The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.     

Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein

A decrease in gap junction-mediated cell-to-cell communication has previously been observed in monolayer cultures of human keratinocytes (HaCaT cells) expressing the human papillomavirus type 16 E5 (HPV16 E5) gene and attributed to the reduced phosphorylation of connexin 43, the most abundant connexin in HaCaT cells. In line with this observation, we have now analyzed the effect of HPV16 E5 on connexin 43 expression in raft cultures produced by transfected HaCaT cells. These keratinocytes transcribe HPV16 E5 under the control of a dexamethasone-inducible promoter. Our results show that treatment with dexamethasone leads to an almost complete disappearance of connexin 43 in rafts expressing the E5 gene but not in control rafts. In our study we discuss the possible effects of this downregulation on cell-cell communication and cellular malignant transformation.     

Activation of epidermal growth factor receptor promotes late terminal differentiation of cell-matrix interaction-disrupted keratinocytes

The biological effects of epidermal growth factor receptor (EGFR) activation may differ between epidermal suprabasal and basal keratinocytes, since growth factors are mitogenic in adherent cells only in the presence of cell-extracellular matrix (ECM) interaction. To investigate biological effects of EGFR activation on keratinocytes without cell-ECM interaction, we cultured normal human keratinocytes on polyhydroxyethylmethacrylate-coated plates, which disrupt cell-ECM but not cell-cell interaction. The cells initially expressed keratin 10 (K10) and then profilaggrin, mimicking sequential differentiation of epidermal suprabasal keratinocytes. The addition of EGF or transforming growth factor-alpha promoted late terminal differentiation (profilaggrin expression, type 1 transglutaminase expression and activity, and cornified envelope formation) of the suspended keratinocytes, while suppressing K10 expression, an early differentiation marker. These effects were attenuated by EGFR tyrosine kinase inhibitor PD153035 or an anti-EGFR monoclonal antibody, whereas protein kinase C inhibitors H7 and bisindolylmaleimide I or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 abolished profilaggrin up-regulation but not K10 suppression. Since the antidifferentiative role of EGFR on cell-ECM interaction-conserved keratinocytes has been well documented, our results indicate that the biological effects of EGFR on keratinocytes are influenced by cell-ECM interaction and suggest that EGFR activation promotes rather than inhibits the terminal differentiation of suprabasal epidermal keratinocytes.     

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

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.     

HB-EGF synthesis and release induced by cholesterol depletion of human epidermal keratinocytes is controlled by extracellular ATP and involves both p38 and ERK1/2 signaling pathways

The heparin-binding EGF-like growth factor (HB-EGF) is an autocrine/paracrine keratinocyte growth factor, which binds to the epidermal growth factor (EGF) receptor family and plays a critical role during the re-epithelialization of cutaneous wound by stimulating the keratinocytes proliferation and migration. In this study, cellular stressing condition in autocrine cultures of human keratinocytes was induced by cholesterol depletion using methyl-beta-cyclodextrin (MβCD). MβCD treatment induces the expression and the release of HB-EGF. By analysis of the culture media, large amounts of cellular ATP were measured particularly after 1 h of MβCD treatment. To investigate whether ATP contributes to the expression of HB-EGF, the nonhydrolyzable ATP analogue, ATP-γ-S, was used to mimic the extracellular ATP released. We report that keratinocytes stimulated with ATP-γ-S induce HB-EGF expression and activate EGFR and ERK1/2. Using an antagonist of P2 purinergic receptors, we demonstrate that HB-EGF synthesis induced by lipid rafts disruption is dependent on ATP interaction with P2 purinergic receptors. Moreover, our data suggest that both MAPKs p38 and ERK1/2 are involved together or independently in the regulation of HB-EGF gene expression. These findings provide new insight into the signaling pathway by which HB-EGF is expressed after lipid rafts disruption. In summary, after lipid raft disruption, keratinocytes release large amount of extracellular ATP. ATP induces HB-EGF synthesis and release by interacting with the P2 purinergic receptor and through p38 and ERK1/2 signaling in response to a challenging environment. A release of ATP acts as an early stress response in keratinocytes.     

Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis

Posttranslational modification-oligomerization, phosphorylation, and proteolytic cleavage-of the human papillomavirus (HPV) E4 protein occurs as the infected keratinocytes migrate up through the suprabasal wart layers. It has been postulated that these events modify E4 function during the virus life cycle. In HPV type 1 (HPV1)-induced warts, N-terminal sequences are progressively cleaved from the full-length E4 protein (E1(wedge)E4) of 17 kDa to produce a series of polypeptides of 16, 11 and 10 kDa. Here, we have shown that in human keratinocytes, a truncated protein (E4-16K), equivalent to the 16-kDa species, mediated a G(2) arrest in the cell cycle that was dependent on a threonine amino acid in a proline-rich domain of the protein. Reconstitution of cyclin B1 expression in E4-16K cells reversed the G(2) arrest. Expression of E4-16K also induced chromosomal rereplication, and this was associated with aberrant nuclear morphology. Perturbation of the mitotic cell cycle was a biological activity specific to the truncated protein. However, coexpression of the full-length E1(wedge)E4 protein and the truncated E4-16K protein inhibited normal cellular proliferation and cellular DNA rereplication but did not prevent cells from arresting in G(2). Our findings provide the first evidence to support the hypothesis that proteolytic cleavage of the E1(wedge)E4 protein modifies its function. Also, different forms of the HPV1 E4 protein cooperate to negatively influence keratinocyte proliferation. We predict that these distinct biological activities of E4 act to support efficient amplification of the viral genome in suprabasal keratinocytes.     

Regulation of interleukin-1alpha expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease

Transgenic mice expressing beta1 integrins in the suprabasal epidermal layers have sporadic skin hyperproliferation and inflammation correlated with activation of extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase and increased interleukin (IL)-1alpha production. We investigated the link between aberrant integrin expression, Erk activation, and expression of IL-1alpha. Transgenic keratinocytes had higher basal Erk activity and IL-1alpha levels than nontransgenic controls and were more sensitive to stimulation of Erk activity and IL-1alpha production by IL-1alpha, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), and serum. Inhibition of Erk in transgenic keratinocytes reduced basal IL-1alpha levels and the stimulation of IL-1alpha production by serum or phorbol ester, demonstrating that Erk could regulate IL-1alpha expression. TPA or IL-1alpha treatment resulted in rapid down-regulation of the EGF receptor in transgenic cells, indicative of transactivation. Inhibition of transactivation blocked basal and TPA or IL-1alpha induced Erk activation, but not IkappaBalpha degradation, and abolished increased IL-1alpha production in transgenic cells. In transgene-negative cells, constitutive activation of IL-1-dependent signaling by wild type or kinase-dead IRAK1 stimulated IL-1alpha production independent of Erk. We conclude that suprabasal integrin expression leads to Erk activation and increased IL-1alpha expression by potentiating activation of the EGF receptor. These results provide a mechanism by which aberrant integrin expression triggers epidermal hyperproliferation and inflammation.     

Effect of human papillomavirus type 16 oncogenes on MAP kinase activity.

The mitogen-activated protein (MAP) kinase signal transduction pathway is an intracellular signaling cascade which mediates cellular responses to growth and differentiation factors. The MAP kinase pathway can be activated by a wide range of stimuli dependent on the cell types, and this is normally a transient response. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells in part by prolonging the activated stage of components within this signaling pathway. The human papillomavirus (HPV) oncogenes E6 and E7 play an essential role in the in vitro transformation of primary human keratinocytes and rodent cells. The HPV type 16 E5 gene has also been shown to have weak transforming activity and may enhance the epidermal growth factor (EGF)-mediated signal transduction to the nucleus. In the present study, we have investigated the effects of the oncogenic HPV type 16 E5, E6, and E7 genes on the induction of the MAP kinase signaling pathway. The E5 gene induced an increase in the MAP kinase activity both in the absence and in the presence of EGF. In comparison, the E6 and E7 oncoproteins do not alter the MAP kinase activity or prolong the MAP kinase activity induced with EGF. These findings suggest that E5 may function, at least in part, to enhance the cell response through the MAP kinase pathway. However, the transforming activity of E6 and E7 is not associated with alterations in the MAP kinase pathway. These findings are consistent with E5 enhancing the response to growth factor stimulation.     

