RESUMO
Members of the transforming growth factor-beta (TGF-beta) superfamily have been found to signal by inducing the formation of hetero-oligomeric complexes of different type I and type II serine/threonine kinase receptors. Recent data indicate that binding of TGF-beta to its constitutively active type II receptor recruits the type I receptor into the complex; the type I receptor is thereafter phosphorylated and activated, processes which are necessary and sufficient for most TGF-beta mediated responses. Recent genetic analyses of Drosophila also indicate a strict requirement for both type I and type II receptors in decapentaplegic signaling in vivo.
Assuntos
Receptores de Ativinas Tipo I , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo IIRESUMO
The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse-chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.
Assuntos
Proteínas de Transporte/metabolismo , Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular , Fibrinolisina/metabolismo , Fibroblastos , Fibrossarcoma/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Ligação a TGF-beta Latente , Peso Molecular , Ligação Proteica , Células Tumorais CultivadasRESUMO
Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais/fisiologia , Transativadores/química , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases , Transporte Ativo do Núcleo Celular/fisiologia , Receptores de Ativinas Tipo I/metabolismo , Animais , Células COS , Células Cultivadas , Proteínas de Ligação a DNA/genética , Deleção de Genes , Ligases/metabolismo , Vison , Mutagênese/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão , Mucosa Respiratória/citologia , Proteína Smad2 , Proteína Smad6 , Proteína Smad7 , Transativadores/genética , Transcrição Gênica/fisiologiaRESUMO
Transforming growth factor-beta (TGF beta) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGF beta as a large latent TGF beta complex containing mature TGF beta, latency associated peptide, and latent TGF beta-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGF beta protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGF beta 1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40-100 nm extracellular fibrillar structure and 5-10-nm microfibrils. The anti-LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGF beta proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGF beta as a large latent TGF beta complex.
Assuntos
Proteínas de Transporte/análise , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mesoderma/citologia , Miocárdio/química , Fator de Crescimento Transformador beta/farmacologia , Animais , Especificidade de Anticorpos , Células COS , Técnicas de Cocultura , Técnicas de Cultura , Endocárdio/citologia , Endocárdio/crescimento & desenvolvimento , Endotélio Vascular/embriologia , Matriz Extracelular/química , Fibrinolisina , Coração/embriologia , Humanos , Proteínas de Ligação a TGF-beta Latente , Camundongos , Camundongos Endogâmicos ICR , Miocárdio/citologia , Transfecção , Fator de Crescimento Transformador beta/análiseRESUMO
The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.
Assuntos
Matriz Óssea/metabolismo , Proteínas de Transporte/metabolismo , Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Crânio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Desenvolvimento Ósseo , Proteínas de Transporte/análise , Células Cultivadas , Imuno-Histoquímica , Proteínas de Ligação a TGF-beta Latente , Ratos , Crânio/embriologia , Fator de Crescimento Transformador beta/análiseRESUMO
Transforming growth factor beta (TGF-beta) is released from cells in a latent form consisting of the mature growth factor associated with an aminoterminal propeptide and latent TGF-beta binding protein (LTBP). The endogenous activation of latent TGF-beta has been described in co-cultures of endothelial and smooth muscle cells. However, the mechanism of this activation remains unknown. Antibodies to native platelet LTBP and to a peptide fragment of LTBP inhibit in a dose-dependent manner the activation of latent TGF-beta normally observed when endothelial cells are cocultured with smooth muscle cells. Inhibition of latent TGF-beta activation was also observed when cells were co-cultured in the presence of an excess of free LTBP. These data represent the first demonstration of a function for the LTBP in the extracellular regulation of TGF-beta activity and indicate that LTBP participates in the activation of latent TGF-beta, perhaps by concentrating the latent growth factor on the cell surface where activation occurs.
