RESUMO
Skeletal muscle is known to regulate bone homeostasis through muscle-bone interaction, although factors that control this activity remain unclear. Here, we newly established Smad3-flox mice, and then generated skeletal muscle-specific Smad2/Smad3 double conditional knockout mice (DcKO) by crossing Smad3-flox with skeletal muscle-specific Ckmm Cre and Smad2-flox mice. We show that immobilization-induced gastrocnemius muscle atrophy occurring due to sciatic nerve denervation was partially but significantly inhibited in DcKO mice, suggesting that skeletal muscle cell-intrinsic Smad2/3 is required for immobilization-induced muscle atrophy. Also, tibial bone atrophy seen after sciatic nerve denervation was partially but significantly inhibited in DcKO mice. Bone formation rate in wild-type mouse tibia was significantly inhibited by immobilization, but inhibition was abrogated in DcKO mice. We propose that skeletal muscle regulates immobilization-induced bone atrophy via Smad2/3, and Smad2/3 represent potential therapeutic targets to prevent both immobilization-induced bone and muscle atrophy.
Assuntos
Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/prevenção & controle , Nervo Isquiático/lesões , Proteína Smad2/genética , Proteína Smad3/genética , Animais , Cruzamentos Genéticos , Feminino , Regulação da Expressão Gênica , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Denervação Muscular/métodos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad3/deficiência , Tíbia/inervação , Tíbia/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Epidermal Langerhans cells (LCs) are skin-resident dendritic cells that are essential for the induction of skin immunity and tolerance. Transforming growth factor-ß 1 (TGFß1) is a crucial factor for LC maintenance and function. However, the underlying TGFß1 signaling pathways remain unclear. Our previous research has shown that the TGFß1/Smad3 signaling pathway does not impact LC homeostasis and maturation. In this study, we generated mice with conditional deletions of either individual Smad2, Smad4, or both Smad2 and Smad4 in the LC lineage or myeloid lineage, to further explore the impact of TGFß1/Smad signaling pathways on LCs. We found that interruption of Smad2 or Smad4 individually or simultaneously in the LC lineage did not significantly impact the maintenance, maturation, antigen uptake, and migration of LCs in vivo or in vitro during steady state. However, the interruption of both Smad2 and Smad4 pathways in the myeloid lineage led to a dramatic inhibition of bone marrow-derived LCs in the inflammatory state. Overall, our data suggest that canonical TGFß1/Smad2/4 signaling pathways are dispensable for epidermal LC homeostasis and maturation at steady state, but are critical for the long-term LC repopulation directly originating from the bone marrow in the inflammatory state.
Assuntos
Proliferação de Células , Dermatite/metabolismo , Epiderme/metabolismo , Células de Langerhans/metabolismo , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem da Célula , Movimento Celular , Células Cultivadas , Dermatite/genética , Dermatite/imunologia , Dermatite/patologia , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/patologia , Feminino , Células de Langerhans/imunologia , Células de Langerhans/patologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad4/deficiência , Proteína Smad4/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Selectins are carbohydrate-binding adhesion molecules that control leukocyte traffic. Induction of selectin ligands on T cells is controlled primarily by cytokines, including TGF-ß1, and requires p38α MAPK, but transcriptional mechanisms that underlie cytokine-driven selectin ligand expression are poorly understood. In this study, we show, using mice with conditional deletions of the TGF-ß1-responsive transcription factors Smad2, Smad3, or Smad4, that induction of selectin ligands on CD4 cells in response to TGF-ß1 requires Smad4 plus either Smad2 or Smad3. Analysis of CD4 cells from mice with only one functional Smad4 allele revealed a sharp gene dosage effect, suggesting the existence of a threshold of TGF-ß1 signal strength required for selectin ligand induction. Both Smad4 plus either Smad2 or Smad3 were selectively required for induction of Fut7 and Gcnt1, glycosyltransferases critical for selectin ligand biosynthesis, but they were not required for St3gal4 or St3gal6 induction. Smad4 plus either Smad2 or Smad3 were also required for induction of Runx transcription factors by TGF-ß1. Enforced expression of Runx2, but not Runx1 or Runx3, in Smad2/Smad3 doubly deficient CD4 cells restored selectin ligand expression to wild-type levels. In contrast, enforced expression of Runx1, Runx2, or Runx3 failed to restore differentiation of TGF-ß1-dependent Th cell lineages, including Th17, Th9, and induced regulatory T cells. These results show that Smads are directly required for Th cell differentiation independent of Runx induction but only indirectly required via Runx2 for TGF-ß1-induced selectin ligand induction on murine CD4 T cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Selectinas/biossíntese , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Smad/deficiência , Proteína Smad2/deficiência , Proteína Smad2/metabolismo , Proteína Smad3/deficiência , Proteína Smad3/metabolismo , Proteína Smad4/deficiência , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/imunologiaRESUMO
IL-9 is a proallergic cytokine produced by a newly proposed Th cell subset, Th9. Th9 cells can be generated by treatment of naive T cells with TGF-ß and IL-4 in vitro. However, it is still not clear how TGF-ß signaling regulates Th9 differentiation. In this study, we demonstrate that Smad2 and Smad4, two transcriptional factors activated by TGF-ß signaling, are required for Th9 differentiation in vitro. Deficiency of Smad2 or Smad4 in T cells resulted in impaired IL-9 expression, which was coincident with enrichment of repressive chromatin modification histone H3 K27 trimethylation and enhanced EZH2 binding to the Il9 locus. Pharmacologic inhibition of EZH2 partially rescued IL-9 production in Smad-deficient Th9 cells. Smad proteins may displace EZH2 directly from the Il9 locus, because Smad2 and Smad4 can bind EZH2. Our data shed light on the molecular mechanisms underlying Th9 cell differentiation, revealing that the TGF-ß-Smad2/4-signaling pathway regulates IL-9 production through an epigenetic mechanism.
