RESUMO
Viral myocarditis (VMC) is a cardiac disease associated with myocardial inflammation and injury induced by virus infection. Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium. Kruppel-like factor 10 (KLF10) is a crucial regulator of various pathological processes and plays different roles in a variety of diseases. However, its role in VMC induced by coxsackievirus B3 (CVB3) infection remains unknown. In this study, we report that cardiac KLF10 confers enhanced protection against viral myocarditis. We found that KLF10 expression was downregulated upon CVB3 infection. KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress. Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC. We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes, and KLF10 cooperated with histone deacetylase 1 (HDAC1) to negatively regulate MCP-1 expression by binding its promoter, leading to activation of MCP-1 transcription and recruitment of Ly6Chigh monocytes/macrophages into the myocardium. This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC.
Assuntos
Infecções por Coxsackievirus , Miocardite , Animais , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/patologia , Miocardite/terapia , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
BACKGROUND: The role and clinical value of ERß1 expression is controversial and recent data demonstrates that many ERß antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERß1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression. METHODS: Nuclear and cytoplasmic expression patterns of ERß1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERß1, we generated multiple ERß1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERß-specific agonist exposure. RESULTS: Nuclear ERß1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERß1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERß-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective. CONCLUSIONS: Using a validated antibody, we have confirmed that nuclear ERß1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERß appears to be a novel therapeutic target. However, the efficacy of SERMs and ERß-specific agonists differ as a function of ERα expression.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Antagonistas de Estrogênios/farmacologia , Receptor beta de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-IdadeRESUMO
Endoxifen has recently been identified as the predominant active metabolite of tamoxifen and is currently being developed as a novel hormonal therapy for the treatment of endocrine sensitive breast cancer. Based on past studies in breast cancer cells and model systems, endoxifen classically functions as an anti-estrogenic compound. Since estrogen and estrogen receptors play critical roles in mediating bone homeostasis, and endoxifen is currently being implemented as a novel breast cancer therapy, we sought to comprehensively characterize the in vivo effects of endoxifen on the mouse skeleton. Two month old ovariectomized C57BL/6 mice were treated with vehicle or 50 mg/kg/day endoxifen hydrochloride via oral gavage for 45 days. Animals were analyzed by dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, micro-computed tomography and histomorphometry. Serum from control and endoxifen treated mice was evaluated for bone resorption and bone formation markers. Gene expression changes were monitored in osteoblasts, osteoclasts and the cortical shells of long bones from endoxifen treated mice and in a human fetal osteoblast cell line. Endoxifen treatment led to significantly higher bone mineral density and bone mineral content throughout the skeleton relative to control animals. Endoxifen treatment also resulted in increased numbers of osteoblasts and osteoclasts per tissue area, which was corroborated by increased serum levels of bone formation and resorption markers. Finally, endoxifen induced the expression of osteoblast, osteoclast and osteocyte marker genes. These studies are the first to examine the in vivo and in vitro impacts of endoxifen on bone and our results demonstrate that endoxifen increases cancellous as well as cortical bone mass in ovariectomized mice, effects that may have implications for postmenopausal breast cancer patients.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Osso e Ossos/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Tamoxifeno/análogos & derivados , Animais , Antineoplásicos Hormonais/farmacologia , Sequência de Bases , Primers do DNA , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Reação em Cadeia da Polimerase , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
TGFß Inducible Early Gene-1 (TIEG1) knockout (KO) mice display a sex-specific osteopenic phenotype characterized by low bone mineral density, bone mineral content, and overall loss of bone strength in female mice. We, therefore, speculated that loss of TIEG1 expression would impair the actions of estrogen on bone in female mice. To test this hypothesis, we employed an ovariectomy (OVX) and estrogen replacement model system to comprehensively analyze the role of TIEG1 in mediating estrogen signaling in bone at the tissue, cell, and biochemical level. Dual-energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro-CT analyses revealed that loss of TIEG1 expression diminished the effects of estrogen throughout the skeleton and within multiple bone compartments. Estrogen exposure also led to reductions in bone formation rates and mineralizing perimeter in wild-type mice with little to no effects on these parameters in TIEG1 KO mice. Osteoclast perimeter per bone perimeter and resorptive activity as determined by serum levels of CTX-1 were differentially regulated after estrogen treatment in TIEG1 KO mice compared with wild-type littermates. No significant differences were detected in serum levels of P1NP between wild-type and TIEG1 KO mice. Taken together, these data implicate an important role for TIEG1 in mediating estrogen signaling throughout the mouse skeleton and suggest that defects in this pathway are likely to contribute to the sex-specific osteopenic phenotype observed in female TIEG1 KO mice.
