RESUMEN
Deregulation of the transforming acidic coiled-coil protein 3 (TACC3), an important factor in the centrosome-microtubule system, has been linked to a variety of human cancer types. We have recently reported on the oncogenic potential of TACC3; however, the molecular mechanisms by which TACC3 mediates oncogenic function remain to be elucidated. In this study, we show that high levels of TACC3 lead to the accumulation of DNA double-strand breaks (DSBs) and disrupt the normal cellular response to DNA damage, at least in part, by negatively regulating the expression of ataxia telangiectasia mutated (ATM) and the subsequent DNA damage response (DDR) signaling cascade. Cells expressing high levels of TACC3 display defective checkpoints and DSB-mediated homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, leading to genomic instability. Importantly, high levels of TACC3 confer cellular sensitization to radiation and poly(ADP-ribose) polymerase (PARP) inhibition. Overall, our findings provide critical information regarding the mechanisms by which TACC3 contributes to genomic instability, potentially leading to cancer development, and suggest a novel prognostic, diagnostic and therapeutic strategy for the treatment of cancer types expressing high levels of TACC3.
Asunto(s)
Daño del ADN , Proteínas Asociadas a Microtúbulos/fisiología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Tolerancia a Radiación , Aberraciones Cromosómicas , Roturas del ADN de Doble Cadena , Humanos , Proteínas Asociadas a Microtúbulos/análisis , Neoplasias/genéticaRESUMEN
The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.
RESUMEN
The protein kinase C (PKC) family represents a large group of phospholipid dependent enzymes catalyzing the covalent transfer of phosphate from ATP to serine and threonine residues of proteins. Phosphorylation of the substrate proteins induces a conformational change resulting in modification of their functional properties. The PKC family consists of at least ten members, divided into three subgroups: classical PKCs (alpha, betaI, betaII, gamma), novel PKCs (delta, epsilon, eta, theta), and atypical PKCs (zeta, iota/lambda). The specific cofactor requirements, tissue distribution, and cellular compartmentalization suggest differential functions and fine tuning of specific signaling cascades for each isoform. Thus, specific stimuli can lead to differential responses via isoform specific PKC signaling regulated by their expression, localization, and phosphorylation status in particular biological settings. PKC isoforms are activated by a variety of extracellular signals and, in turn, modify the activities of cellular proteins including receptors, enzymes, cytoskeletal proteins, and transcription factors. Accordingly, the PKC family plays a central role in cellular signal processing. Accumulating data suggest that various PKC isoforms participate in the regulation of cell proliferation, differentiation, survival and death. These findings have enabled identification of abnormalities in PKC isoform function, as they occur in several cancers. Specifically, the initiation of squamous cell carcinoma formation and progression to the malignant phenotype was found to be associated with distinct changes in PKC expression, activation, distribution, and phosphorylation. These studies were recently further extended to transgenic and knockout animals, which allowed a more direct analysis of individual PKC functions. Accordingly, this review is focused on the involvement of PKC in physiology and pathology of the skin.
