RESUMO
OBJECTIVE: Ageing is the main risk factor for osteoarthritis (OA). We investigated if expression of transforming growth factor ß (TGFß)-family components, a family which is crucial for the maintenance of healthy articular cartilage, is altered during ageing in cartilage. Moreover, we investigated the functional significance of selected age-related changes. DESIGN: Age-related changes in expression of TGFß-family members were analysed by quantitative PCR in healthy articular cartilage obtained from 42 cows (age: ¾-10 years). To obtain functional insight of selected changes, cartilage explants were stimulated with TGFß1 or bone morphogenetic protein (BMP) 9, and TGFß1 and BMP response genes were measured. RESULTS: Age-related cartilage thinning and loss of collagen type 2a1 expression (â¼256-fold) was observed, validating our data set for studying ageing in cartilage. Expression of the TGFß-family type I receptors; bAlk2, bAlk3, bAlk4 and bAlk5 dropped significantly with advancing age, whereas bAlk1 expression did not. Of the type II receptors, expression of bBmpr2 decreased significantly. Type III receptor expression was unaffected by ageing. Expression of the ligands bTgfb1 and bGdf5 also decreased with age. In explants, an age-related decrease in TGFß1-response was observed for the pSmad3-dependent gene bSerpine1 (P = 0.016). In contrast, ageing did not affect BMP9 signalling, an Alk1 ligand, as measured by expression of the pSmad1/5 dependent gene bId1. CONCLUSIONS: Ageing negatively affects both the TGFß-ALK5 and BMP-BMPR signalling routes, and aged chondrocytes display a lowered pSmad3-dependent response to TGFß1. Because pSmad3 signalling is essential for cartilage homeostasis, we propose that this change contributes to OA development.
Assuntos
Envelhecimento , Animais , Receptores de Proteínas Morfogenéticas Ósseas , Cartilagem Articular , Bovinos , Condrócitos , Transdução de Sinais , Fator de Crescimento Transformador betaRESUMO
BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Colorretais/metabolismo , Proteína Smad4/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Via de Sinalização Wnt , Proteínas Morfogenéticas Ósseas/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Transdução de Sinais , Transfecção , Proteína Supressora de Tumor p53/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
OBJECTIVE: In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorß1 (TGFß1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFß1 signaling was also studied. DESIGN: Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFß1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFß/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. RESULTS: In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFß1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFß/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFß1. CONCLUSIONS: This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFß1. This observation underlines the importance of TGFß1 in maintenance of chondrocyte phenotype.
Assuntos
Condrócitos/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Fator 2 de Diferenciação de Crescimento/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Bovinos , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/antagonistas & inibidores , Hipertrofia , Ligantes , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/metabolismo , Proteína Smad2/metabolismo , Proteína Smad5/metabolismoRESUMO
BACKGROUND: The transforming growth factor-ß (TGF-ß) pathway has dual effects on tumor growth. Seemingly, discordant results have been published on the relation between TGF-ß signaling markers and prognosis in breast cancer. Improved prognostic information for breast cancer patients might be obtained by assessing interactions among TGF-ß signaling biomarkers. PATIENTS AND METHODS: The expression of nuclear Smad4, nuclear phosphorylated-Smad2 (p-Smad2), and the membranous expression of TGF-ß receptors I and II (TßRI and TßRII) was determined on a tissue microarray of 574 breast carcinomas. Tumors were stratified according to the Smad4 expression in combination with p-Smad2 expression or Smad4 in combination with the expression of both TGF-ß receptors. RESULTS: Tumors with high expression of TßRII, TßRI and TßRII, and p-Smad2 (P = 0.018, 0.005, and 0.022, respectively), and low expression of Smad4 (P = 0.005) had an unfavorable prognosis concerning progression-free survival. Low Smad4 expression combined with high p-Smad2 expression or low expression of Smad4 combined with high expression of both TGF-ß receptors displayed an increased hazard ratio of 3.04 [95% confidence interval (CI) 1.390-6.658] and 2.20 (95% CI 1.464-3.307), respectively, for disease relapse. CONCLUSIONS: Combining TGF-ß biomarkers provides prognostic information for patients with stage I-III breast cancer. This can identify patients at increased risk for disease recurrence that might therefore be candidates for additional treatment.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Biomarcadores Tumorais/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Receptor ErbB-2/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Proteína Smad2/biossíntese , Proteína Smad4/biossíntese , Análise Serial de TecidosRESUMO
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 Lefty2 in left-right patterning was investigated by analysis of mutant mice that lack asymmetric expression of lefty2. These animals exhibited various situs defects including left isomerism. The asymmetric expression of nodal was prolonged and the expression of Pitx2 was upregulated in the mutant embryos. The absence of Lefty2 conferred on Nodal the ability to diffuse over a long distance. Thus, Nodal-responsive genes, including Pitx2, that are normally expressed on the left side were expressed bilaterally in the mutant embryos, even though nodal expression was confined to the left side. These results suggest that Nodal is a long-range signaling molecule but that its range of action is normally limited by the feedback inhibitor Lefty2.
