Activation of the aryl hydrocarbon receptor sensitises human keratinocytes for CD95L- and TRAIL-induced apoptosis.

In this study, we have analysed the apoptotic effects of the ubiquitous environmental toxin benzo[a]pyrene (BP) in HaCaT cells and human keratinocytes. Although prolonged exposure to BP was not cytotoxic on its own, a strong enhancement of CD95 (Fas)-mediated apoptosis was observed with BP at concentrations activating the aryl hydrocarbon receptor (AhR). Importantly, the ultimately mutagenic BP-metabolite, that is, (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE), failed to enhance CD95-mediated cell death, suggesting that the observed pro-apoptotic effect of BP is neither associated with DNA adducts nor DNA-damage related signalling. CD95-induced apoptosis was also enhanced by ß-naphtoflavone, a well-known agonist of the AhR that does not induce DNA damage, thus suggesting a crucial role for AhR activation. Consistently, BP failed to sensitise for CD95L-induced apoptosis in AhR knockdown HaCaT cells. Furthermore, inhibition of CYP1A1 and/or 1B1 expression did not affect the pro-apoptotic crosstalk. Exposure to BP did not increase expression of CD95, but led to augmented activation of caspase-8. Enhancement of apoptosis was also observed with the TRAIL death receptors that activate caspase-8 and apoptosis by similar mechanisms as CD95. Together, these observations indicate an interference of AhR signalling with the activity of receptor-associated signalling intermediates that are shared by CD95 and TRAIL receptors. Our data thus suggest that AhR agonists can enhance cytokine-mediated adversity upon dermal exposure.

TNF-like weak inducer of apoptosis inhibits proinflammatory TNF receptor-1 signaling.

Soluble TNF-like weak inducer of apoptosis (TWEAK) trimers induce, in a variety of cell lines, translocation of cytosolic tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) to a triton X-100-insoluble compartment without changes in the total cellular TRAF2 content. TWEAK-induced TRAF2 translocation is paralleled by a strong increase in nuclear factor kappaB 2 (NFkappaB2)/p100 processing to p52, indicating that TRAF2 redistribution is sufficient for activation of the alternative NFkappaB pathway. In accordance with the crucial role of TRAF2 in proinflammatory, anti-apoptotic TNF receptor-1 (TNFR1) signaling, we observed that TWEAK-primed cells have a reduced capacity to activate the classical NFkappaB pathway or JNK (cJun N-terminal kinase) in response to TNF. Furthermore, TWEAK-primed cells are sensitized for the TNFR1-mediated induction of apoptotic and necrotic cell death. Notably, the expression of the NFkappaB-regulated, TRAF2-interacting TRAF1 protein can attenuate TWEAK-induced depletion of the triton X-100-soluble TRAF2 fraction and improve TNFR1-induced NFkappaB signaling in TWEAK-primed cells. Taken together, we demonstrate that soluble TWEAK desensitizes cells for proinflammatory TNFR1 signaling and thus identify TWEAK as a modifier of TNF signaling.

Tumor necrosis factor receptor-associated factor-1 enhances proinflammatory TNF receptor-2 signaling and modifies TNFR1-TNFR2 cooperation.

It has been shown that tumor necrosis factor receptor-2 (TNFR2) stimulation leads to degradation of TNF receptor associated factor-2 (TRAF2) and inhibition of TNFR1-induced activation of NFkappaB and JNK. Here, we show that TRAF1 inhibits TNFR2-induced proteasomal degradation of TRAF2 and relieves TNFR1-induced activation of NFkappaB from the inhibitory effect of TNFR2. TRAF1 co-recruited with TRAF2 to both TNF receptors. Despite lacking an amino-terminal RING/zinc-finger domain, TRAF1 did not interfere with TNFR1-induced activation of JNK and NFkappaB. It is noted that physiological expression levels of TRAF1 enhanced NFkappaB activation and interleukin-8 (IL8) production induced by TNFR2. Thus, TRAF1 shifts the quality of integrated TNFR1-TNFR2 signaling from apoptosis induction to proinflammatory NFkappaB signaling.

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis.

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.

Subcellular localisation of the X protein in HBV infected hepatocytes.