Cottontail rabbit papillomavirus E8 protein is essential for wart formation and provides new insights into viral pathogenesis

The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin. The scarce warts induced showed very slow growth, despite sustained expression of E6 and E7 oncogenes. This points to an essential role of E8 in disturbing epidermal homeostasis. Using a yeast two-hybrid screen, we found that E8 interacted with the zinc transporter ZnT1, protocadherin 1 (PCDH1), and AHNAK/desmoyokin, three proteins as yet unrelated to viral pathogenesis or cell transformation. HPV16 E5 also interacted with these proteins in two-hybrid assay. CRPV E8 mainly localized to the Golgi apparatus and the early endosomes of transfected keratinocytes and colocalized with ZnT1, PCDH1, and AHNAK. We showed that ZnT1 and PCDH1 formed a complex and that E8 disrupted this complex. CRPV E8, like HPV16 E5, increased epidermal growth factor (EGF)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and both the EGF-dependent and the EGF-independent activity of activating protein-1 (AP-1). Competition experiments with a nonfunctional truncated ZnT1 protein showed that E8-ZnT1 interaction was required for AP-1 activation. Our data identify CRPV E8 as a key player in papilloma induction and unravel novel cellular targets for inducing the proliferation of keratinocytes.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

The human papillomavirus type 16 E5 protein impairs TRAIL- and FasL-mediated apoptosis in HaCaT cells by different mechanisms

The effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis was investigated by using the polyclonal HaCaT-cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference. Apoptosis was triggered either by Fas ligand (FasL) or by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and was monitored by detection of cleavage of procaspase-8 and procaspase-3, as well as their substrate poly(ADP-ribose) polymerase (PARP). In contrast to the HaCaT/pMSG control cells we found that apoptosis induced by either of the two ligands is strongly suppressed in the E5-expressing keratinocytes. Fas expression is reduced by about a factor of two in HaCaT/E5 cells, which could be part of the mechanisms that protect the cells from FasL-induced apoptosis. For the TRAIL receptors, no such downregulation was observed. Here, E5 impairs the formation of the death-inducing signaling complex triggered by TRAIL. Apparently, E5 employs different mechanisms to inhibit death receptor signaling. This effect is not restricted to HaCaT/E5 cells since we found that the mouse fibroblast cell line A31-E5 is protected from TRAIL-induced apoptosis, as well but not the E5-lacking control cells A31-Neo. However, no such protection was observed upon FasL-induced apoptosis. Presumably, some of the antiapoptotic mechanisms employed by E5 of the human pathogenic HPV-16 are cell type specific. We propose that inhibition of ligand-mediated apoptosis in human keratinocytes is a primary function of the HPV-16 E5 protein needed to prevent apoptosis at early stages of viral infection.     

Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.

In this study we used a dominant-negative FGF receptor mutant to block FGF function in a specific tissue of transgenic mice. The mutant receptor, which is known to block signal transduction in cells when co-expressed with wild-type receptors, was targeted to suprabasal keratinocytes using a keratin 10 promoter. The transgene was expressed specifically in the skin and highest expression levels were found in the tail. Expression of the mutant receptor disrupted the organization of epidermal keratinocytes, induced epidermal hyperthickening and resulted in an aberrant expression of keratin 6. This suggests that FGF is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation. Our study demonstrates that dominant-negative growth factor receptors can be used to block selectively the action of a growth factor in specific tissues of transgenic mice.     

Feedback regulation between orphan nuclear receptor TR2 and human papilloma virus type 16

The human TR2 orphan receptor (TR2), initially isolated from testis and prostate cDNA libraries, is a member of the steroid receptor superfamily. TR2 can regulate several target genes via binding to a consensus response element (AGGTCA) in direct repeat orientation (AGGTCAX((n))AGGTCA, n = 0-6). Here we show that TR2 is able to induce the expression of human papilloma virus type 16 (HPV-16) genes via binding to a DR4 response element in the long control region of HPV-16. Additionally, one of the HPV-16 gene products, the E6 oncogene, regulates TR2 gene expression. A likely mechanism for this regulation involves E6-mediated degradation of the tumor suppressor p53, a protein known to suppress TR2 expression. Together our data provide evidence for feedback regulation between TR2 and HPV-16, which represents a novel regulatory pathway involving a member of the steroid receptor superfamily and the HPV-16 DNA tumor virus.