RESUMO
The p63 transcription factor, homolog to the p53 tumor suppressor gene, plays a crucial role in epidermal and limb development, as its mutations are associated to human congenital syndromes characterized by skin, craniofacial and limb defects. While limb and skin-specific p63 transcriptional targets are being discovered, little is known of the post-translation modifications controlling ΔNp63α functions. Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions. Furthermore we show that Fibroblast Growth Factor-8 (FGF8), a morphogenetic signaling molecule essential for embryonic limb development, increases the binding of ΔNp63α to the tyrosine kinase c-Abl as well as the levels of ΔNp63α acetylation. Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway. This mutant ΔNp63α protein displays promoter-specific loss of DNA binding activity and consequent altered expression of development-associated ΔNp63α target genes. Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity. Hence, limb malformation-causing p63 mutations, such as the K193E mutation, are likely to result in aberrant limb development via the combined action of altered protein stability and altered promoter occupancy.
Assuntos
Anormalidades Congênitas/genética , Fator 8 de Crescimento de Fibroblasto/genética , Proteínas Proto-Oncogênicas c-abl/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Fatores de Transcrição de p300-CBP/genética , Animais , Linhagem Celular , Anormalidades Congênitas/embriologia , Anormalidades Congênitas/patologia , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Fator 8 de Crescimento de Fibroblasto/biossíntese , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/biossíntese , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Fatores de Transcrição de p300-CBP/biossíntese , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The transcription factor interferon regulatory factor 6 (IRF6) regulates craniofacial development and epidermal proliferation. We recently showed that IRF6 is a component of a regulatory feedback loop that controls the proliferative potential of epidermal cells. IRF6 is transcriptionally activated by p63 and induces its proteasome-mediated down-regulation, thereby limiting keratinocyte proliferative potential. We hypothesized that IRF6 may also be involved in skin carcinogenesis. Hence, we analyzed IRF6 expression in a large series of squamous cell carcinomas (SCCs) and found a strong down-regulation of IRF6 that correlated with tumor invasive and differentiation status. IRF6 down-regulation in SCC cell lines and primary tumors correlates with methylation on a CpG dinucleotide island located in its promoter region. To identify the molecular mechanisms regulating IRF6 potential tumor suppressive activity, we performed a genome-wide analysis by combining ChIP sequencing for IRF6 binding sites and gene expression profiling in primary human keratinocytes after siRNA-mediated IRF6 depletion. We observed dysregulation of cell cycle-related genes and genes involved in differentiation, cell adhesion, and cell-cell contact. Many of these genes were direct IRF6 targets. We also performed in vitro invasion assays showing that IRF6 down-regulation promotes invasive behavior and that reintroduction of IRF6 into SCC cells strongly inhibits cell growth. These results indicate a function for IRF6 in suppression of tumorigenesis in stratified epithelia.
Assuntos
Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Fatores Reguladores de Interferon/fisiologia , Proteínas Supressoras de Tumor , Fenômenos Fisiológicos Celulares/genética , Proliferação de Células , Metilação de DNA , Humanos , Fatores Reguladores de Interferon/genética , Queratinócitos/patologia , Invasividade Neoplásica/genética , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Psoriasis (Ps) is a common and stigmatizing chronic inflammatory skin disease that may cause other chronic inflammatory conditions with overlapping pathology, such as rheumatoid arthritis (RA). Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a pivotal role in chronic inflammatory and autoimmune diseases such as uveitis, multiple sclerosis, systemic lupus, arthritis, Ps, and Crohn's disease. The TNF superfamily and receptors represent active targets for drug development. Anti-TNF biological therapies, such as infliximab, adalimumab (ADL), and etanercept, are effective in treating RA, spondyloarthritis, Ps, and inflammatory bowel diseases, but long-term treatment can induce anti-drug antibody (ADA) formation associated with lower drug levels and clinical nonresponse. An investigation of the relationship between serum ADL/anti-adalimumab antibody (AAA) concentration, and clinical response in moderate to severe Ps, confirmed an association between ADL and AAA levels and response. Although the detection of ADAs can be used to determine the cause of nonresponse and aid therapy decisions, the contrary observation of long-term responders with low drug levels and detectable ADA suggests that another mechanism is also involved.