Assuntos
Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/biossíntese , Estrogênios/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/biossíntese , Animais , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/patologia , Osso e Ossos/patologia , Proteínas de Ligação a DNA/genética , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
Endoxifen, a cytochrome P450 mediated tamoxifen metabolite, is being developed as a drug for the treatment of estrogen receptor (ER) positive breast cancer. Endoxifen is known to be a potent anti-estrogen and its mechanisms of action are still being elucidated. Here, we demonstrate that endoxifen-mediated recruitment of ERα to known target genes differs from that of 4-hydroxy-tamoxifen (4HT) and ICI-182,780 (ICI). Global gene expression profiling of MCF7 cells revealed substantial differences in the transcriptome following treatment with 4HT, endoxifen and ICI, both in the presence and absence of estrogen. Alterations in endoxifen concentrations also dramatically altered the gene expression profiles of MCF7 cells, even in the presence of clinically relevant concentrations of tamoxifen and its metabolites, 4HT and N-desmethyl-tamoxifen (NDT). Pathway analysis of differentially regulated genes revealed substantial differences related to endoxifen concentrations including significant induction of cell cycle arrest and markers of apoptosis following treatment with high, but not low, concentrations of endoxifen. Taken together, these data demonstrate that endoxifen's mechanism of action is different from that of 4HT and ICI and provide mechanistic insight into the potential importance of endoxifen in the suppression of breast cancer growth and progression.
Assuntos
Antagonistas de Estrogênios/metabolismo , Tamoxifeno/análogos & derivados , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Análise por Conglomerados , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligação Proteica , Reprodutibilidade dos Testes , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
Bone marrow-derived macrophages (BMMs) treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), differentiate into GM-CSF-induced mouse bone marrow-derived macrophages (GM-BMMs) or M-CSF-induced mouse bone marrow-derived macrophages (M-BMMs), which have an M1 or M2 profile, respectively. GM-BMMs produce large amounts of proinflammatory cytokines and mediate resistance to pathogens, whereas M-BMMs produce antiinflammatory cytokines that contribute to tissue repair and remodeling. M-BMMs stimulated with lipopolysaccharide (LPS) are in an antiinflammatory state, with an IL-12(low) IL-10(high) phenotype. However, the regulation of this process remains unclear. Klf10 belongs to the family of Krüppel-like transcription factors and was initially described as a TGF-ß inducible early gene 1. IL-12p40 is upregulated in LPS-stimulated M-BMMs from Klf10-deficient mice, but downregulated during Klf10 overexpression. Klf11, another member of the Krüppel-like factor family, can also repress the production of IL-12p40. Furthermore, Klf10 binds to the CACCC element of the IL-12p40 promoter and inhibits its transcription. We have therefore identified Klf10 as a transcription factor that regulates the expression of IL-12p40 in M-BMMs.
