RESUMO
BACKGROUND & AIMS: In injured liver, TGF-ß affects all hepatic cell types and participates in wound healing and fibrogenesis. TGF-ß downstream signaling is highly complex and cell type dependent, involving Smad and non-Smad signaling cascades thus requiring tight regulation. Endocytosis has gained relevance as important mechanism to control signaling initiation and termination. In this study, we investigated endocytic mechanisms for TGF-ß mediated Smad and non-Smad signaling in hepatocytes. METHODS: Endocytosis in hepatocytes was elucidated using chemical inhibitors, RNAi, viral gene transfer and caveolin-1-/- mice. TGF-ß signaling was monitored by Western blot, reporter assays and gene expression analysis. RESULTS: In hepatocytes, Smad activation is to a large degree accomplished AP-2 complex dependent on the hepatocyte surface without the necessity of clathrin coated pit formation or an endocytic step. In contrast, non-Smad/AKT pathway activation required functional dynamin mediated endocytosis and the presence of caveolin-1, an essential protein for caveolae formation. Furthermore, these two TGF-ß signaling initiation platforms discriminate distinct signaling routes that integrate at the transcriptional level as shown for TGF-ß target genes, Id1, Smad7, and CTGF. Endocytosis inhibition increased canonical Smad signaling and culminated in a superinduction of Id1 and Smad7 expression, whereas caveolin-1 mediated AKT pathway activation was required for maximal CTGF induction. CONCLUSIONS: Endocytosis is critical for TGF-ß signaling regulation in hepatocytes and determines gene expression signature and (patho)physiological outcome.
Assuntos
Endocitose/fisiologia , Hepatócitos/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Caveolina 1/deficiência , Caveolina 1/genética , Caveolina 1/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: The transmembrane protein caveolin-1 (CAV1) is an essential component of caveolae, small membrane invaginations involved in vesicle formation. CAV1 plays a role in signal transduction, tumor suppression and oncogene transformation. Previous studies with CAV1 knockout mice and CAV1 knockdown in pancreatic tumor cells implicated CAV1 in mediating radioresistance. The aim of this work was to test the effect of CAV1 overexpression after irradiation in human cells lacking endogenous CAV1 expression. MATERIAL AND METHODS: Human CAV1 was overexpressed in lymphoblastoid TK6 cells (TK6-wt) using a eukaryotic expression plasmid, pCI-CAV1, or a lentiviral SIN (self-inactivating) vector, HR'SIN-CAV1. CAV1 expression was verified in TK6 cells with Western blot analysis or intracellular FACS (fluorescence-activated cell sorting) staining. The effect of CAV1 on proliferation kinetics after irradiation of TK6 cells was measured with a growth assay. RESULTS: TK6-wt showed no detectable endogenous CAV1 expression. Lentivirally mediated transduction with HR'SIN-CAV1 (TK6-CAV1) resulted in a considerably stronger CAV1 expression in comparison to TK6 cells electroporated with pCI-CAV1. Intracellular FACS analysis showed that 90% of transduced cells expressed CAV1. CAV1 enhanced early proliferation of TK6 cells after irradiation with a dose of 2 Gy, whereas proliferation of unirradiated cells was not affected. CAV1 also protected cells after irradiation with 4 Gy. This radioprotective effect was supported by a reduction of radiation-induced apoptosis. CONCLUSION: A model system for expression of exogenous CAV1 by stable lentiviral transduction of TK6 cells was established. Functional assays demonstrated enhanced early proliferation by CAV1 expression in TK6 cells after irradiation with clinically relevant doses supporting the role of CAV1 as a prosurvival factor.