Asunto(s)
Proteína Quinasa C/fisiología , Transducción de Señal/fisiología , Neoplasias Cutáneas/enzimología , Piel/enzimología , Animales , Epitelio/enzimología , Humanos , IsoenzimasRESUMEN
Protein kinase C (PKC)-delta is proapoptotic in human keratinocytes, and is downregulated or inactivated in keratinocytes expressing the activated Ha-ras oncogene, making it a candidate tumor suppressor gene for squamous cell carcinoma (SCC). We evaluated the significance of PKC-delta loss in transformed human keratinocytes using tumorigenic HaCaT Ras II-4 cells that have significantly reduced PKC-delta levels. Re-expression of PKC-delta by retrovirus transduction caused an increase in apoptosis and growth inhibition in culture. The growth inhibition induced by PKC-delta could be partially reversed by Bcl-x(L) expression, indicating that apoptosis was in part responsible for PKC-delta-induced growth inhibition. PKC-delta re-expression suppressed the tumorigenicity of HaCaT Ras II-4 cells in nude mice (P<0.05), and the small tumors that did form contained elevated levels of activated caspase-3, indicating increased apoptosis. In addition, we found that 29% (12/42) of human Bowen's disease (squamous carcinoma in situ) or SCC cases had absent or reduced PKC-delta when compared to the surrounding normal epidermis. These results indicate that PKC-delta inhibits transformed keratinocyte growth by inducing apoptosis, and that PKC-delta may function as a tumor suppressor in human SCCs where its loss in cells harboring activated ras could provide a growth advantage by conferring resistance to apoptosis.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Genes Supresores de Tumor , Proteína Quinasa C-delta/genética , Animales , Apoptosis , Carcinoma de Células Escamosas/genética , Línea Celular , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Quinasa C-delta/metabolismoRESUMEN
Keratinocyte apoptosis induced by UV radiation is a major protective mechanism from skin photocarcinogenesis. The induction of apoptosis by UV radiation, as well as a variety of genotoxic stimuli, involves the activation of PKC-delta by caspase-3-mediated cleavage in its hinge region, thus generating a constitutively active catalytic fragment. To determine the role of PKC-delta cleavage in UV apoptosis signaling, we introduced a caspase-resistant PKC-delta mutant (D330A) into human keratinocytes by retrovirus transduction. Overexpression of PKC-delta(D330A) protected keratinocytes from UV-induced apoptosis and enhanced long-term survival. PKC-delta(D330A) partially prevented the release of cytochrome c from the mitochondria and the loss of Mcl-1, a key antiapoptotic protein downregulated during UV apoptosis. Thus, the cleavage and activation of PKC-delta are critical components of UV-induced apoptosis in human keratinocytes, and the inactivation of PKC-delta can promote the survival of keratinocytes exposed to UV radiation.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Queratinocitos/citología , Proteína Quinasa C/metabolismo , Rayos Ultravioleta/efectos adversos , Caspasa 3 , Inhibidores de Caspasas , Dominio Catalítico , Supervivencia Celular , Células Cultivadas , Citocromos c/antagonistas & inhibidores , Regulación hacia Abajo , Activación Enzimática , Citometría de Flujo , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Potenciales de la Membrana , Mitocondrias/fisiología , Mitocondrias/efectos de la radiación , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/biosíntesis , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Proteína Quinasa C-delta , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesisRESUMEN
Establishing an effective epidermal barrier requires a series of coordinated molecular events involving keratinocytes (KCs) within a stratified epithelium. Epidermal maturation depends on convergence of pathways involving components of NF-kappaB and peroxisome proliferator activated receptor (PPAR) signaling systems that promote terminal differentiation and production of a stratum corneum. The Notch-1 receptor and its ligand Delta-1 have been proposed by others to participate in early events in KC differentiation. Here, we establish differential expression patterns for several Notch receptors and ligands in normal human skin. These immunolocalization findings, together with functional studies demonstrating increased levels of Notch ligand/receptors occurring during the onset of differentiation, prompted use of a soluble Notch ligand, a peptide derived from the most conspicuously expressed ligand in skin, Jagged-1. Exposing submerged KC monolayers to this peptide (JAG-1) in co-presence of elevated calcium ion concentration, produced stratification with loricrin expression. Using a living human epidermal equivalent (EE) model system, when submerged cultures were raised to an air/liquid interface to generate a fully mature epidermis, activation of Notch signaling was detected. Addition of JAG-1 peptide to submerged EEs was sufficient to induce epidermal maturation. Moreover, a soluble decoy Notch inhibitor prevented such differentiation and corneogenesis in human EEs exposed to either an air/liquid interface or to the JAG-1 peptide. In KC monolayers, addition of JAG-1 peptide induced IKKalpha mediated NF-kappaB activation, as well as increased PPARgamma expression. Immunoprecipitation/Western blot analysis revealed a physical association between the p65 subunit of NF-kappaB and PPARgamma. These results indicate that activation of Notch signaling is necessary for maturation of human epidermis, and activation by a soluble Notch ligand is sufficient to trigger complete KC differentiation including cornification.