Assuntos
Interleucina-9/biossíntese , Complexo Repressor Polycomb 2/metabolismo , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Histonas/metabolismo , Interleucina-9/genética , Camundongos , Camundongos Transgênicos , Complexo Repressor Polycomb 2/antagonistas & inibidores , Regiões Promotoras Genéticas , Transdução de Sinais/imunologia , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad4/deficiência , Proteína Smad4/genética , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
The TGF-ß/Smad3 pathway plays a major role in tissue fibrosis, but the precise mechanisms are not fully understood. Here we identified microRNA miR-433 as an important component of TGF-ß/Smad3-driven renal fibrosis. The miR-433 was upregulated following unilateral ureteral obstruction, a model of aggressive renal fibrosis. In vitro, overexpression of miR-433 enhanced TGF-ß1-induced fibrosis, whereas knockdown of miR-433 suppressed this response. Furthermore, Smad3, but not Smad2, bound to the miR-433 promoter to induce its expression. Delivery of an miR-433 knockdown plasmid to the kidney by ultrasound microbubble-mediated gene transfer suppressed the induction and progression of fibrosis in the obstruction model. The antizyme inhibitor Azin1, an important regulator of polyamine synthesis, was identified as a target of miR-433. Overexpression of miR-433 suppressed Azin1 expression, while, in turn, Azin1 overexpression suppressed TGF-ß signaling and the fibrotic response. Thus, miR-433 is an important component of TGF-ß/Smad3-induced renal fibrosis through the induction of a positive feedback loop to amplify TGF-ß/Smad3 signaling, and may be a potential therapeutic target in tissue fibrosis.
Assuntos
Proteínas de Transporte/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , MicroRNAs/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Doença Antimembrana Basal Glomerular/genética , Doença Antimembrana Basal Glomerular/metabolismo , Doença Antimembrana Basal Glomerular/patologia , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular , Modelos Animais de Doenças , Doxorrubicina , Fibrose , Rim/patologia , Nefropatias/etiologia , Nefropatias/genética , Nefropatias/patologia , Nefropatias/prevenção & controle , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Regiões Promotoras Genéticas , Interferência de RNA , Ratos , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/deficiência , Proteína Smad3/genética , Proteína Smad7/genética , Proteína Smad7/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta1/genética , Regulação para Cima , Obstrução Ureteral/complicaçõesRESUMO
RATIONALE: Vascular smooth muscle cell (VSMC) differentiation from neural crest cells (NCCs) is critical for cardiovascular development, but the mechanisms remain largely unknown. OBJECTIVE: Transforming growth factor-ß (TGF-ß) function in VSMC differentiation from NCCs is controversial. Therefore, we determined the role and mechanism of a TGF-ß downstream signaling intermediate Smad2 in NCC differentiation to VSMCs. METHODS AND RESULTS: By using Cre/loxP system, we generated a NCC tissue-specific Smad2 knockout mouse model and found that Smad2 deletion resulted in defective NCC differentiation to VSMCs in aortic arch arteries during embryonic development and caused vessel wall abnormality in adult carotid arteries where the VSMCs are derived from NCCs. The abnormalities included 1 layer of VSMCs missing in the media of the arteries with distorted and thinner elastic lamina, leading to a thinner vessel wall compared with wild-type vessel. Mechanistically, Smad2 interacted with myocardin-related transcription factor B (MRTFB) to regulate VSMC marker gene expression. Smad2 was required for TGF-ß-induced MRTFB nuclear translocation, whereas MRTFB enhanced Smad2 binding to VSMC marker promoter. Furthermore, we found that Smad2, but not Smad3, was a progenitor-specific transcription factor mediating TGF-ß-induced VSMC differentiation from NCCs. Smad2 also seemed to be involved in determining the physiological differences between NCC-derived and mesoderm-derived VSMCs. CONCLUSIONS: Smad2 is an important factor in regulating progenitor-specific VSMC development and physiological differences between NCC-derived and mesoderm-derived VSMCs.