Assuntos
Proteínas de Transporte/metabolismo , Endotélio Vascular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Músculo Liso Vascular/metabolismo , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Bovinos , Células Cultivadas , Meios de Cultura , Endotélio Vascular/citologia , Fibrinolisina/metabolismo , Técnicas In Vitro , Proteínas de Ligação a TGF-beta Latente , Músculo Liso Vascular/citologiaRESUMO
Proteins in the TGF-beta superfamily transduce their effects through binding to type I and type II serine/threonine kinase receptors. Osteogenic protein-1 (OP-1, also known as bone morphogenetic protein-7 or BMP-7), a member of the TGF-beta superfamily which belongs to the BMP subfamily, was found to bind activin receptor type I (ActR-I), and BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB) in the presence of activin receptors type II (ActR-II) and type IIB (ActR-IIB). The binding affinity of OP-1 to ActR-II was two- to threefold lower than that of activin A. A transcriptional activation signal was transduced after binding of OP-1 to the complex of ActR-I and ActR-II, or that of BMPR-IB and ActR-II. These results indicate that ActR-II can act as a functional type II receptor for OP-1, as well as for activins. Some of the known biological effects of activin were observed for OP-1, including growth inhibition and erythroid differentiation induction. Compared to activin, OP-1 was shown to be a poor inducer of mesoderm in Xenopus embryos. Moreover, follistatin, an inhibitor of activins, was found to inhibit the effects of OP-1, if added at a 10-fold excess. However, certain effects of activin, like induction of follicle stimulating hormone secretion in rat pituitary cells were not observed for OP-1. OP-1 has overlapping binding specificities with activins, and shares certain but not all of the functional effects of activins. Thus, OP-1 may have broader effects in vivo than hitherto recognized.
Assuntos
Proteínas Morfogenéticas Ósseas , Inibinas/farmacologia , Proteínas/farmacologia , Receptores de Fatores de Crescimento/metabolismo , Receptores de Ativinas , Ativinas , Animais , Proteína Morfogenética Óssea 7 , Células Cultivadas , Chlorocebus aethiops , Indução Embrionária/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Hormônio Foliculoestimulante/metabolismo , Folistatina , Glicoproteínas/farmacologia , Inibidores do Crescimento/farmacologia , Técnicas In Vitro , Mesoderma/citologia , Vison , Adeno-Hipófise/metabolismo , Ligação Proteica , Proteínas/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Proteínas de XenopusRESUMO
We previously demonstrated that bone morphogenetic proteins (BMPs) induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1. Transcription factors Smads mediate transforming growth factor-beta signaling and the ATF/CREB family transcription factor ATF-2 has recently been shown to act as a common target of the Smad and the TAK1 pathways. We here examined the role of Smads and ATF-2 in cardiomyocyte differentiation of P19CL6, a clonal derivative of murine P19 cells. Although P19CL6 efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide, P19CL6noggin, a P19CL6 cell line constitutively overexpressing the BMP antagonist noggin, did not differentiate into cardiomyocytes. Cooverexpression of Smad1, a ligand-specific Smad, and Smad4, a common Smad, restored the ability of P19CL6noggin to differentiate into cardiomyocytes, whereas stable overexpression of Smad6, an inhibitory Smad, completely blocked differentiation of P19CL6, suggesting that the Smad pathway is necessary for cardiomyocyte differentiation. ATF-2 stimulated the betaMHC promoter activity by the synergistic manner with Smad1/4 and TAK1 and promoted terminal cardiomyocyte differentiation of P19CL6noggin, whereas overexpression of the dominant negative form of ATF-2 reduced the promoter activities of several cardiac-specific genes and inhibited differentiation of P19CL6. These results suggest that Smads, TAK1, and their common target ATF-2 cooperatively play a critical role in cardiomyocyte differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fibras Musculares Esqueléticas/citologia , Miocárdio/citologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fator 2 Ativador da Transcrição , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Expressão Gênica/fisiologia , Fibras Musculares Esqueléticas/enzimologia , Proteínas/genética , Proteínas Smad , Proteína Smad6 , Transativadores/genéticaRESUMO
Transforming growth factor-beta (TGF-beta) and activin exert their effects by binding to heteromeric complexes of type I and type II receptors. The type II receptors for TGF-beta and activin are transmembrane serine-threonine kinases; a series of related receptors, denoted activin receptor-like kinase (ALK) 1 to 5, have recently been identified, and ALK-6 is described here. ALK-5 has been shown to be a functional TGF-beta type I receptor. A systematic analysis revealed that most ALKs formed heteromeric complexes with the type II receptors for TGF-beta and activin after overexpression in COS cells; however, among the six ALKs, only ALK-5 was a functional TGF-beta type I receptor for activation of plasminogen activator inhibitor-1, and only ALK-2 and ALK-4 bound activin with high affinity.