Assuntos
Anticorpos Monoclonais Humanizados , Anticorpos/sangue , Antirreumáticos , Psoríase/sangue , Psoríase/tratamento farmacológico , Adalimumab , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/sangue , Antirreumáticos/imunologia , Antirreumáticos/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Humanos , Psoríase/imunologia , Qualidade de Vida , Índice de Gravidade de Doença , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
The transforming growth factor type beta-1 (TGF-beta) signaling pathway is a major tumor suppressor during early carcinogenesis, and its growth-suppressive activity is commonly lost during early tumor progression. IkappaB kinase alpha (IKKalpha) also acts as a tumor suppressor in stratified epithelia, and its expression and nuclear localization are progressively down-regulated during malignant progression of squamous cell carcinoma (SCC) and acquisition of an invasive phenotype. A critical role for IKKalpha in TGF-beta signaling in stratified epithelia was identified recently during normal keratinocyte differentiation, and both IKKalpha and components of the TGF-beta signaling pathway are required for induction of antiproliferative Myc antagonists in such cells. We now describe that the interaction between IKKalpha and the TGF-beta signaling pathway is also important in a subset of SCCs. In SCCs that are unable to shuttle IKKalpha to the nucleus, defective TGF-beta-induced growth arrest was rescued by introduction of a constitutively nuclear IKKalpha variant. These results suggest that the tumor-suppressive activity of IKKalpha in stratified epithelia may be exerted in part via the TGF-beta signaling pathway.
Assuntos
Proliferação de Células , Epitélio/metabolismo , Genes Supressores de Tumor , Quinase I-kappa B/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinoma de Células Escamosas , Regulação para Baixo , Epitélio/enzimologia , Humanos , Quinase I-kappa B/análise , Quinase I-kappa B/genética , Queratinócitos/metabolismo , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas c-myc , Fator de Crescimento Transformador beta/genéticaRESUMO
Dominant mutations in TP63 cause ankyloblepharon ectodermal dysplasia and clefting (AEC), an ectodermal dysplasia characterized by skin fragility. Since DeltaNp63alpha is the predominantly expressed TP63 isoform in postnatal skin, we hypothesized that mutant DeltaNp63alpha proteins are primarily responsible for skin fragility in AEC patients. We found that mutant DeltaNp63alpha proteins expressed in AEC patients function as dominant-negative molecules, suggesting that the human AEC skin phenotype could be mimicked in mouse skin by downregulating DeltaNp63alpha. Indeed, downregulating DeltaNp63 expression in mouse epidermis caused severe skin erosions, which resembled lesions that develop in AEC patients. In both cases, lesions were characterized by suprabasal epidermal proliferation, delayed terminal differentiation, and basement membrane abnormalities. By failing to provide structural stability to the epidermis, these defects likely contribute to the observed skin fragility. The development of a mouse model for AEC will allow us to further unravel the genetic pathways that are normally regulated by DeltaNp63 and that may be perturbed in AEC patients. Ultimately, these studies will not only contribute to our understanding of the molecular mechanisms that cause skin fragility in AEC patients, but may also result in the identification of targets for novel therapeutic approaches aimed at treating skin erosions. (c) 2009 Wiley-Liss, Inc.
Assuntos
Anormalidades Múltiplas/patologia , Fenda Labial/patologia , Fissura Palatina/patologia , Modelos Animais de Doenças , Displasia Ectodérmica/patologia , Pálpebras/anormalidades , Pele/patologia , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Animais , Diferenciação Celular , Fenda Labial/diagnóstico , Fenda Labial/genética , Fissura Palatina/diagnóstico , Fissura Palatina/genética , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Células Epidérmicas , Pé/patologia , Mãos/patologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Síndrome , Transativadores/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genéticaRESUMO
Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis and vasculopathy. CXCL4 represents an early serum biomarker of severe SSc and likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc, in which CXCL4 organizes "self" and microbial DNA into liquid crystalline immune complexes that amplify TLR9-mediated plasmacytoid dendritic cell (pDC)-hyperactivation and interferon-α production. Surprisingly, this activity does not require CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Thus, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and outline a paradigm in which chemokines can drastically modulate innate immune receptors without being direct agonists.