Assuntos
Diferenciação Celular , Desenvolvimento Muscular , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Crista Neural/metabolismo , Proteína Smad2/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Aorta Torácica/anormalidades , Aorta Torácica/metabolismo , Sítios de Ligação , Artérias Carótidas/anormalidades , Artérias Carótidas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Músculo Liso Vascular/anormalidades , Miócitos de Músculo Liso/patologia , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad2/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
Although transforming growth factor (TGF)-ß1 is a well-known immunosuppressive cytokine, little is known about the role of its downstream transcription factors, Smad2 and Smad3, in the suppression of macrophage activation. Previous studies have demonstrated that Smad3 is critical for the suppression of LPS-mediated inducible nitric oxide (NO) synthase (iNOS) induction, although the role of Smad2 remains to be investigated. In this study, we found that iNOS induction was enhanced in Smad2-deficient bone marrow-derived macrophages (BMDMs) and peritoneal macrophages in vitro and tumor-associated macrophages in vivo, compared with wild-type (WT) macrophages. However, TGF-ß1 still suppressed iNOS induction in Smad2-deficient macrophages. In Smad2/3 double knockout (KO) (Smad2/3 DKO) BMDMs, LPS-mediated NO/iNOS induction was more strongly elevated than in Smad2 or Smad3 single KO BMDMs, and its suppression by exogenous TGF-ß1 was severely impaired. These data suggest that Smad2 and Smad3 redundantly regulate iNOS induction. Similarly, the production of IL-6 and TNFα, but not IL-10 was augmented in Smad2/3 DKO BMDMs, suggesting that Smad2 and Smad3 also redundantly suppressed some cytokines production. In Smad2/3 DKO macrophages, TLR3- as well as TLR4-mediated IRF3 activation and IFN-ß production were strongly augmented, which resulted in hyper STAT1 phosphorylation. Furthermore, IFN-ß- and IFN-γ-induced iNOS induction in the absence of TLR signaling and STAT1 transcriptional activity were augmented in Smad2/3 DKO BMDMs. These results suggest that Smad2 and Smad3 negatively regulate iNOS induction in macrophages by suppressing multiple steps in the IRF3-IFN-ß-STAT1 pathway.
Assuntos
Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Animais , Células da Medula Óssea/imunologia , Células Cultivadas , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/metabolismo , Interferon gama/metabolismo , Interleucina-10 , Interleucina-6/biossíntese , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad3/deficiência , Proteína Smad3/genética , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Transcrição Gênica , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
TGF-ß modulates immune response by suppressing non-regulatory T (Treg) function and promoting Treg function. The question of whether TGF-ß achieves distinct effects on non-Treg and Treg cells through discrete signaling pathways remains outstanding. In this study, we investigated the requirements of Smad-dependent and -independent TGF-ß signaling for T-cell function. Smad2 and Smad3 double deficiency in T cells led to lethal inflammatory disorder in mice. Non-Treg cells were spontaneously activated and produced effector cytokines in vivo on deletion of both Smad2 and Smad3. In addition, TGF-ß failed to suppress T helper differentiation efficiently and to promote induced Treg generation of non-Treg cells lacking both Smad2 and Smad3, suggesting that Smad-dependent signaling is obligatory to mediate TGF-ß function in non-Treg cells. Unexpectedly, however, the development, homeostasis, and function of Treg cells remained intact in the absence of Smad2 and Smad3, suggesting that the Smad-independent pathway is important for Treg function. Indeed, Treg-specific deletion of TGF-ß-activated kinase 1 led to failed Treg homeostasis and lethal immune disorder in mice. Therefore, Smad-dependent and -independent TGF-ß signaling discretely controls non-Treg and Treg function to modulate immune tolerance and immune homeostasis.