Hepatitis B virus X protein (HBx) is a multifunctional protein that exerts its effects primarily by acting as a transcriptional transactivator of viral and multiple host cell genes. HBx is thought to be essential for maintaining viral replication and has been implicated in the development of hepatocellular carcinoma in patients chronically infected with hepatitis B virus. Very little is known about its functional mechanisms and although interactions with several nuclear and cytoplasmic proteins have been demonstrated in vitro, there is no clear consensus as to where HBx localises in infected hepatocytes. In this study, the expression and intracellular distribution of HBx were examined in human liver biopsies using an anti-HBx rabbit polyclonal antiserum. HBx was detected in a high proportion (69%) of samples from patients with chronic HBV infection. Detection of HBx correlated with the absence of cirrhosis and the presence of serum e-antigen. HBx was detected predominantly in the cytoplasm; however, it was also found in the nuclei of up to 20% of positively stained hepatocytes, either exclusively nuclear or localised both in the nucleus and cytoplasm within the same cell. Furthermore, the intracellular distribution of HBx was analysed in transfected Huh-7 cells by confocal microscopy, using the monoclonal antibody 16F1. In these experiments, a substantial nuclear detection was confirmed in a significant proportion of HBx expressing cells. The data indicate a high functional significance of nuclear HBx, consistent with the concept that transactivation may involve interactions with nuclear proteins.

The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators.

The TNF-receptor-associated factor (TRAF) family is a phylogenetically conserved group of scaffold proteins that link receptors of the IL-1R/Toll and TNF receptor family to signalling cascades, leading to the activation of NF-kappaB and mitogen-activated protein kinases. Furthermore, TRAF proteins serve as a docking platform for a variety of regulators of these signalling pathways and are themselves often regulated at the transcriptional and posttranslational level. In this review, we address the structural and molecular basis of TRAF protein functions and highlight their role in cytokine signalling.

p53 upregulates cFLIP, inhibits transcription of NF-kappaB-regulated genes and induces caspase-8-independent cell death in DLD-1 cells.

One of the main functions of the tumor suppressor p53 is the induction of programmed cell death. Here we investigated in detail the molecular mechanisms that underlay p53 transactivation-dependent apoptosis in the human colon cancer cell line DLD-1. Although p53 upregulated the death receptors Fas, TRAIL-R1 and TRAIL-R2 in this cell line, p53-induced cell death occurred without detectable caspase-8 activation whereas, activation of caspase-9 and caspase-3 was readily observed. In addition to the upregulation of death receptors, p53 induced the pro-apoptotic Bcl-2 family members Bik and Bak and downregulated the anti-apoptotic Bcl-xL protein. Moreover, in RNase protection assay analyses as well as in reporter gene analyses we found a p53-dependent upregulation of the death receptor-inhibitory protein cFLIP. Together, these data argue for a p53-mediated activation of the mitochondrial pathway of apoptosis. In contrast to recently published data obtained in different cellular systems, there was no evidence for an essential role of NF-kappaB in p53-induced cell death. Moreover, induction of p53 interfered with TNF-induced NF-kappaB activation independently from apoptosis-induction.

Trangenic misexpression of the differentiation-specific desmocollin isoform 1 in basal keratinocytes.

Keratinocytes undergoing terminal differentiation are characterized by well-defined changes in protein expression, which contribute towards the transformation of cytoarchitecture and epithelial morphology. Characteristic patterns of desmosomal cadherins are tightly regulated and distinct isoforms are expressed during development and differentiation of epithelial tissues. Desmocollin-1 is strictly confined to suprabasal layers of epidermis, but it is absent in mitotically active, basal keratinocytes. This raises the question of whether basal desmocollin-1 could alter desmosomal functions and compromise keratinocyte proliferation, stratification, or early differentiation in skin. In this study, we misexpressed human desmocollin-1 in mouse epidermis, under control of the keratin-14 promoter. Transgenic animals were generated, which showed a specific expression of transgenic human desmocollin-1 in epidermal basal cells. High level transgenic expression, which was equal to or greater than endogenous protein levels, was observed in mice with multiple copy integration of the transgene. A punctate distribution of desmocollin-1 was demonstrated at the cell membrane by indirect immunofluorescence. Transgenic human desmocollin-1 colocalized with endogenous desmosomal marker proteins, indicating efficient incorporation into desmosomes. Transgenic mice did not display any obvious abnormalities, either in the histology of skin and hair follicles, or in the ultrastructure of desmosomes. These observations suggest that desmocollin-1 can function as a desmosomal cadherin both in basal and suprabasal cells. We propose that the differentiation-specific desmocollin isoforms desmocollin-1 and desmocollin-3 are functionally equivalent in basal epidermal cells and suggest that their changing expression patterns are markers, but not regulators, of the initial steps in keratinocyte differentiation.