Assuntos
Células da Medula Óssea/imunologia , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/imunologia , Regulação da Expressão Gênica/genética , Mediadores da Inflamação/metabolismo , Subunidade p40 da Interleucina-12/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Transcrição Gênica/imunologia , Transgenes/genéticaRESUMO
Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease. A recent study showed that male KLF10-encoded TGFß Inducible Early Gene-1 knock-out mice (TIEG-/-) develop HCM with 13-fold up-regulation of PTTG1-encoded pituitary tumor-transforming gene 1. We hypothesized TIEG1 could be a novel candidate gene in the pathogenesis of genotype negative HCM in humans, possibly through a loss of its repression on PTTG1 expression. A cohort of 923 unrelated patients from two independent HCM centers was analyzed for mutations in TIEG's four translated exons using DHPLC and direct DNA-sequencing. Site directed mutagenesis was performed to clone novel variants. The effect of TIEG1 mutations on SMAD7 and PTTG1 promoters was studied using transient transfection and luciferase-assays. Altered expression of PTTG1 in cardiac tissue was studied by immunohistochemistry (IHC) to determine levels of PTTG1 protein in hypertrophic diseases. Six novel TIEG1 missense mutations were discovered in six patients (two males/four females, mean age at diagnosis 56.2±23 years, MLVWT 20.8±4 mm). Compared to WT TIEG1, five TIEG1 mutants significantly increased PTTG1 promoter function similar to TIEG1-/--mice. By IHC, PTTG1-protein expression was significantly increased in multiple models of hypertrophic cardiac disease, including TIEG1-mutation positive HCM compared to normal hearts. This is the first article to associate mutations in TIEG1 to human disease with the discovery of six novel, HCM-associated variants. Functional assays suggest a role for PTTG1 in the pathogenesis of TIEG1-mediated HCM. Up-regulation of PTTG1 seems to be a common pathway in hypertrophic heart disease, including TIEG1-mediated HCM.
Assuntos
Cardiomiopatia Hipertrófica/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas de Neoplasias/genética , Proteína Smad7/genética , Sequência de Bases , Cardiomiopatia Hipertrófica/patologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Mutação , Mutação de Sentido Incorreto , Regiões Promotoras Genéticas , Securina , Análise de Sequência de DNA , Fator de Crescimento Transformador beta/genéticaRESUMO
The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERß and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERß and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERß antibodies are insensitive for ERß and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERß antibody is shown to be highly specific and sensitive for detection of full-length ERß and its variant forms. Strong and variable staining patterns for endogenous levels of ERß protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERß was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERß antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERß in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERß, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.
Assuntos
Anticorpos Monoclonais Murinos/biossíntese , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/metabolismo , Receptor beta de Estrogênio/imunologia , Animais , Especificidade de Anticorpos , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Masculino , Camundongos , Especificidade de Órgãos , Próstata/metabolismo , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Testículo/metabolismoRESUMO
Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-ß1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, â¼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-ß responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-ß1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-ß1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular , Fatores de Transcrição de Resposta de Crescimento Precoce/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Neovascularização Fisiológica , Transdução de Sinais , Fator de Crescimento Transformador beta1/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/fisiologia , Membro Posterior , Isquemia/metabolismo , Isquemia/patologia , Isquemia/fisiopatologia , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/fisiologia , Doença Arterial Periférica/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fluxo Sanguíneo Regional , Fator de Crescimento Transformador beta1/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The remodeling of extracellular matrix is a crucial mechanism in tendon development and the proliferation of fibroblasts is a key factor in this process. The purpose of this study was to further elucidate the role of TIEG1 in mediating important tenocyte properties throughout the aging process. Wildtype and TIEG1 knockout tenocytes adhesion, spreading and proliferation were characterized on different substrates (fibronectin, collagen type I, gelatin and laminin) and the expression levels of various genes known to be involved with tendon development were analyzed by RT-PCR. The experiments revealed age-dependent and substrate-dependent properties for both wildtype and TIEG1 knockout tenocytes. Taken together, our results indicate an important role for TIEG1 in regulating tenocytes adhesion, spreading, and proliferation throughout the aging process. Understanding the basic mechanisms of TIEG1 in tenocytes may provide valuable information for treating multiple tendon disorders.