Asunto(s)
Diferenciación Celular/fisiología , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Piel/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Adulto , Proteínas de Unión al Calcio , Compartimento Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Células Epidérmicas , Epidermis/metabolismo , Humanos , Quinasa I-kappa B , Inmunohistoquímica , Recién Nacido , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Masculino , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Receptor Notch1 , Receptor Notch2 , Receptor Notch4 , Receptores de Superficie Celular/metabolismo , Receptores Notch , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Piel/citología , Piel/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The induction of apoptosis in human keratinocytes by UV radiation involves caspase-mediated cleavage and activation of protein kinase C delta (PKCdelta). Here we examined the role of PKC activation in caspase activation and disruption of mitochondria function by UV radiation. Inhibition of PKC partially blocked UV radiation-induced cleavage of PKCdelta, pro-caspase-3, and pro-caspase-8, and the activation of these caspases. PKC inhibition also blocked the UV-induced loss of mitochondria membrane potential, but did not block the release of cytochrome c from mitochondria. Expression of the active catalytic domain of PKCdelta was sufficient to induce apoptosis and disrupt mitochondrial membrane potential, however a kinase inactive PKCdelta catalytic domain did not. Furthermore, the PKCdelta catalytic fragment generated following UV radiation localized to the mitochondria fraction, as did ectopically expressed PKCdelta catalytic domain. These results identify a functional role for PKC activation in potentiating caspase activation and disrupting mitochondrial function during UV-induced apoptosis.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Mitocondrias/efectos de la radiación , Proteína Quinasa C/metabolismo , Rayos Ultravioleta , Apoptosis/fisiología , Línea Celular , Activación Enzimática , Humanos , Isoenzimas/metabolismo , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Potenciales de la Membrana/efectos de la radiación , Mitocondrias/fisiología , Proteína Quinasa C-deltaRESUMEN
OBJECTIVE: The t(2;5)(p23;q35) translocation creates a fusion gene NPM-ALK (p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma (ALCL). Our study was aimed to analyze tyrosine kinase activity and phosphotyrosine in ALCLs. We were also interested in determining the effect of tyrosine kinase inhibitors on survival of ALCL. METHODS: Eleven cases of ALCL and three ALCL cell lines with t(2;5)(Karpas-299, SUPM2, SU-DHL-1) and 10 Hodgkin's disease (HD) samples were stained with anti-phosphotyrosine antibody. The tyrosine kinase activity, p80 phosphorylation, and the apoptotic effects of two tyrosine kinase inhibitors, herbimycin A and STI-571, were determined on ALCL cell lines. RESULTS: Herbimycin A had showed both a time- and dose-dependent apoptotic effect on all three cell lines, while STI-571 demonstrated a minimal effect. Following herbimycin A treatment, a decrease in tyrosine kinase activity in the ALCL cell lines and inhibition in NPM-ALK (p80) autophosphorylation was demonstrated by immunoprecipitation and Western blotting. Herbimycin A-induced apoptosis was accompanied by caspase-3 activation. Furthermore, apoptosis induced by herbimycin A was blocked by both z-VAD-FMK and z-DEVD-FMK, suggesting a critical role of caspases. CONCLUSIONS: These findings indicate that tyrosine kinase activity is a common characteristic of ALCLs and necessary for ALCL cell survival. These findings further suggest that therapies targeting tyrosine kinases, including p80, may have clinical utility.