Assuntos
DNA Bacteriano/metabolismo , Interferon-alfa/metabolismo , Fator Plaquetário 4/metabolismo , Escleroderma Sistêmico/imunologia , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Biópsia , Estudos de Casos e Controles , DNA Bacteriano/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Interferon-alfa/imunologia , Cristais Líquidos , Masculino , Pessoa de Meia-Idade , Fator Plaquetário 4/imunologia , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismo , Escleroderma Sistêmico/microbiologia , Escleroderma Sistêmico/patologia , Pele/citologia , Pele/imunologia , Pele/microbiologia , Pele/patologia , Receptor Toll-Like 9/imunologiaRESUMO
Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA via unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Artrite Psoriásica/imunologia , Autoanticorpos/imunologia , Líquido Sinovial/imunologia , Adolescente , Adulto , Peptídeos Catiônicos Antimicrobianos/metabolismo , Artrite Psoriásica/metabolismo , Autoanticorpos/metabolismo , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Carbamilação de Proteínas/imunologia , Líquido Sinovial/metabolismo , CatelicidinasRESUMO
The p53 protein plays a pivotal role in determining the quality of the response to DNA damage through its transcriptional activity. Upon DNA damage, p53 is activated by post-translational modifications, binds its cognate sequences on the promoters of its target genes and stimulates transcription. In proliferating keratinocytes, the activity of p53 is blunted by its inhibitor DeltaNp63alpha. Here, we describe a novel mechanism through which DeltaNp63 functions in order to prevent the survival and propagation of ultraviolet (UV)-damaged keratinocytes. We found that UVB stimulation induces the rapid phosphorylation of DeltaNp63, which precedes DeltaNp63 transcriptional downregulation and protein degradation, which is mediated by the p38 MAPK. Phosphorylated DeltaNp63 has a lower affinity for p53REs and detaches from cell cycle arrest and apoptotic promoters, thus allowing the rapid activation of p53-dependent transcriptional apoptotic program.
Assuntos
Apoptose/efeitos da radiação , DNA/metabolismo , Queratinócitos/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Humanos , Queratinócitos/efeitos da radiação , Fosforilação , Ligação Proteica , Fatores de Transcrição , Proteínas Supressoras de TumorRESUMO
OBJECTIVES: An observational study to evaluate the relationship between serum concentrations of adalimumab and disease activity in patients receiving long-term adalimumab treatment for psoriatic arthritis. METHODS: Serum adalimumab and adalimumab antidrug antibodies were quantified by enzyme linked immunosorbent assay. Disease activity was assessed using Disease Activity Score (44 joint measures). Serum C-reactive protein was quantified using standard methods. RESULTS: A total of 30 patients were recruited. There were significant inverse correlations between serum adalimumab concentration and serum C-reactive protein (CRP) concentration [r = -0.43], the number of tender joints (r = -0.4), and Disease Activity Score (DAS44)-CRP (r = -0.36). Mean serum adalimumab levels were significantly higher in patients with DAS44-CRP <1.6 than in patients with DAS44-CRP ≥1.6. CONCLUSIONS: Serum adalimumab could be an important tool that may improve the management of psoriatic arthritis in patients responding to long-term treatment.
RESUMO
Tazarotene, a member of the new class of acetylenic retinoids, has been shown to be effective in the treatment of several hyperproliferative skin diseases, including non-melanoma skin cancer. Its effectiveness is thought to rely on the ability to activate retinoic acid receptors beta and gamma and to induce a number of downstream anti-proliferative genes. Here, we show that the p53-related gene p73 is a target of tazarotene. Indeed, tazarotene modulates the expression of the p73 gene in immortalized keratinocyte cell lines by inducing the pro-apoptotic and anti-proliferative TAp73 isoforms and by repressing the anti-apoptotic and pro-proliferative DeltaNp73 isoforms. This occurs at the transcriptional level through a coordinated action on P1p73 and P2p73 promoters that control the expression of TA and DeltaN isoforms, respectively. The selective downregulation of DeltaNp73 expression by small interfering RNA led to an enhancement of tazarotene-induced bax activation and apoptosis, whereas the downregulation of both TA and DeltaN isoforms impairs tazarotene-mediated apoptosis. These results indicate the relevance of p73 gene products in tazarotene-induced growth inhibition and effectiveness in the treatment of skin tumors.
Assuntos
Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Queratinócitos/fisiologia , Ceratolíticos/farmacologia , Ácidos Nicotínicos/farmacologia , Proteínas Nucleares/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Genes Supressores de Tumor , Humanos , Queratinócitos/citologia , Camundongos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno , Receptores do Ácido Retinoico/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Receptor gama de Ácido RetinoicoRESUMO
Psoriasis is a common T-cell-mediated skin disease with 2-3% prevalence worldwide. Psoriasis is considered to be an autoimmune disease, but the precise nature of the autoantigens triggering T-cell activation remains poorly understood. Here we find that two-thirds of patients with moderate-to-severe plaque psoriasis harbour CD4(+) and/or CD8(+) T cells specific for LL37, an antimicrobial peptide (AMP) overexpressed in psoriatic skin and reported to trigger activation of innate immune cells. LL37-specific T cells produce IFN-γ, and CD4(+) T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and may be tracked in patients blood by tetramers staining. Presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. Thus, we uncover a role of LL37 as a T-cell autoantigen in psoriasis and provide evidence for a role of AMPs in both innate and adaptive immune cell activation.