Assuntos
Adesão Celular , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/fisiologia , Expressão Gênica , Tendões/citologia , Tendões/metabolismo , Fatores de Transcrição/fisiologia , Fatores Etários , Animais , Western Blotting , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traumatismos dos Tendões , CicatrizaçãoRESUMO
Deletion of TIEG1/KLF10 in mice results in a gender specific osteopenic skeletal phenotype with significant defects in both cortical and trabecular bone, which are observed only in female animals. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display reduced expression levels of multiple bone related genes, including Runx2, and exhibit significant delays in their mineralization rates relative to wildtype controls. These data suggest that TIEG1 plays an important role in regulating Runx2 expression in bone and that decreased Runx2 expression in TIEG1 KO mice is in part responsible for the observed osteopenic phenotype. In this manuscript, data is presented demonstrating that over-expression of TIEG1 results in increased expression of Runx2 while repression of TIEG1 results in suppression of Runx2. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Runx2 promoter. The zinc finger containing domain of TIEG1 is necessary for this regulation supporting that activation occurs through direct DNA binding. A role for the ubiquitin/proteasome pathway in fine tuning the regulation of Runx2 expression by TIEG1 is also implicated in this study. Additionally, the regulation of Runx2 expression by cytokines such as TGFß1 and BMP2 is shown to be inhibited in the absence of TIEG1. Co-immunoprecipitation and co-localization assays indicate that TIEG1 protein associates with Runx2 protein resulting in co-activation of Runx2 transcriptional activity. Lastly, Runx2 adenoviral infection of TIEG1 KO calvarial osteoblasts leads to increased expression of Runx2 and enhancement of their ability to differentiate and mineralize in culture. Taken together, these data implicate an important role for TIEG1 in regulating the expression and activity of Runx2 in osteoblasts and suggest that decreased expression of Runx2 in TIEG1 KO mice contributes to the observed osteopenic bone phenotype.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição de Resposta de Crescimento Precoce/fisiologia , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Osteoblastos/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , DNA/química , Feminino , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Osteoblastos/citologia , Regiões Promotoras Genéticas , Ligação Proteica , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-ß-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-ß-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Transferência Adotiva , Animais , Western Blotting , Núcleo Celular/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Imunoprecipitação , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , TYK2 Quinase/metabolismo , Células Th17/metabolismo , Fator de Crescimento Transformador beta/metabolismo , UbiquitinaçãoRESUMO
TGF-ß Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-ß treatment. As reported previously, TIEG1(-/-) mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1(-/-) osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1(-/-) precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1(-/-) osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1(-/-) osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1(-/-) cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1(-/-) precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Osteoclastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligante RANK/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1RESUMO
INTRODUCTION: We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERß in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERß and examine its effectiveness as an anti-estrogenic agent in these cell lines. METHODS: MCF7, Hs578T and U2OS cells were stably transfected with full-length ERß. ERß protein stability, dimer formation with ERα and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERß-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes. RESULTS: Our data demonstrate that endoxifen stabilizes ERß protein, unlike its targeted degradation of ERα, and induces ERα/ERß heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERß is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20 to 40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERß, whereas much higher endoxifen concentrations are needed when ERß is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40 nM) when comparing MCF7 cells which express ERß to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations. CONCLUSIONS: Taken together, these data demonstrate that the presence of ERß enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERα/ß heterodimers. These findings underscore the need to further elucidate the role of ERß in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERß expression.