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/farmacología , Linfoma de Células B Grandes Difuso/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Antineoplásicos/farmacología , Benzamidas , Benzoquinonas , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 5 , Inhibidores Enzimáticos/farmacología , Enfermedad de Hodgkin/patología , Humanos , Mesilato de Imatinib , Inmunohistoquímica , Lactamas Macrocíclicas , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosforilación , Piperazinas/farmacología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Quinonas/farmacología , Proteínas Tirosina Quinasas Receptoras , Rifabutina/análogos & derivados , Translocación Genética , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
In mammalian epidermis, expression of the alpha6beta4 integrin is restricted to the hemidesmosome complexes, which connect the proliferative basal cell layer with the underlying basement membrane. Keratinocyte differentiation is associated with down-regulation of alpha6beta4 expression and detachment of keratinocytes from the basement membrane. Neoplastic keratinocytes delay maturation, proliferate suprabasally, and retain the expression of the alpha6beta4 integrin in suprabasal cells disassociated from the hemidesmosomes. We now show that the alpha6beta4 integrin is a substrate for serine phosphorylation by protein kinase C in keratinocytes. Furthermore, protein kinase C-mediated phosphorylation of alpha6beta4 is associated with redistribution of this integrin from the hemidesmosome to the cytosol. Specifically, in vitro kinase assays identified the protein kinase Cdelta as the primary isoform phosphorylating alpha6 and beta4 integrin subunits. Using recombinant protein kinase C adenoviruses, overexpression of protein kinase Cdelta but not protein kinase Calpha in primary keratinocytes increased beta4 serine phosphorylation, decreased alpha6beta4 localization to the hemidesmosome complexes, and reduced keratinocyte attachment. Taken together, these results establish a link between protein kinase Cdelta-mediated serine phosphorylation of alpha6beta4 integrin and its effects on alpha6beta4 subcellular localization and keratinocyte attachment to the laminin underlying matrix.
Asunto(s)
Antígenos de Superficie/metabolismo , Hemidesmosomas/metabolismo , Integrinas/metabolismo , Isoenzimas/metabolismo , Queratinocitos/metabolismo , Proteína Quinasa C/metabolismo , Animales , Antígenos de Superficie/fisiología , Adhesión Celular/fisiología , Activación Enzimática , Hemidesmosomas/fisiología , Homeostasis/fisiología , Integrina alfa6beta4 , Integrinas/fisiología , Isoenzimas/fisiología , Queratinocitos/citología , Queratinocitos/enzimología , Ratones , Ratones Endogámicos BALB C , Fosforilación , Proteína Quinasa C/fisiología , Proteína Quinasa C-deltaRESUMEN
The Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells. As demonstrated for differentiation-specific granular markers, Dlx3 is activated in primary mouse keratinocytes cultured in vitro by increasing the level of the extracellular Ca(2+). This activation is mediated through a protein kinase C-dependent (PKC) pathway. In this study, we investigated whether PKC can modulate the activity of murine Dlx3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the Dlx3 protein. Using keratinocyte nuclear extracts for the kinase reaction, we determined that Dlx3 protein is phosphorylated, and the phosphorylation is inhibited by the PKC-specific inhibitor GF109203X, suggesting that Dlx3 is phosphorylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested alpha, delta, epsilon and zeta. Dlx3 is primarily phosphorylated by PKC alpha. By deletion and mutational analysis, we show that the serine residue S(138), located in the homeodomain of Dlx3 protein, was specifically phosphorylated by PKC. The phosphorylation of purified Dlx3 proteins by PKC partially inhibited formation of complexes between Dlx3 protein and DNA. These results suggest that Dlx3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of Dlx3.