Assuntos
Apoptose/efeitos da radiação , Caveolina 1/genética , Divisão Celular/efeitos da radiação , Expressão Gênica/efeitos da radiação , Apoptose/genética , Western Blotting , Divisão Celular/genética , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Expressão Gênica/genética , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Plasmídeos , Proteção Radiológica , Transdução Genética/métodosRESUMO
Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors linking extracellular signals to target gene transcription. Hematopoietic cells express two highly conserved STAT5-isoforms (STAT5A/STAT5B), and STAT5 is directly activated by JAK2 downstream of several cytokine receptors and the oncogenic BCR-ABL tyrosine kinase. Using an IL-3-dependent cell line with inducible BCR-ABL-expression we compared STAT5-activation by IL-3 and BCR-ABL in a STAT5-isoform specific manner. RNAi targeting of STAT5B strongly inhibits BCR-ABL-dependent cell proliferation, and STAT5B but not STAT5A is essential for BCL-XL-expression in the presence of BCR-ABL. Although BCR-ABL induces STAT5-tyrosine phosphorylation independent of JAK2-kinase activity, BCR-ABL is less efficient in inducing active STAT5A:STAT5B-heterodimerization than IL-3, leaving constitutive STAT5A and STAT5B-homodimerization unaffected. In comparison to IL-3, nuclear accumulation of a STAT5A-eGFP fusion protein is reduced by BCR-ABL, and BCR-ABL tyrosine kinase activity induces STAT5A-eGFP translocation to the cell membrane and co-localization with the IL-3 receptor. Furthermore, BCR-ABL-dependent phosphorylation of Y682 in STAT5A was detected by mass-spectrometry. Finally, RNAi targeting STAT5B but not STAT5A sensitizes human BCR-ABL-positive cell lines to imatinib-treatment. These data demonstrate differences between IL-3 and BCR-ABL-mediated STAT5-activation and isoform-specific effects, indicating therapeutic options for isoform-specific STAT5-inhibition in BCR-ABL-positive leukemia.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Benzamidas , Linhagem Celular , Proliferação de Células/fisiologia , Dimerização , Imunofluorescência , Vetores Genéticos/genética , Humanos , Mesilato de Imatinib , Immunoblotting , Imunoprecipitação , Interleucina-3/metabolismo , Lentivirus , Espectrometria de Massas , Fosforilação , Piperazinas , Pirimidinas , Interferência de RNA , Transdução de Sinais/genéticaRESUMO
Genome-wide association studies (GWAS) identified several genetic risk factors for breast cancer, however, most of them were validated among women of European ancestry. This study examined single-nucleotide polymorphisms (SNPs) contributing to breast cancer in Chinese (984 cases and 2206 controls) and German (311 cases and 960 controls) populations. Eighteen SNPs significantly associated with breast cancer, previously identified in GWAS were genotyped. Twelve SNPs passed quality control and were subjected to statistical analysis. Seven SNPs were confirmed to be significantly associated with breast cancer in the Chinese population, reflecting three independent loci (ESR1, FGFR2, TOX3) and five of these were also confirmed in the German population. The strongest association was identified for rs2046210 in the Chinese (odds ratio (OR)=1.42, 95% confidence interval (CI)=1.28-1.59, P=1.9 × 10(-10)) and rs3803662 in the German population (OR=1.43, 95% CI=1.17-1.74, P=4.01 × 10(-4)), located upstream of the ESR1 and TOX3 gene, respectively. For the first time, rs3757318 at 6q25.1, located next to the gene encoding estrogen receptor α (ESR1) was found to be strongly associated with breast cancer (OR=1.33, 95% CI=1.18-1.49, P=1.94 × 10(-6)) in the Chinese population. The frequency of this variant was markedly lower in the German population and the association was not significant. Despite the genetic differences, essentially the same risk loci were identified in the Chinese and the German populations. Our study suggested the existence of common genetic factors as well as disease susceptibility differences for breast cancer in both populations and highlighted the importance of performing comparison analyses for disease susceptibility within ethnic populations.
Assuntos
Povo Asiático/genética , Neoplasias da Mama/genética , Genes Neoplásicos/genética , Predisposição Genética para Doença , Variação Genética , Idade de Início , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/patologia , Estudos de Casos e Controles , China , Receptor alfa de Estrogênio/genética , Feminino , Loci Gênicos/genética , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Alemanha , Haplótipos/genética , Proteínas de Grupo de Alta Mobilidade , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo Único/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptores de Progesterona/genética , TransativadoresRESUMO
Tumor radiotherapy with large-field irradiation results in an increase of p53-dependent apoptosis of the radiosensitive hematopoietic stem cells. Proapoptotic PUMA is a transcriptional target of p53. Thus suppression of PUMA expression by gene therapy with the transcription repressor SNAI2 as transgene might be a potential approach for normal tissue protection during radiotherapy. SNAI2 cDNA was cloned in a lentiviral SIN vector in a bicistronic expression cassette followed by a floxed IRES-EMCV linker and EGFP as selection gene. Wild-type p53 TK6 cells were used as the cellular model system. We could demonstrate the significant radioprotective effect of SNAI2 overexpression in a cytotoxicity assay after irradiation with 0-5 Gy compared with untransduced or control vector (inverse oriented SNAI2 cDNA)-transduced cells. Additionally, TK6-SNAI2 compared to TK6-SNAI2inv cells showed a survival advantage in a clonogenic assay after irradiation with 0-3 Gy. Determination of the proportion of sub-G(1) cells in TK6-SNAI2 cells revealed an approximately 50% reduction in apoptosis compared with both control entities. In this study using a bicistronic lentiviral vector, we were able to provide proof of principle that lentiviral overexpression of SNAI2 might be used for radioprotective gene therapy to widen the therapeutic range in radiotherapy.