Assuntos
Autoantígenos/imunologia , Catelicidinas/imunologia , Psoríase/imunologia , Linfócitos T/imunologia , Peptídeos Catiônicos Antimicrobianos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica , Índice de Gravidade de DoençaRESUMO
The human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the DeltaNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasome-mediated DeltaNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.
Assuntos
Regulação da Expressão Gênica , Fatores Reguladores de Interferon/metabolismo , Mutação , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Displasia Ectodérmica , Humanos , Queratinócitos/citologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Fenótipo , Pele/patologia , Fatores de TranscriçãoRESUMO
The transcription factor p63 plays a pivotal role in the development and differentiation of the epidermis and epithelial appendages. Indeed, mutations in p63 are associated with a group of ectodermal dysplasias characterized by skin, limb, and craniofacial defects. It was hypothesized that p63 exerts its functions by activating specific genes during epidermal development, which in turn regulate epidermal stratification and differentiation. We have identified I-kappaB kinase alpha (IKKalpha) as a direct transcriptional target of p63 that is induced at early phases of terminal differentiation of primary keratinocytes. We show that the DeltaNp63 isoform is required for IKKalpha expression in differentiating keratinocytes and that mutant p63 proteins expressed in ectodermal dysplasia patients exhibit defects in inducing IKKalpha. Furthermore, we observed reduced IKKalpha expression in the epidermis of an ankyloblepharon ectodermal dysplasia clefting patient. Our data demonstrate that a failure to properly express IKKalpha may play a role in the development of ectodermal dysplasias.
Assuntos
Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Transativadores/fisiologia , Transcrição Gênica , Proteínas Supressoras de Tumor/fisiologia , Linhagem Celular Tumoral , Displasia Ectodérmica/patologia , Regulação Enzimológica da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Modelos Biológicos , Mutação , Isoformas de Proteínas , Interferência de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição , TransfecçãoRESUMO
The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of DeltaNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (-80%). DeltaNp63alpha binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a -1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon-Ectodermal dysplasia-Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias.
Assuntos
Epitélio/embriologia , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/fisiologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Claudina-1 , Regulação para Baixo , Ectoderma/metabolismo , Epiderme/metabolismo , Genes Reporter , Queratinócitos/citologia , Camundongos , Modelos Biológicos , Mutação , Isoformas de Proteínas , Pele/metabolismoRESUMO
Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (DeltaN) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. DeltaNp63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that DeltaNp63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two DeltaNp63alpha target genes that mediate these processes. We propose that DeltaNp63alpha initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal-dermal interface at the basement membrane. Subsequently, induction of IkappaB kinase-alpha by DeltaNp63alpha initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of DeltaNp63alpha in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.
Assuntos
Epiderme/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Queratinócitos/citologia , Morfogênese/genética , Fosfoproteínas/genética , Transativadores/genética , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Primers do DNA , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Morfogênese/fisiologia , Potoroidae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização/fisiologiaRESUMO
Herpes simplex viruses (HSVs) are able to hijack the host-cell IkappaB kinase (IKK)/NF-kappaB pathway, which regulates critical cell functions from apoptosis to inflammatory responses; however, the molecular mechanisms involved and the outcome of the signaling dysregulation on the host-virus interaction are mostly unknown. Here we show that in human keratinocytes HSV-1 attains a sophisticated control of the IKK/NF-kappaB pathway, inducing two distinct temporally controlled waves of IKK activity and disrupting the NF-kappaB autoregulatory mechanism. Using chromatin immunoprecipitation we demonstrate that dysregulation of the NF-kappaB-response is mediated by a virus-induced block of NF-kappaB recruitment to the promoter of the IkappaBalpha gene, encoding the main NF-kappaB-inhibitor. We also show that HSV-1 redirects NF-kappaB recruitment to the promoter of ICP0, an immediate-early viral gene with a key role in promoting virus replication. The results reveal a new level of control of cellular functions by invading viruses and suggest that persistent NF-kappaB activation in HSV-1-infected cells, rather than being a host response to the virus, may play a positive role in promoting efficient viral replication.