Asunto(s)
Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Proteína Quinasa C/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Sitios de Unión/fisiología , Diferenciación Celular , Núcleo Celular/química , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Homeodominio/genética , Indoles/farmacología , Isoenzimas/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Maleimidas/farmacología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/análisis , Fosforilación , Pruebas de Precipitina , Proteína Quinasa C/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
The transcriptional activation and proper regulation of NF-kappaB is known to be important to the apoptotic resistant phenotype of epidermal-derived keratinocytes. By comparing and contrasting the responses of normal foreskin-derived keratinocytes versus an immortalized skin-derived keratinocyte cell line (i.e. HaCaT cells), several molecular defects involving NF-kappaB signaling pathway were delineated in the immortalized keratinocytes. While exposure to IFN-gamma plus TPA produces growth arrest in both normal and immortalized keratinocytes, with rapid phosphorylation of MEKKI and recruitment of distinctive protein kinase C isoforms into the signalosome complex, subsequent molecular events necessary for NF-kappaB activation were abnormal in HaCaT cells. This disrupted NF-kappaB activation in HaCaT cells was accompanied by enhanced susceptibility to UV-light induced apoptosis, which was associated with elevated levels of E2F-1 and decreased TRAF1/TRAF2 levels. Additional defects in HaCaT cells included markedly diminished levels of IKKbeta (and lack of induction of kinase activity) in response to inflammatory stimuli, a failure of p21(WAF1/CIP1) to associate with CDK2, and a decreased association between p65 and p300. These studies suggest caution in using HaCaT cells as a substitute for normal keratinocytes to study apoptosis in the skin. Thus, it appears that while the immortalized cells can escape cell cycle checkpoints by elevated levels of E2F-1, an adverse biological consequence of such dysregulated cell cycle control is the inability to activate the anti-apoptotic NF-kappaB signaling pathway. Therefore, exploiting this apoptosis vulnerability in pre-malignant, or immortalized cells, prior to acquiring a death-defying phenotype characteristic of more advanced malignant cell types, provides the basis for an early interventional therapeutic strategy for cutaneous oncologists.
Asunto(s)
Apoptosis , Queratinocitos/metabolismo , Queratinocitos/patología , FN-kappa B/metabolismo , Transducción de Señal , Línea Celular Transformada , HumanosRESUMEN
Acute promyelocytic leukaemia (APL;M3) is a unique form of acute myelogenous leukaemia characterized by t(15;17) translocation. The induction of apoptosis via inhibiting protein kinase C (PKC) has been recently viewed as a promising tool for the eradication of several malignant disorders. In the present study, we investigated the effect of two different protein kinase C inhibitors, Gö6976 and safingol, on the induction of apoptosis in the APL cell line NB4 and its all trans retinoic acid (ATRA)-resistant variant NB4.306. The effect of the PKC inhibitors on leukaemic cells obtained from three APL patients was also studied. We also evaluated the possible involvement of the caspases in apoptosis induced by PKC inhibitors. Significant time- and concentration-dependent apoptotic changes were demonstrated using Gö6976 and safingol. In addition, our results demonstrated that the caspases were involved in the apoptosis induced by the PKC inhibitors. In conclusion, our study illustrates that the PKC inhibitors Gö6976 and safingol induce apoptosis in APL and hence could be potential therapeutic agents for the treatment of this disease.
Asunto(s)
Apoptosis/efectos de los fármacos , Carbazoles/uso terapéutico , Indoles/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Proteína Quinasa C/antagonistas & inhibidores , Esfingosina/análogos & derivados , Caspasas/metabolismo , Línea Celular , Fragmentación del ADN , Inhibidores Enzimáticos/uso terapéutico , Citometría de Flujo , Genisteína/uso terapéutico , Humanos , Esfingosina/uso terapéuticoRESUMEN
Defining the molecular basis responsible for regulating the proliferative potential of keratinocytes has important implications for normal homeostasis and neoplasia of the skin. Under current culture conditions, neonatal foreskin-derived human keratinocytes possess a relatively short replicative lifespan. Recently it was reported that forced overexpression of the helix-loop-helix protein Id-1 was capable of immortalizing keratinocytes, secondary to activation of telomerase activity and suppression of p16/Rb-mediated growth arrest pathways. To investigate the relationship between Id-1, telomerase activity, telomere length, p16, Rb cell cycle regulators, and senescence, whole populations of keratinocytes were infected with a retrovirus to induce overexpression of Id-1. In these unselected cultures, enhanced Id-1 levels clearly extended the lifespan of keratinocytes, but Id-1 did not prevent the onset of replicative senescence. Under these experimental conditions, Id-1 expression did not trigger induction of telomerase activity, and there was progressive shortening of the telomeres that was accompanied by elevated p16 levels and prevalence of active Rb. The ability of Id-1 to postpone, but not prevent, senescence may be related to partial inhibition of p16 expression, as the Id-1-overexpressing cultures displayed a decreased capacity for 12-O-tetradecanoylphorbol-13-acetate-mediated p16 induction. Thus, while no immortalization was observed, Id-1 could delay the onset of replicative senescence in unselected human keratinocyte populations.