Assuntos
Transdução de Sinais/imunologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Deleção de Genes , Homeostase/imunologia , Inflamação/patologia , Integrases/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Proteína Smad2/deficiência , Proteína Smad3/deficiência , Linfócitos T/citologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Transforming growth factor-ß (TGF-ß) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-ß through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-ß via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-ß in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-ß via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation.
Assuntos
Proteína Smad2/imunologia , Proteína Smad3/imunologia , Proteínas com Domínio T/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Sítios de Ligação , Diferenciação Celular , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad3/deficiência , Células Th17/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor beta1 (TGFbeta1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFbeta1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFbeta signaling pathway, is a master regulator of ADAM12 expression in response to TGFbeta1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFbeta1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFbeta1-induced expression of ADAM12. In a panel of TGFbeta1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFbeta1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFbeta1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.
Assuntos
Proteínas ADAM/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteínas ADAM/biossíntese , Proteína ADAM12 , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/biossíntese , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteína Smad2/deficiência , Proteína Smad2/metabolismo , Proteína Smad3/deficiência , Proteína Smad3/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Whereas TGF-beta is essential for the development of peripherally induced Foxp3(+) regulatory T cells (iTreg cells) and Th17 cells, the intracellular signaling mechanism by which TGF-beta regulates development of both cell subsets is less understood. In this study, we report that neither Smad2 nor Smad3 gene deficiency abrogates TGF-beta-dependent iTreg induction by a deacetylase inhibitor trichostatin A in vivo, although the loss of the Smad2 or Smad3 gene partially reduces iTreg induction in vitro. Similarly, SMAD2 and SMAD3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTreg cells, whereas the p38 pathway predominately modulated Th17 and experimental autoimmune encephalomyelitis induction. Therefore, selective targeting of these intracellular TGF-beta signaling pathways during iTreg and Th17 cell development might lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.
Assuntos
Diferenciação Celular/imunologia , Interleucina-17/biossíntese , Transdução de Sinais/imunologia , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Fatores de Transcrição Forkhead/biossíntese , Técnicas de Introdução de Genes , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad3/deficiência , Proteína Smad3/genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.
Assuntos
Neoplasias Colorretais/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Animais , Divisão Celular , Neoplasias Colorretais/genética , Progressão da Doença , Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Fígado/fisiologia , Camundongos , Camundongos Knockout , Estadiamento de Neoplasias , Fosforilação , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad3/deficiência , Proteína Smad3/genéticaRESUMO
Transforming growth factor beta (TGF-beta) superfamily members are critical in maintaining cell growth and differentiation in the ovary. Although signaling of activins, TGF-betas, growth differentiation factor 9, and nodal converge preferentially to SMAD2 and SMAD3, the in vivo functions and redundancy of these SMADs in the ovary and female reproduction remain largely unidentified. To circumvent the deleterious phenotypic aspects of ubiquitous deletion of Smad2 and Smad3, a conditional knockout strategy was formulated to selectively inactivate Smad2, Smad3, or both Smad2 and Smad3 in ovarian granulosa cells. While granulosa cell ablation of individual Smad2 or Smad3 caused insignificant changes in female fertility, deletion of both Smad2 and Smad3 led to dramatically reduced female fertility and fecundity. These defects were associated with the disruption of multiple ovarian processes, including follicular development, ovulation, and cumulus cell expansion. Furthermore, the impaired expansion of cumulus cells may be partially associated with altered cumulus expansion-related transcripts that are regulated by SMAD2/3 signaling. Our results indicate that SMAD2 and SMAD3 function redundantly in vivo to maintain normal female fertility and further support the involvement of an intraovarian SMAD2/3 pathway in mediating oocyte-produced signals essential for coordinating key events of the ovulatory process.
Assuntos
Células da Granulosa/patologia , Ovário/patologia , Proteína Smad2/fisiologia , Proteína Smad3/fisiologia , Animais , Células do Cúmulo/patologia , Feminino , Infertilidade Feminina/etiologia , Camundongos , Camundongos Knockout , Ovulação , Proteína Smad2/deficiência , Proteína Smad3/deficiênciaRESUMO
TGF-beta and its signaling mediators, Smad2, -3, and -4, are involved with tumor suppression and promotion functions. Smad4-/- mouse epidermis develops spontaneous skin squamous cell carcinomas (SCCs), and Smad3-/- mice are resistant to carcinogen-induced skin cancer; however, the role of Smad2 in skin carcinogenesis has not been explored. In the present study, we found that Smad2 and Smad4, but not Smad3, were frequently lost in human SCCs. Mice with keratinocyte-specific Smad2 deletion exhibited accelerated formation and malignant progression of chemically induced skin tumors compared with WT mice. Consistent with the loss of Smad2 in poorly differentiated human SCCs, Smad2-/- tumors were poorly differentiated and underwent epithelial-mesenchymal transition (EMT) prior to spontaneous Smad4 loss. Reduced E-cadherin and activation of its transcriptional repressor Snail were also found in Smad2-/- mouse epidermis and occurred more frequently in Smad2-negative human SCCs than in Smad2-positive SCCs. Knocking down Snail abrogated Smad2 loss-associated EMT, suggesting that Snail upregulation is a major mediator of Smad2 loss-associated EMT. Furthermore, Smad2 loss led to a significant increase in Smad4 binding to the Snail promoter, and knocking down either Smad3 or Smad4 in keratinocytes abrogated Smad2 loss-associated Snail overexpression. Our data suggest that enhanced Smad3/Smad4-mediated Snail transcription contributed to Smad2 loss-associated EMT during skin carcinogenesis.