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells.

Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV-HBx reporter plasmid and the pCMV-CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in approximately 50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and approximately 25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.

Erk-independent partial activation of AP-1 sites by the hepatitis B virus HBx protein.

The hepatitis B virus X protein (HBx) is suggested to regulate transcription by stimulation of intracellular signalling pathways. We have analysed the effects of HBx on activation of the MAP kinase (Erk) and JNK/SAPK signalling pathways and confirm a stimulation of the Erk/MAP kinase in quiescent cells. However, a substantial Erk-independent activation of AP-1, and phosphorylation of c-Jun (serine-63), but not Erk-2, was induced by HBx in dividing, serum-maintained cells. These data suggest that HBx promiscuously activates Erk and JNK responsive pathways and that its overall effect on signalling may be influenced by external mitogenic stimuli.

Hepatitis B virus transcriptional activators: mechanisms and possible role in oncogenesis.

The hepatitis B virus (HBV) genome encodes a 154 amino acid protein termed X (HBx, hepatitis B x protein), which is a promiscuous transcriptional activator of polymerase II and III promoters. HBx upregulates a wide range of cellular and viral genes and is thought to facilitate viral pregenome and mRNA transcription; however, its precise role in the viral replication cycle remains to be elucidated. The functional mechanisms of HBx appear very complex. It was shown to activate transcription factors AP-1 and NF-kappa B vis cytoplasmic pathways including ras-MAP kinase. In contrast, nuclear HBx is thought to activate the transcriptional machinery directly. A second transcriptional activator protein (Mst, middle s transactivator) is encoded by 3'-truncated preS2/S sequences of integrated HBV DNA, but not by the intact viral gene. HBx and Mst may contribute to the pathogenicity of chronic hepatitis B and are suggested to promote hepatocyte transformation via upregulation of cellular proto-oncogenes. Further, HBx may enhance HBV related carcinogenesis by inactivation of the tumour suppressor gene product p53.

Mutant p53 but not hepatitis B virus X protein is present in hepatitis B virus-related human hepatocellular carcinoma.

Hepatitis B virus (HBV) X protein is thought to play an important role in the development of hepatocellular carcinoma. Recent studies on a transgenic mouse tumor model suggest that HBV X protein may contribute to transformation by binding to and inactivating the cellular growth suppressor protein p53. We have studied 31 hepatocellular carcinoma tissues from Chinese patients for the possible occurrence of such interactions. Although most of the samples contained markers of HBV infection, including free and/or integrated HBV DNA, there was no detectable expression of HBV X protein by Western blot, immunoprecipitation, or histochemical staining. There was also no evidence of HBV X protein associated with p53 immunoprecipitated from the tumors. These observations suggest that, in naturally occurring human hepatocellular carcinoma, such interactions are uncommon and, therefore, unlikely to be of relevance in the latter stages of tumor development. On the other hand, 29 of 31 (93%) samples contained mutated forms of p53, as determined by various antibodies that detect wild-type or mutant p53 or both, and by the association of heat shock protein 70 with immunoprecipitated p53. These results show that conformationally altered p53 protein is present in tumors at a much higher frequency than is suggested by the presence of known mutations in the gene. This mutant p53 is functionally inactive, as suggested by the lack of expression of the p53-induced M(r) 21,000 Cip1/Waf1 protein in the tumors. Because this inactivation of p53 was not correlated with the expression of HBV X protein, any interaction of HBV X protein with p53 may be relevant only during acute infection. Such an interaction could serve to relax cell growth control at a time when virus replication requires hepatocyte destruction to be balanced by regeneration.

A mitochondrial elongation factor-like protein is over-expressed in tumours and differentially expressed in normal tissues.

The tissue-specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43. Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have levels of oxidative metabolism. Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues. The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17. The deduced amino acid sequence of P43 is remarkably similar to that of E. coli EF-Tu and the mitochondrial EF-Tu of S. cerevisiae with the structurally and functionally important amino acids of EF-Tu being completely conserved in P43. A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43. Taken together these results suggest that P43 is a human mitochondrial elongation factor.