Asunto(s)
Senescencia Celular/fisiología , Proteínas de Unión al ADN/fisiología , Queratinocitos/citología , Queratinocitos/fisiología , Proteínas Represoras , Factores de Transcripción/fisiología , Apoptosis , Ciclo Celular , División Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Genes de Retinoblastoma , Secuencias Hélice-Asa-Hélice , Humanos , Recién Nacido , Proteína 1 Inhibidora de la Diferenciación , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Recombinantes/metabolismo , Piel/citología , Telomerasa/metabolismo , Factores de Transcripción/genética , Transfección , Proteína bcl-XRESUMEN
The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.
Asunto(s)
Receptores ErbB/metabolismo , Isoenzimas/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Proteína Quinasa C/metabolismo , Animales , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Células Epidérmicas , Epidermis/metabolismo , Receptores ErbB/genética , Queratinocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Fosforilación , Proteína Quinasa C-delta , Receptor Cross-Talk , Factor de Crecimiento Transformador alfa/biosíntesis , Tirosina/metabolismoRESUMEN
Several studies point to a role for NF-kappaB in modulating epidermal thickness and apoptotic susceptibility of keratinocytes. When phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) are topically applied, prominent epidermal thickening occurs, and exposure to interferon (IFN)-gamma promotes increased epidermal thickness producing psoriatic lesions. While keratinocytes derived from psoriatic plaque resist apoptosis, and combination of TPA and IFN-gamma activates NF-kappaB, the molecular mechanism linking NF-kappaB activation and keratinocyte apoptosis resistance was unknown. Therefore, we examined the ability of IFN-gamma plus TPA to influence NF-kappaB activity, gene expression, and response to UV light-induced apoptosis. These responses in normal keratinocytes were compared with immortalized keratinocytes (HaCaT cells). Exposure of normal keratinocytes to IFN-gamma plus TPA produced a synergistic activation of NF-kappaB, compared with when each reagent was used individually. Normal keratinocytes when exposed to IFN-gamma plus TPA acquired a resistance to UV light-induced apoptosis, which was dependent on NF-kappaB because expression of a dominant negative form of IkappaBalpha overcame the resistance. Compared with normal keratinocytes, HaCaT cells have a dysfunctional constitutive NF-kappaB signaling pathway not induced by IFN-gamma and TPA, rendering HaCaT cells highly susceptible to UV-induced apoptosis. Thus, immortalized HaCaT cells have an abnormal constitutive and dysfunctional NF-kappaB signaling system. These results provide evidence that activation and proper regulation of NF-kappaB is essential for acquisition of an apoptotic-resistant phenotype for epidermal-derived keratinocytes.
Asunto(s)
Apoptosis/fisiología , Queratinocitos/metabolismo , FN-kappa B/fisiología , Apoptosis/efectos de la radiación , Secuencia de Bases , Transporte Biológico , Células Cultivadas , ADN/metabolismo , Cartilla de ADN , Humanos , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , FN-kappa B/metabolismo , Fenotipo , Acetato de Tetradecanoilforbol/farmacología , Rayos UltravioletaRESUMEN
Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-kappaB were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-kappaB is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-kappaB, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-kappaB but not early differentiation.