Assuntos
Neoplasias da Mama/metabolismo , Moduladores de Receptor Estrogênico/farmacologia , Receptor beta de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP2D6/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Estrogênios/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Multimerização Proteica , Tamoxifeno/farmacologiaRESUMO
JARID1B/KDM5B (jumonji AT-rich interactive domain 1B/lysine-specific demethylase 5B) is an enzyme that efficiently removes methyl residues from trimethylated lysine 4 on histone H3, a pivotal mark for active chromatin. TIEG1/KLF10 (transforming growth factor-ß inducible early gene-1/Krüppel-like transcription factor 10) is a zinc-finger transcription factor that is involved in bone metabolism and exerts antiproliferative activity. Here, we found that TIEG1 interacts with JARID1B. In particular, the repression domains of TIEG1 bind to the C-terminus of JARID1B. Moreover, overexpression of JARID1B augments TIEG1 to repress transcription of Smad7, an inhibitor of the TGF-ß (transforming growth factor-ß) signaling pathway. Conversely, JARID1B knock-down leads to increased Smad7 mRNA levels. Thus, TIEG1 and JARID1B may cooperate to suppress tumorigenesis by enhancing TGF-ß signaling. Notably, both TIEG1 and JARID1B are downregulated in melanomas, suggesting that they indeed cooperate physiologically. In conclusion, JARID1B is the first TIEG1 corepressor identified, explaining how TIEG1 represses transcription through inducing histone H3 lysine 4 demethylation, which may be important for TIEG1 function in both normal and cancer cells.
Assuntos
Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas Nucleares/genética , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
BACKGROUND: Numerous transcription factors are involved in the establishment and maintenance of the osteoblastic phenotype, such as Runx2, osterix and Dlx5. The transcription factor retinoblastoma binding protein-1 (RBP1) was recently identified as an estrogen regulated gene in an osteosarcoma cell model. Since the function of RBP1 in osteoblastic differentiation and mineralization is unknown, we investigated the role of RBP1 in these processes. METHODS: To create a cell model with suppressed RBP1 expression, primary calvarial osteoblasts were infected with a shRNA lentiviral vector specific for mouse RBP1 (CalOB-DeltaRBP1) or a scrambled control shRNA lentivirus (CalOB-Control). Stable cell lines were generated and their mineralization potential was determined using osteoblastic differentiation medium, Alizarin Red staining, and quantitative PCR (QPCR) analyses. Runx2 coactivation by RBP1 was determined through the use of transient transfection assays. RESULTS: Stable expression of the RBP1 shRNA lentivirus in CalOB-DeltaRBP1 cells resulted in a 65-70% suppression of RBP1 expression. Osteoblastic mineralization assays demonstrated that suppression of RBP1 results in a potent delay in osteoblastic nodule formation in the CalOB-DeltaRBP1 cells with a concomitant decrease in the expression of the osteogenic transcription factors Runx2 and osterix, along with decreases in BMP2, alkaline phosphatase, osteocalcin and bone sialoprotein. Regulation of Runx2 expression by RBP1 was shown to be mediated through the proximal P2 Runx2 promoter. Furthermore, RBP1 was demonstrated to be a potent coactivator of Runx2 transcriptional activity on two known Runx2 reporter constructs. These data suggest that the expression and activity of Runx2 is critically dependent on the presence of RBP1. CONCLUSIONS: This study is the first to demonstrate that RBP1 is an important mediator of the osteoblastic phenotype and clearly defines RBP1 as a novel transcription factor involved in the well known Runx2-osterix transcriptional cascade. As such, the effects of RBP1 on these processes are mediated through both regulation of Runx2 expression and transcriptional activity.
