Human Papillomavirus E6 Regulates the Cytoskeleton Dynamics of Keratinocytes through Targeted Degradation of p53

Abstract

The attachment and spreading of keratinocyte cells result from interactions between integrins and immobilized extracellular matrix molecules. Human papillomavirus type 16 (HPV-16) E6 augmented the kinetics of cell spreading, while E6 genes from HPV-11 or bovine papillomavirus type 1 did not. The ability of E6 to interact with the E6AP ubiquitin ligase and target p53 degradation was required to augment cell-spreading kinetics; dominant negative p53 alleles also enhanced the kinetics of cell spreading and the level of attachment of cells to hydrophobic surfaces. The targeted degradation of p53 by E6 may contribute to the invasive phenotype exhibited by cervical cells that contain high-risk HPV types.

Human papillomaviruses (HPVs) produce benign epitheliomas, but with a subset of HPV types, infection can eventually lead to invasive cancers (14). HPVs with this property are termed “high-risk” types, and the HPV most frequently isolated from cervical cancers is HPV-16 (23). In cancer cells, the HPV genome is integrated into the host chromosome such that the intact E6 and E7 oncoproteins are expressed. It is as yet unclear whether the expression of E6 and E7 directly contributes to an invasive-cancer phenotype or whether the properties of E6 and E7 (which include the abrogation of normal cell cycle checkpoint controls) contribute to a mutator phenotype, with the subsequent selection for invasive cells.

HPV-16 E6 (16E6) is the prototype high-risk HPV E6 protein. 16E6 directly interacts with a cellular ubiquitin ligase termed E6AP by binding a LXXLL motif of E6AP; the complex of 16E6 together with E6AP binds to the p53 tumor suppressor protein, resulting in ubiquitin-mediated degradation of p53 by the proteasome (7, 8, 22). The in vivo degradation of p53 requires sequences at the amino terminus of E6 that interact with p53 (3). The six amino acids at the carboxy terminus of 16E6 contain a PDZ ligand that interacts with a subset of cellular proteins containing one or more PDZ domains; as with p53, the interaction of E6 with these PDZ-containing cellular proteins may lead to their E6AP-dependent degradation (4, 5, 9, 10, 12, 15). Some evidence supports the involvement of ubiquitin ligases other than E6AP (17, 24).

We and others had previously observed that the E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) E6 binds to paxillin and that paxillin was required for the tyrosine phosphorylation of focal adhesion kinase (FAK), a kinase that plays a regulatory role in cell migration (26-29). These findings prompted us to examine whether expression of E6 proteins might modulate signaling from integrin receptors to the cytoskeleton. We observed that keratinocytes retrovirally transduced with E6 from genital cancer-associated HPV-16 induced a marked increase in early cell spreading after attaching to a matrix-coated surface, while keratinocytes transduced with E6 proteins from low-risk HPV-11 and BPV-1 did not (Fig. 1A). Cultures of normal keratinocyte cell (NIKS cells) (1) were washed twice with phosphate-buffered saline, lightly trypsinized at room temperature, and then rinsed twice to remove feeder fibroblasts, leaving the epithelial cells. A second trypsinization at 37°C detached keratinocytes that were collected by centrifugation, and they were resuspended at 5 × 10⁵ cells/ml in complete media (18) and then incubated with periodic gentle swirling for 20 min at 37°C. A cell suspension mixture (0.2 ml) was added to 24-well, matrix-coated microtiter plates and incubated at 37°C. At various time points, the media were gently removed and the plates were gently rinsed with cold, serum-free media to remove unattached cells. The cells were immediately fixed, stained, and scored as spread if the cytoplasms exceeded twice the diameter of the nuclei (Fig. 1A and 2A and B) or digitally analyzed to determine the areas of the cell surfaces (Fig. 1C). Assays to determine the levels of cell attachment and spreading were performed as previously described (29).

FIG. 1.

HPV-16 E6 enhances early cell spreading after attachment of the cells to matrix-coated surfaces. (A) Cell spreading is altered by cancer-associated HPV E6. Only HPV-16 E6 enhanced early cell spreading after attachment. The values shown are the averages of the results of three assays described in the text. (B) 16E6 enhances early cell spreading. The cells shown were fixed and stained with formalin as described in the text. (C) Quantitation of the enhancement of cell spreading by 16E6. The total cell surface area was quantitated and compared by Image analysis software (NIH Image) 20 min after plating. Student's t test was used to determine the P value for a 300-cell population of each cell type. The error bars indicate the standard errors of the means.

FIG. 2.

The effects of matrix and E6 mutations on cell spreading. (A) 16E6 augments cell spreading but not cell attachment. NIKS cells transduced with either empty retroviral vectors or 16E6 were assayed to determine the levels of cell attachment (top panel) and cell spreading (bottom panel) 15, 30, or 60 min after being plated on fibronectin-coated plates (10 μg/ml) as previously described (28). OD, optical density. (B) Enhanced spreading is independent of the matrix type and requires amino-terminal sequences of 16E6. NIKS cells transduced with empty retroviral vectors, 16E6 (shown as WT), or the indicated 16E6 mutants were seeded on plastic plates coated with either collagen type 1 or fibronectin for 20, 60, or 180 min. At least 300 cells were counted at each time point in three representative experiments.