Assuntos
Regulação da Expressão Gênica , Genes Virais , Proteínas I-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Simplexvirus/metabolismo , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Primers do DNA/química , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Inflamação , Queratinócitos/metabolismo , Modelos Genéticos , Inibidor de NF-kappaB alfa , Plasmídeos/metabolismo , Prostaglandinas A/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transfecção , Ubiquitina-Proteína Ligases/metabolismo , Raios UltravioletaRESUMO
The p53 family of transcription factors plays a pivotal role in the control of the cellular response to DNA damaging agents. In addition to pro-apoptotic molecules such as p53, TAp73 and TAp63, this gene family also encodes for the anti-apoptotic molecules deltaNp73, deltaNp63, deltaNp53, and p53 mutants are often found in tumor cells, that have the role to limit and to modulate the pro-apoptotic side of the family. The ratio between the different members of the family is critical to make the life or death decision following DNA damage and is tightly regulated by post-translational and transcriptional mechanisms. In this study we have uncovered a novel positive feedback that involves the transcriptional activation of the anti-apoptotic molecule deltaNp63 by the anti-apoptotic molecules deltaNp73 and mutant p53, and that is put into motion upon treatment with a subset of DNA damaging agents such as Doxorubicin and 5-FU. DeltaNp73 and mutant p53 associate with the deltaNp63 promoter inducing its transcription and this is enhanced by doxorubicin treatment. Furthermore we have observed that deltaNp73- and mutp53-mediated activation of the deltaNp63 promoter requires the functionality of the proximal CCAAT boxes of this promoter, being hampered by mutation of CCAAT boxes or by dominant negative NFYA expression. This mechanism may serve as an additional control of the response of a normal cell to DNA damage or as an anti-apoptotic barrier of cancer cells subjected to DNA damage.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/biossíntese , Doxorrubicina/farmacologia , Genes p53 , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transativadores/biossíntese , Transativadores/metabolismo , Fatores de Transcrição , Ativação Transcricional/efeitos dos fármacos , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/biossínteseRESUMO
In the present study, we aimed to demonstrate that CD4 may represent a critical turning point that governs the apoptotic and survival programs in T cells, without modifying the physical association with the TCR-CD3 complex. To address this issue, we have explored the possibility that the activation of CD4 may transduce apoptotic signals unless signaling effectors neutralize them. Our data show that in Jurkat T cells CD4 engagement by Leu3a mAb results in a rapid and strong increase of Lck kinase activity, subsequent alterations of mitochondrial membrane potential, and apoptosis. Critical parameters are coassociation of CD4/Lck with TCR/CD3 and up-regulation of the proapoptotic protein Bax. Indeed, Leu3a-mediated Lck activation failed to induce apoptotic features in Jurkat cells either defective for TCR/CD3 or overexpressing the antiapoptotic protein Bcl-2. Furthermore, we demonstrate that Leu3a treatment of Jurkat cells overexpressing Vav results in the inhibition of mitochondrial damage and apoptosis; this rescue effect is accompanied with a significant decrease of Bax expression observed in apoptotic cells. Our evidence that the activation of Lck activates in T cells apoptotic pathways which are counteracted by Vav, a signaling molecule that cooperates with CD28 to boost TCR signals, suggests a novel role for costimulation in protecting T cells from CD4-mediated cell death.
Assuntos
Antígenos CD4/fisiologia , Proteínas de Ciclo Celular , Fatores de Troca do Nucleotídeo Guanina , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas/biossíntese , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Anticorpos Monoclonais/metabolismo , Apoptose/imunologia , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Regulação para Baixo/imunologia , Ativação Enzimática/imunologia , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Membranas Intracelulares/imunologia , Membranas Intracelulares/patologia , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Potenciais da Membrana/imunologia , Mitocôndrias/imunologia , Permeabilidade , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas c-vav , Transdução de Sinais/imunologia , Transfecção , Proteína X Associada a bcl-2RESUMO
Several findings support the importance of GM1-enriched lipid microdomains of plasma membrane and of Vav, an essential regulator of actin cytoskeletal rearrangement, in the regulation of T cell activation. Moreover, a functional link among lipid microdomains, Vav and the HIV product Nef has been described. These observations suggest that Nef can modify plasma membrane GM1, affecting the behavior of HIV-infected cells towards antigen recognition and Vav towards counteracting such an effect. We observed that Nef expression, either following viral infection or ectopic expression, significantly decreased the level of plasma membrane GM1 in unstimulated T cells. This down-regulation was associated with the inhibition of NF-AT activation, but not with NF-kappaB activation induced by TCR engagement. Dissecting the signaling pathway that regulates NF-AT activation, we found that Nef inhibited exclusively the Ca(2+)/calcineurin cascade, whereas the JNK cascade and AP-1 transcriptional activity were not affected. Our evidence that Vav overexpression counteracted both the Nef-induced decrease of GM1 expression and the inhibition of NF-AT activity, suggests a novel mechanism by which Nef may interfere with TCR-mediated activation through the modulation of intracellular trafficking and clustering of GM1-enriched microdomains at the cell surface.