Fig. 6) and function (Fig. 4) demonstrate that this particular function of E7 may be conserved and suggest that dysregulation of SRC-1 localization and function by E7 may be of general importance for the biology of HPVs. This hypothesis is currently under investigation in our laboratory.
Because of the synergy between HPV and steroid hormones in cervical cancer, down-regulation of SRC-1 function by HPV E7 may seem counterintuitive initially. Hormone-dependent gene expression, however, is a complex, exquisitely regulated process. In a recent report demonstrating the complexity of SRC-mediated gene regulation, SRC family members and corepressors (histone deacetylases, silencing mediator of retinoid and thyroid receptors [SMRT] and NcoR) were recently shown to be recruited simultaneously to the same promoter; the final regulation results from a dynamic balance between coactivators and corepressors (13). Additionally, SRC-1 has been reported to act not only as a coactivator of steroid hormone-dependent gene expression but also as a coregulator of nonsteroid receptors (34). Similarly, results reported here indicate that E7 expression interferes with SRC-1-mediated transcription for hormonally regulated (Fig. 3A) and non-hormonally regulated (Fig. 3B and 4) reporters. It is therefore plausible that relocalization of SRC-1 by HPV E7 may disrupt the balance of coactivators and corepressors on promoters, leading to dysregulation of SRC-1-mediated transcription. It will be important in the future to determine target genes that are affected by this dysregulation.
We report here the association between an HPV oncoprotein and a component of steroid hormone signaling. Our data demonstrate that the HPV E7 oncoprotein dysregulates SRC-1 localization and function. Since SRC-1 is a regulator of hormone-dependent gene expression, dysregulation of SRC-1 by the HPV E7 oncoprotein likely dysregulates hormone-dependent gene expression, possibly contributing to the process of cervical carcinogenesis.
ACKNOWLEDGMENTS
We thank Myles Brown (Dana Farber Cancer Institute, Boston, MA) and Bert O'Malley (Baylor College of Medicine, Houston, TX) for kindly providing the SRC-1 expression vectors and Azad Bonni (Pathology Department, Harvard Medical School, Boston, MA) for providing the IL-8 luciferase reporter construct.
This work was supported by National Institutes of Health grants F32CA112978-01A1 (A.B.), T32-CAO9031-28 (A.B.), and R01CA66980 (K.M.).
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