Quantitation of early cell spreading showed that 16E6 expression enhanced the early-spread total cell surface area by about 2.5-fold (Fig. 1B and C). The attachment of keratinocytes to the matrix was not altered by 16E6, but subsequent early cell spreading was markedly increased by 16E6 (Fig. 2A) on surfaces coated with fibronectin, laminin, collagen, and vitronectin (Fig. 2B and data not shown). This analysis also demonstrated that three different 16E6 mutants that alter the amino-terminal p53 interaction motif of 16E6 abrogated the enhancement of cell spreading (16E6_K11E, 16E6_R8Q, and 16E6_F2V are defective for degrading p53 [3]), as did mutations that eliminate the ability of 16E6 to interact with the LXXLL motif of E6AP (16E6_Y79N and 16E6_E75G [3]) (Fig. 2B). Surprisingly, deletion of the PDZ ligand of 16E6 (16E6_ΔC) had less effect upon cell spreading (Fig. 2B).

The cell-spreading assay employed measures integrin signaling to the cytoskeleton in the initial minutes after cell attachment (20). To determine whether E6 might also have a prolonged effect upon keratinocyte attachment and spreading, we plated keratinocytes onto a hydrophobic surface, a more-difficult substrate for the cells to attach to. Keratinocytes transduced with 16E6 but not 16E7 had enhanced levels of cell spreading on bacterial plates at 24 and 48 h postplating (Fig. 3). A 16E6 mutant defective for degrading p53 (16E6_F2V) did not augment cell spreading, while 16E6 from cells expressing either a 16E6 mutant with the PDZ ligand deleted or a p53 dominant negative mutant appeared indistinguishable from wild-type 16E6 (Fig. 4A). The expression levels of wild-type and mutant E6 proteins were similar (Fig. 4B) (the 16E6_F2V mutation destroys the 6F4 epitope). These results indicate that interference with wild-type p53 by either targeted degradation or transdominant interference enhances cell spreading.

FIG. 3.

16E6 augments cell spreading on hydrophobic surfaces. (A) Spread of NIKS cells on bacterial plates 24 h after plating. NIKS cells were stably transduced with vectors or the indicated oncogenes, seeded on bacterial plates, and photographed 24 h later.

FIG. 4.

p53 regulates spreading on hydrophobic surfaces. (A) NIKS cells transduced with the indicated 16E6 genes or a dominant negative p53 gene (p53 DD) (25) were photographed 48 h after being plated on bacterial plates. (B) Expression levels of 16E6 and 16E6 mutants in NIKS cells. NIKS cells transduced with retrovirus vectors and lysed in sodium dodecyl sulfate were analyzed by Western blotting, using monoclonal antibodies (MAb) to 16E6 (11). Monoclonal antibody 3F8 recognizes an epitope common to 16E6 and all the mutants, while monoclonal antibody 6F4 recognizes the amino terminus of 16E6; this epitope is disrupted by the 16E6_F2V mutation.

We performed our experiments using a nontransformed but immortalized human keratinocyte cell line (NIKS cells) that supports the full HPV life cycle in organotypic culture but is nontransformed and requires feeder cells, epidermal growth factor, insulin, and serum in the media (1). These cells were chosen so that vector- and 16E6-transduced keratinocytes would retain similar proliferation and differentiation characteristics such that cytoskeleton changes would not be an artifact of the two populations having different degrees of proliferative potential or terminal differentiation. We obtained similar results with transient transfections of NIKS cells with 16E6, 16E6_F2V, and mutant p53 cDNAs (data not shown).

Our experiments examining cell spreading on hydrophobic surfaces were prompted by the intriguing observation that primary keratinocytes that are cultured on bacterial plates have extended proliferative potential (6). Most NIKS keratinocytes fail to attach to a hydrophobic surface, and among those that do attach, most fail to spread well (Fig. 3 and 4). However, the population of cells that do attach and spread can then be passaged upon bacterial plates (6; our unpublished observations). It appears that culturing on hydrophobic plates selects a subpopulation of keratinocytes that both attach to and spread on hydrophobic surfaces and have enhanced proliferative potential. Both 16E6 and dominant negative p53 were able to induce enhanced spreading of NIKS cells on hydrophobic plates (Fig. 4). Dominant negative p53 has recently been shown to cooperate with p16 inhibition to extend the life spans of primary keratinocytes (19).

Mouse fibroblasts null for p53 exhibit enhanced cell spreading and migration, and the mechanisms involved appear complex (21). In studies not shown here, we did not observe a clear and consistent effect of 16E6 upon the tyrosine phosphorylation of FAK or the nucleotide loading of Rac1, Cdc42, or Rho in NIKS cells (data not shown). A previous study of FAK phosphorylation showed enhanced activation of FAK tyrosine phosphorylation in cells expressing HPV-18 E6 and E7 compared to that for vector-transduced primary keratinocytes (13); however, in our experience, there are substantial differences in differentiation and proliferation between these two cell populations which could confound the interpretation. Migration is complex, involving attachment, spreading, polarization, directed cytoskeletal extension, and release of the caudal cell part from the substrate (20). The initial stage of cell spreading that is the subject of this study is a crucial component of migration but is only a part. This report extends the observation of the role of p53 in altering cell spreading to keratinocytes, where the targeted degradation of p53 correlates with HPV types associated with invasive cancers. It is interesting that neither the E6 proteins of HPV-11 nor those of BPV-1 markedly altered early cell spreading, despite the ability of both of these E6 proteins to interact with E6AP and, in the case of HPV-11, to target associated proteins for degradation (2, 16). Neither BPV-1 E6 nor HPV-11 E6 targets the degradation of p53, and upon the mutation of the p53 interaction motif of 16E6, the effect of 16E6 on cell spreading is largely lost. Therefore, it is possible that the degradation of p53 by 16E6 contributes in part to the abnormal invasion and migration that are characteristic of cervical cancer cells.