Assuntos
Inibinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Receptores de Ativinas , Ativinas , Sequência de Aminoácidos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Linhagem Celular , Ligantes , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento Transformadores beta/químicaRESUMO
Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways.
Assuntos
Calcitriol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Fatores de Crescimento , Transativadores/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas , Proteínas Morfogenéticas Ósseas/farmacologia , Células COS , Calcitriol/farmacologia , Núcleo Celular/metabolismo , Histona Acetiltransferases , Ligantes , Coativador 1 de Receptor Nuclear , Fosforilação , Receptor Cross-Talk , Receptores de Superfície Celular/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Transdução de Sinais , Proteína Smad3 , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 were found to act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors.
Assuntos
Citocinas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Interleucina-6 , Proteínas Nucleares/metabolismo , Receptores de Fatores de Crescimento , Transdução de Sinais , Transativadores/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta , Animais , Astrócitos/citologia , Proteína Morfogenética Óssea 2 , Receptores de Proteínas Morfogenéticas Ósseas , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Células COS , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Proteína p300 Associada a E1A , Proteína Glial Fibrilar Ácida/genética , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Linfocinas/metabolismo , Linfocinas/farmacologia , Camundongos , Regiões Promotoras Genéticas , Receptores de Superfície Celular/metabolismo , Receptores de Citocinas/metabolismo , Receptores de OSM-LIF , Fator de Transcrição STAT3 , Deleção de Sequência , Proteínas Smad , Proteína Smad1 , Células-Tronco/citologia , Células-Tronco/metabolismo , Telencéfalo/embriologia , Telencéfalo/metabolismoRESUMO
Mitogen-activated protein (MAP) kinase cascades are activated in response to various extracellular stimuli, including growth factors and environmental stresses. A MAP kinase kinase kinase (MAPKKK), termed ASK1, was identified that activated two different subgroups of MAP kinase kinases (MAPKK), SEK1 (or MKK4) and MKK3/MAPKK6 (or MKK6), which in turn activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and p38 subgroups of MAP kinases, respectively. Overexpression of ASK1 induced apoptotic cell death, and ASK1 was activated in cells treated with tumor necrosis factor-alpha (TNF-alpha). Moreover, TNF-alpha-induced apoptosis was inhibited by a catalytically inactive form of ASK1. ASK1 may be a key element in the mechanism of stress- and cytokine-induced apoptosis.
Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células COS , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 6 , MAP Quinase Quinase Quinases , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Recent studies have consolidated the pivotal role of Smads as intracellular effectors of TGF-beta family members. Upon binding to their specific type I and type II serine/threonine kinase receptors, each family member activates a particular subset of Smad proteins. Activated, receptor-regulated Smads form hetero-oligomeric complexes with common-partner Smads that translocate into the nucleus, where they control the expression of target genes in a cell-type-specific manner. Smads appear to function not only as nuclear effectors for TGF-beta family members, but as signal integrators within an extensive intracellular network.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , DNA/metabolismo , Proteína Smad2 , Proteína Smad3 , Proteína Smad6RESUMO
Transforming growth factor-beta (TGF-beta) binds to two different types of serine/threonine kinase receptors termed type II (TbetaR-II) and type I (TbetaR-I). TGF-beta is unable to bind to TbetaR-I in the absence of TbetaR-II, and initiates receptor assembly by binding with high affinity to TbetaR-II. Previous structural analysis of the TGF-beta3-TbetaR-II complex has suggested that two charged amino acid residues, D55 and E142 of TbetaR-II, are binding sites of TGF-beta. In the present study, we have shown that mutations of the amino-acid residues, D55 and E142 of TbetaR-II, resulted in loss of TGF-beta binding and downstream signaling activity. Moreover, we found that 3,5,7,2',4'-pentahydroxyflavone (Morin) inhibits TGF-beta binding to TbetaR-II, and suppresses phosphorylation of Smad2 and expression of a TGF-beta target gene Smad7 induced by TGF-beta. Our findings may thus provide useful information for designing therapeutic agents for various diseases induced by TGF-beta, including advanced cancers.
