RESUMO
Signal transducer and activator of transcription 3 (Stat3) is the major mediator of interleukin-6 (IL-6) family cytokines. In addition, Stat3 is known to be involved in the pathophysiology of many malignancies. Here, we show that the cis-trans peptidyl-prolyl isomerase cyclophilin (Cyp) B specifically interacts with Stat3, whereas the highly related CypA does not. CypB knockdown inhibited the IL-6-induced transactivation potential but not the tyrosine phosphorylation of Stat3. Binding of CypB to Stat3 target promoters and alteration of the intranuclear localization of Stat3 on CypB depletion suggested a nuclear function of Stat3/CypB interaction. By contrast, CypA knockdown inhibited Stat3 IL-6-induced tyrosine phosphorylation and nuclear translocation. The Cyp inhibitor cyclosporine A (CsA) caused similar effects. However, Stat1 activation in response to IL-6 or interferon-gamma was not affected by Cyp silencing or CsA treatment. As a result, Cyp knockdown shifted IL-6 signaling to a Stat1-dominated pathway. Furthermore, Cyp depletion or treatment with CsA induced apoptosis in IL-6-dependent multiple myeloma cells, whereas an IL-6-independent line was not affected. Thus, Cyps support the anti-apoptotic action of Stat3. Taken together, CypA and CypB both play pivotal roles, yet at different signaling levels, for Stat3 activation and function. These data also suggest a novel mechanism of CsA action.
Assuntos
Ciclofilina A/metabolismo , Ciclofilinas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Apoptose , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Imunoprecipitação da Cromatina , Ciclofilina A/genética , Ciclofilinas/genética , Citometria de Fluxo , Humanos , Immunoblotting , Interleucina-6/farmacologia , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT3/genética , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
BCL3 is a proto-oncogene affected by chromosomal translocations in some patients with chronic lymphocytic leukemia. It is an IkappaB family protein that is involved in transcriptional regulation of a number of NF-kappaB target genes. In this study, interleukin (IL)-6-induced BCL3 expression and its effect on survival of multiple myeloma (MM) cells were examined. We demonstrate the upregulation of BCL3 by IL-6 in INA-6 and other MM cell lines. Sequence analysis of the BCL3 gene locus revealed four potential signal transducer and activator of transcription (Stat) binding sites within two conserved intronic enhancers regions: one located within enhancer HS3 and three within HS4. Chromatin immunoprecipitation experiments showed increased Stat3 binding to both enhancers upon IL-6 stimulation. Silencing Stat3 expression by small interfering RNA (siRNA) abrogated BCL3 expression by IL-6. Using reporter gene assays, we demonstrate that BCL3 transcription depends on HS4. Mutation of the Stat motifs within HS4 abolished IL-6-dependent BCL3 induction. Furthermore, BCL3 transcription was inhibited by its own gene product. This repressive feedback is mediated by NF-kappaB sites within the promoter and HS3. Finally, we show that overexpression of BCL3 increases apoptosis, whereas BCL3-specific siRNA does not affect the viability of INA-6 cells suggesting that BCL3 is not essential for the survival of these cells.
