Expression of the HPV11 E2 gene in transgenic mice does not result in alterations of the phenotypic pattern.

The E2 early protein of human papillomaviruses (HPV) has been found associated with the mitotic spindle therefore being implicated in the partition of the replicated viral DNA to daughter cells. In addition, E2 proteins bind to the upstream regulatory region of the virus and to cellular promoters modulating thereby cellular transcription and differentiation. In many cervical cancers, the E2 reading frame is interrupted upon incorporation of the viral genome into the host DNA. This results in the loss of the E2 mediated transcriptional repression and uncontrolled expression of the viral oncogenes. All these results have been obtained in transfected cells but no information is available on the E2 effects in the context of the entire organism. Transgenic mice were generated expressing the E2 protein of HPV11 under the control of the Ubiquitin C promoter. E2 mRNA is present in all mice tissues analysed and the E2 protein expressed in the skin (the target tissue of HPV11) was shown by Western blotting, albeit at a very low level. Analysis of the transgenic mice shows no major histological changes in the skin or all other tissues investigated. These data indicate that in transgenic mice the human papillomavirus type 11 E2 does not grossly modulate cellular proliferation or differentiation events.

Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein.

Using chemical inhibitors and small interfering RNA (siRNA), we have confirmed roles for cathepsin B (CatB) and cathepsin L (CatL) in Ebola virus glycoprotein (GP)-mediated infection. Treatment of Ebola virus GP pseudovirions with CatB and CatL converts GP1 from a 130-kDa to a 19-kDa species. Virus with 19-kDa GP1 displays significantly enhanced infection and is largely resistant to the effects of the CatB inhibitor and siRNA, but it still requires a low-pH-dependent endosomal/lysosomal function. These and other results support a model in which CatB and CatL prime GP by generating a 19-kDa intermediate that can be acted upon by an as yet unidentified endosomal/lysosomal enzyme to trigger fusion.

The HPV-16 E5 protein inhibits TRAIL- and FasL-mediated apoptosis in human keratinocyte raft cultures.

By using raft cultures of the polyclonal HaCaT cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference, we investigated the effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis. In comparison to conventional monolayer cultures this model system allows analysis of apoptosis under more tissue-like conditions by mimicking the stratified organization of a normal surface epithelium. Apoptosis was triggered either by FasL or TRAIL. Execution of the death program was checked at early and late stages by monitoring procaspase-3 cleavage and DNA fragmentation, respectively. Rafts of E5-expressing keratinocytes were completely protected from apoptosis and showed a background of apoptotic cells as low as the untreated cultures. In contrast, the HaCaT/pMSG cultures revealed a dramatic increase in apoptotic cells upon ligand treatment throughout the epithelial compartment. We conclude that the presence of the HPV-16 E5 protein in our tissue-like model prevents FasL- or TRAIL-mediated apoptosis.

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.

Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitol.

Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 alpha and beta kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway.

Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process.

Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental evidence that in these cells, cPLA(2) is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E(2) formation was detected. Elucidating the mechanism of cPLA(2) upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR) activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA(2) phosphorylation. Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress kinase, inhibited cPLA(2) phosphorylation, indicating that the ras-raf-MEK cascade is the major signalling pathway involved in cPLA(2) phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA(2) phosphorylation, suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress phosphorylates cPLA(2) in human keratinocytes by an EGFR-mediated process.

The human papillomavirus type 16 (HPV-16) E5 protein sensitizes human keratinocytes to apoptosis induced by osmotic stress.

We have studied the role of the HPV-16 E5 protein in apoptosis, using HaCaT cell lines stably transfected with either E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as control. When subjected to a hyperosmolar concentration of sorbitol, HaCaT/E5 cells respond with cytochrome c release, activation of caspase-3, -8, and -9, and PARP-cleavage, showing that the mitochondria and death-receptor mediated apoptotic pathways are involved in subsequent cell death. Similar effects are observed for the control cells only after extended sorbitol treatment. Thus, E5-expressing cells are more sensitive to osmotic stress, perhaps because of modifications of the cellular membranes caused by this strongly hydrophobic molecule.