Assuntos
Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Sequência Conservada , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/genética , Alinhamento de SequênciaAssuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga a MutS , Proteínas de Neoplasias/genética , Proteínas Nucleares , Linhagem , Proteínas Proto-Oncogênicas/genéticaRESUMO
Bone morphogenic protein (BMP)-4 inhibits proliferation and induces the apoptosis of myeloma cells. However, little is known about the molecular mechanisms of how BMP-4 executes this apoptosis. In this report, we investigated the roles of p53 and the endoplasmic reticulum (ER) in BMP-4-induced apoptosis of mouse hybridoma HS-72 cells. We found that 3 ng/ml of BMP-4 is sufficient to induce the expression of proapoptotic proteins, puma and bax, in a p53-dependent mechanism, and facilitate Ca(2+) release from the ER to the cytosol, resulting in the activation of caspase-12 and ER dysfunction. Similarly to HS-72 cells, multiple myeloma cells with wild-type p53 genes show much higher sensitivity to BMP-4-induced apoptosis than cells without wild-type p53 genes, suggesting that wild-type p53 status is required for dysfunction of the ER during BMP-4-induced apoptosis in ER-enriched cells, such as hybridoma and myeloma cells. These findings demonstrate that the presence of wild-type p53 genes and enrichment of the ER determines the sensitivity to effective apoptosis by BMP-4, and suggest that ER stress-inducing agents would be valuable in the treatment of multiple myeloma.
Assuntos
Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/farmacologia , Retículo Endoplasmático/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Linfócitos B/metabolismo , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Citometria de Fluxo , Humanos , Hibridomas/efeitos dos fármacos , Hibridomas/metabolismo , Immunoblotting , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mieloma Múltiplo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/efeitos dos fármacosRESUMO
TGF-beta plays an important role in lung fibrosis, which is a major cause of suffering and death seen in pulmonary disease. Smad7 has been recently identified as an antagonist of TGF-beta signaling. To investigate whether this novel molecule can be exploited for therapy of lung fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant human type 5 adenovirus vector, on bleomycin-induced lung fibrosis in mice. C57BL/6 mice with bleomycin-induced lungs received an intratracheal injection of a recombinant adenovirus carrying mice Smad7 cDNA. These mice demonstrated suppression of type I precollagen mRNA, reduced hydroxyproline content, and no morphological fibrotic responses in the lungs when compared with mice administered adenovirus carrying Smad6 cDNA. In addition, we found that expression of Smad7 transgene blocked Smad2 phosphorylation induced by bleomycin in mouse lungs. These data indicated that gene transfer of Smad7 (but not Smad6) prevented bleomycin-induced lung fibrosis, suggesting that Smad7 may have applicability in the treatment of pulmonary fibrosis.
Assuntos
Bleomicina/toxicidade , Proteínas de Ligação a DNA/fisiologia , Terapia Genética , Fibrose Pulmonar/prevenção & controle , Transativadores/fisiologia , Transfecção , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adenoviridae/genética , Animais , Líquido da Lavagem Broncoalveolar/química , Colágeno/biossíntese , Colágeno/genética , Citomegalovirus/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/fisiologia , Proteína Smad6 , Proteína Smad7 , Transativadores/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Vascular proliferative disorders are characterized by the proliferation of vascular smooth muscle cells (SMCs) and excessive extracellular matrix synthesis. We found that bone morphogenetic protein-2 (BMP-2) inhibited serum-stimulated increases in DNA synthesis and cell number of cultured rat arterial SMCs in a fashion quite different from that in the case of transforming growth factor-beta1 (TGF-beta1). In addition, TGF-beta1 stimulated collagen synthesis in SMCs, whereas BMP-2 did not. In an in vivo rat carotid artery balloon injury model, the adenovirus-mediated transfer of the BMP-2 gene inhibited injury-induced intimal hyperplasia. These results indicate that BMP-2 has the ability to inhibit SMC proliferation without stimulating extracellular matrix synthesis, and suggest the possibility of therapeutic application of BMP-2 for the prevention of vascular proliferative disorders.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Inibidores do Crescimento/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Adenoviridae/genética , Animais , Aorta , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Artérias Carótidas/citologia , Lesões das Artérias Carótidas , Cateterismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , DNA/biossíntese , Técnicas de Transferência de Genes , Inibidores do Crescimento/genética , Humanos , Músculo Liso Vascular/citologia , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes , Fator de Crescimento Transformador beta/farmacologiaRESUMO
We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-DeltaN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal apoptosis.