Assuntos
Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/fisiologia , Osteogênese/fisiologia , Proteínas Celulares de Ligação ao Retinol/metabolismo , Animais , Calcificação Fisiológica/fisiologia , Linhagem Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Celulares de Ligação ao Retinol/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , TransfecçãoRESUMO
Evidence has emerged that the clinical benefit of tamoxifen is related to the functional status of the hepatic metabolizing enzyme cytochrome P450 2D6 (CYP2D6). CYP2D6 is the key enzyme responsible for the generation of the potent tamoxifen metabolite, endoxifen. Multiple studies have examined the relationship of CYP2D6 status to breast cancer outcomes in tamoxifen-treated women; the majority of studies demonstrated that women with impaired CYP2D6 metabolism have lower endoxifen concentrations and a greater risk of breast cancer recurrence. As a result, practitioners must be aware that some of the most commonly prescribed medications coadministered with tamoxifen interfere with CYP2D6 function, thereby reducing endoxifen concentrations and potentially increasing the risk of breast cancer recurrence. After reviewing the published data regarding tamoxifen metabolism and the evidence relating CYP2D6 status to breast cancer outcomes in tamoxifen-treated patients, we are providing a guide for the use of medications that inhibit CYP2D6 in patients administered tamoxifen.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocromo P-450 CYP2D6/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Estudos de Coortes , Citocromo P-450 CYP2D6/genética , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Mastectomia Segmentar/métodos , Preparações Farmacêuticas/administração & dosagem , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Medição de Risco , Tamoxifeno/administração & dosagem , Tamoxifeno/análise , Resultado do TratamentoRESUMO
The purpose of this study was to characterize the effect of TIEG1 on the molecular structure of collagen within tail tendon fibers using 3-mo-old female C57BL/6 wild-type (WT) and TIEG1 KO mice. Synchrotron X-ray microdiffraction experiments were carried out on single tendon fibers extracted from the WT and TIEG1 KO dorsal tail tendon. The fibers were scanned in the radial direction, and X-ray patterns were obtained. From these patterns, the meridional direction was analyzed through X-ray intensity profile. In addition, collagen content was investigated using hydroxyproline assays, and qualitative real-time PCR experiments were performed on RNA isolated from fibroblasts to examine specific gene expression changes. The results showed different X-ray diffraction patterns between WT and TIEG1 KO tendon fibers, indicating a disorganization of the collagen structure for the TIEG1 KO compared with WT mice. Furthermore, the analyses of the X-ray intensity profiles exhibited a higher (23 A) period of collagen for the TIEG1 KO compared with the WT mice. The results of the hydroxyproline assays revealed a significant decrease in the TIEG1 KO compared with WT mice, leading to a decrease in the total amount of collagen present within the TIEG1 KO tendons. Moreover, qualitative real-time PCR results showed differences in the expression profiles of specific genes known to play important roles in tendon fiber development. These data further elucidate the role of TIEG1 on tendon structure and could explain the previous defects in the structure-function relationship found for TIEG1 KO tendon fibers.
Assuntos
Colágeno/química , Colágeno/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Refratometria/métodos , Tendões/química , Tendões/ultraestrutura , Fatores de Transcrição/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Ligação a DNA/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação Molecular , Proteínas Repressoras , Síncrotrons , Cauda/química , Cauda/ultraestrutura , Fatores de Transcrição/genéticaRESUMO
The circadian timing system coordinates many aspects of mammalian physiology and behavior in synchrony with the external light/dark cycle. These rhythms are driven by endogenous molecular clocks present in most body cells. Many clock outputs are transcriptional regulators, suggesting that clock genes primarily control physiology through indirect pathways. Here, we show that Krüppel-like factor 10 (KLF10) displays a robust circadian expression pattern in wild-type mouse liver but not in clock-deficient Bmal1 knockout mice. Consistently, the Klf10 promoter recruited the BMAL1 core clock protein and was transactivated by the CLOCK-BMAL1 heterodimer through a conserved E-box response element. Profiling the liver transcriptome from Klf10(-/-) mice identified 158 regulated genes with significant enrichment for transcripts involved in lipid and carbohydrate metabolism. Importantly, approximately 56% of these metabolic genes are clock controlled. Male Klf10(-/-) mice displayed postprandial and fasting hyperglycemia, a phenotype accompanied by a significant time-of-day-dependent upregulation of the gluconeogenic gene Pepck and increased hepatic glucose production. Consistently, functional data showed that the proximal Pepck promoter is repressed directly by KLF10. Klf10(-/-) females were normoglycemic but displayed higher plasma triglycerides. Correspondingly, rhythmic gene expression of components of the lipogenic pathway, including Srebp1c, Fas, and Elovl6, was altered in females. Collectively, these data establish KLF10 as a required circadian transcriptional regulator that links the molecular clock to energy metabolism in the liver.