Asunto(s)
Apoptosis , División Celular , Senescencia Celular , Queratinocitos/citología , Apoptosis/efectos de la radiación , Secuencia de Bases , Diferenciación Celular , Línea Celular Transformada , Células Cultivadas , Cartilla de ADN , Humanos , Interferón gamma/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiación , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Rayos UltravioletaRESUMEN
The elimination of ultraviolet (UV) radiation-damaged keratinocytes via apoptosis is an important mechanism for the protection of the skin from sunlight, an ubiquitous environmental carcinogen. Due to the pleiotropic nature of UV radiation, the molecular mechanisms of UV-induced apoptosis are poorly understood. Protein kinase C (PKC) is a family of enzymes critically involved in the regulation of differentiation in the epidermis, and is associated with the induction of apoptosis by ionizing radiation in other cell types. In normal human keratinocytes, the induction of apoptosis by UV exposure correlated with generation of the catalytic domain of PKCdelta in the soluble fraction. In contrast, phorbol ester 12-O-tetradecanoylphorbol-13-acetate caused translocation of PKCdelta from the soluble to the particulate fraction without inducing apoptosis. The effect of UV radiation on PKCdelta was isoform specific, as UV exposure did not stimulate the cleavage, or effect the subcellular distribution of any other PKC isoform. The soluble, catalytic domain of PKCdelta induced by UV exposure was associated with an increase in soluble PKCdelta activity. Proteases of the caspase family are activated during UV-induced apoptosis. Inhibition of caspases blocked the UV-induced cleavage of PKCdelta and apoptosis. In addition, inhibition of PKC activity specifically inhibited UV-induced apoptosis of keratinocytes, without affecting the G0/G1 cell cycle block induced by UV exposure. These results indicate that PKC activation is involved in the UV-induced death effector pathway of keratinocytes undergoing apoptosis, and defines a novel role for this enzyme in epidermal homeostasis.
Asunto(s)
Apoptosis/efectos de la radiación , Cisteína Endopeptidasas/metabolismo , Isoenzimas/metabolismo , Queratinocitos/efectos de la radiación , Proteína Quinasa C/metabolismo , Relación Dosis-Respuesta en la Radiación , Activación Enzimática , Fase G1 , Humanos , Hidrólisis , Isoenzimas/antagonistas & inhibidores , Queratinocitos/enzimología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-delta , Rayos UltravioletaRESUMEN
Three desmoglein (Dsg) isoforms are expressed in a differentiation-specific fashion in the epidermis, with Dsg2 being basal, Dsg3 (pemphigus vulgaris antigen) basal and spinous, and Dsg1 (pemphigus foliaceus antigen) predominantly granular. To better understand the mechanism(s) regulating Dsg isoform expression, we examined the expression pattern of Dsg1, Dsg2, and Dsg3 in normal human epidermal keratinocytes (NHEKs), the immortalized, nontumorigenic HaCaT cell line, and several squamous cell carcinoma cell lines (SCC-9, SCC-12F, SCC-13, and SCC-25). In all cells, the accumulation of high Dsg protein levels required calcium and was not observed in low calcium (0.05-0.07 mM) media. NHEKs expressed Dsg1 in all media tested, consistent with their normal differentiation capacity. HaCaT and SCC-25 also expressed Dsg1; however, the presence of serum in the media dramatically decreased Dsg1 protein levels. Serum also inhibited Dsg1 mRNA levels in HaCaT cells. Dsg1 was not detected in extracts from SCC-9, SCC-12F, and SCC-13 under any conditions. Since activation of protein kinase C (PKC) is involved in keratinocyte differentiation, we evaluated the effects of PKC down-regulation on Dsg isoform expression. Long-term treatment with either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or bryostatin 1 inhibited levels of Dsg1 and Dsg3, but not Dsg2 in NHEKs and HaCaT cells. Chronic TPA also decreased Dsg1 and Dsg3 mRNA levels in NHEKs, further supporting a role for PKC activation in the expression of the suprabasal Dsg1 and Dsg3. These results identify several regulatory mechanisms by which the differentiation-specific pattern of desmosomal cadherins is established in the epidermis.