Assuntos
Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/patologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Proteína Smad2/deficiência , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Células Cultivadas , Progressão da Doença , Epitélio/patologia , Técnica Direta de Fluorescência para Anticorpo , Deleção de Genes , Humanos , Queratinócitos/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/metabolismo , Estudos Retrospectivos , Neoplasias Cutâneas/genética , Proteína Smad2/genética , Proteína Smad2/metabolismoRESUMO
Phosphorylation-dependent activation of the transcription factors Smad2 and Smad3 plays an important role in TGFbeta-dependent signal transduction. Following phosphorylation of Smad2 and Smad3, these molecules are translocated to the nucleus where they interact with coactivators and/or corepressors, including p300, CBP, and P/CAF, and regulate the expression of TGFbeta target genes. In the current study, we demonstrate that both Smad2 and Smad3 are acetylated by the coactivators p300 and CBP in a TGFbeta-dependent manner. Smad2 is also acetylated by P/CAF. The acetylation of Smad2 was significantly higher than that of Smad3. Lys(19) in the MH1 domain was identified as the major acetylated residue in both the long and short isoform of Smad2. Mutation of Lys(19) also reduced the p300-mediated acetylation of Smad3. By generating acetyl-Lys(19)-specific antibodies, we demonstrate that endogenous Smad2 is acetylated on this residue in response to TGFbeta signaling. Acetylation of the short isoform of Smad2 improves its DNA binding activity in vitro and enhances its association with target promoters in vivo, thereby augmenting its transcriptional activity. Acetylation of Lys(19) also enhanced the DNA binding activity of Smad3. Our data indicate that acetylation of Lys(19) induces a conformational change in the MH1 domain of the short isoform of Smad2, thereby making its DNA binding domain accessible for interactions with DNA. Thus, coactivator-mediated acetylation of receptor-activated Smad molecules could represent a novel way to regulate TGFbeta signaling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição de p300-CBP/fisiologia , Acetilação , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa , Humanos , Lisina/metabolismo , Camundongos , Regiões Promotoras Genéticas , Conformação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Proteína Smad2/deficiência , Proteína Smad2/genética , Proteína Smad3/genética , Transcrição Gênica , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
Smad2 is a member of the intracellular mediators that transduce signals from TGF-beta receptors and activin receptors. Targeted inactivation of Smad2 in mice leads to early lethality before gastrulation. It was shown previously that TGF-betaRII deficiency in vivo leads to defects in B cell homeostasis, Ag responsiveness, and IgA class switch recombination of B cells. To investigate the importance of Smad2-mediated signaling in B lymphocytes, we generated a B cell-specific inactivation of Smad2 in mice (bSmad2(-/-)). bSmad2(-/-) mice had normal B cell numbers in the spleen but showed a reduced population of marginal zone B cells. In contrast, B cells in Peyer's patches and peritoneal B-1a cells of bSmad2(-/-) mice were increased in numbers. bSmad2(-/-) mice showed a reduced number of surface-IgA(+) B cells and of IgA-secreting cells in Peyer's patches, decreased levels of IgA in serum, and, after immunization with a T cell-dependent Ag, a reduced IgA response. Class switch recombination to IgA was impaired in Smad2-deficient B cells, when stimulated in vitro with LPS in the presence of TGF-beta. The growth-inhibitory effects of TGF-beta in LPS-stimulated B cells were not affected in Smad2-deficient B cells. In summary, our data indicate a crucial role of Smad2 in mediating signals for the TGF-beta-directed class switch to IgA and the induction of IgA responses in vivo. Other B cell functions like growth-inhibitory signaling, which are known to be regulated by signals via the TGF-betaR, are not affected in Smad2-deficient B cells.