Asunto(s)
Calcio/farmacología , Proteínas del Citoesqueleto/biosíntesis , Queratinocitos/metabolismo , Proteína Quinasa C/metabolismo , Piel/citología , Sangre , Brioestatinas , Carcinoma de Células Escamosas , Moléculas de Adhesión Celular/biosíntesis , Diferenciación Celular , Línea Celular Transformada , Células Cultivadas , Medios de Cultivo , Desmogleína 1 , Desmogleína 2 , Desmogleínas , Desmoplaquinas , Desmosomas/efectos de los fármacos , Desmosomas/fisiología , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Recién Nacido , Queratinocitos/citología , Cinética , Lactonas/farmacología , Macrólidos , Masculino , Proteína Quinasa C/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell-cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell-cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin-plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.
Asunto(s)
Cadherinas/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Desmosomas/metabolismo , Estructura Terciaria de Proteína , Animales , Cadherinas/química , Cadherinas/genética , Citoplasma/química , Citoplasma/metabolismo , Desmocolinas , Desmogleína 1 , Desmogleínas , Desmoplaquinas , Desmosomas/química , Desmosomas/genética , Espacio Extracelular/química , Espacio Extracelular/genética , Humanos , Células L , Sustancias Macromoleculares , Ratones , Péptidos/metabolismo , Péptidos/fisiología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/fisiología , alfa Catenina , gamma CateninaRESUMEN
We have assessed the role of epidermal growth factor receptor (EGFR) signaling in biological responses to the v-ras(Ha) oncogene using primary keratinocytes from Egfr -/- mice and wild-type littermates. On the basis of several criteria, Egfr -/- keratinocytes were unresponsive to either acute or chronic exposure to several EGFR ligands but were stimulated to proliferate in response to several other mitogens. Although conditioned medium from primary keratinocytes transduced with v-ras(Ha) retrovirus (v-ras(Ha) keratinocytes) was a potent mitogen for wild-type but not Egfr -/- keratinocytes, v-ras(Ha) transduction of primary keratinocytes of either genotype resulted in a strong mitogenic response, arguing against an obligatory role for EGFR activation in v-ras(Ha)-mediated stimulation of keratinocyte proliferation. Infection with high-titer v-ras(Ha) retrovirus altered the keratin expression pattern in keratinocytes of both genotypes, suppressing differentiation-specific keratins K1 and K10 while activating aberrant expression of K8 and K18. In wild-type but not Egfr -/- cultures, K1 and K10 were also suppressed following infection at lower retroviral titers, presumably as a result of paracrine EGFR activation on uninfected cells present in these cultures. Squamous papillomas produced by grafting Egfr -/- v-ras(Ha) keratinocytes onto nude mice were only 21% of the size of wild-type v-ras(Ha) tumors, and a striking redistribution of S-phase cells was detected by immunostaining for bromodeoxyuridine. In Egfr -/- v-ras(Ha) papillomas, the fraction of total labeled nuclei detected in suprabasal layers was increased from 19 to 39%. In contrast, the basal layer labeling index of Egfr -/- papillomas was reduced to 34%, compared to 43% in wild-type tumors. Our results indicate that, although autocrine EGFR signaling is not required for keratinocyte responses to oncogenic ras in culture or benign tumor formation in nude mouse grafts, disruption of this pathway impairs growth of v-ras(Ha) papillomas by a mechanism that may involve alterations in keratinocyte cell cycle progression and/or migration in vivo.