Assuntos
Padronização Corporal/fisiologia , Proteínas Nucleares , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Difusão , Retroalimentação Fisiológica/fisiologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Determinação Direita-Esquerda , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Nodal , Fosforilação , Transdução de Sinais/fisiologia , Proteína Smad2 , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
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
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
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
Fibrodysplasia ossificans progressiva (FOP) is a very rare devastating heterotopic ossification disorder, classically caused by a heterozygous single point mutation (c.617G>A) in the ACVR1gene, encoding the Bone morphogenetic protein (BMP) type I receptor, also termed activin receptor-like kinase (ALK)2. FOP patients develop heterotopic ossification episodically in response to inflammatory insults, thereby compromising tissue sampling and the development of in vitro surrogate models for FOP. Here we describe the generation and characterization of a control and a classical FOP induced pluripotent stem cell (iPSC) line derived from periodontal ligament fibroblast cells using Sendai virus vectors.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular/patologia , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Ligamento Periodontal/patologia , Adulto , Sequência de Bases , Feminino , Humanos , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.
Assuntos
Benzamidas/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Receptores de Ativinas Tipo I/antagonistas & inibidores , Administração Oral , Animais , Benzamidas/administração & dosagem , Modelos Animais de Doenças , Fibrose , Humanos , Camundongos , Camundongos Endogâmicos , Fosforilação/efeitos dos fármacos , Pirazóis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Transforming growth factor-beta (TGF-beta) elicits a potent growth inhibitory effect on many normal cells by binding to specific serine/threonine kinase receptors and activating specific Smad proteins, which regulate the expression of cell cycle genes, including the p21 cyclin-dependent kinase (CDK) inhibitor gene. Interestingly, cancer cells are often insensitive to the anti-mitogenic effects of TGF-beta for which the molecular mechanisms are not well understood. In this study, we found that the candidate prostate cancer susceptibility gene ELAC2 potentiates TGF-beta/Smad-induced transcriptional responses. ELAC2 associates with activated Smad2; the C-terminal MH2 domain of Smad2 interacts with the N-terminal region of ELAC2. Small interfering siRNA-mediated knock-down of ELAC2 in prostate cells suppressed TGF-beta-induced growth arrest. Moreover, ELAC2 was shown to specifically associate with the nuclear Smad2 partner, FAST-1 and to potentiate the interaction of activated Smad2 with transcription factor Sp1. Furthermore, activation of the p21 CDK inhibitor promoter by TGF-beta is potentiated by ELAC2. Taken together our data indicate an important transcriptional scaffold function for ELAC2 in TGF-beta/Smad signaling mediated growth arrest.
Assuntos
Divisão Celular/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Sequência de Bases , Células COS , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Primers do DNA , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , RNA Interferente Pequeno , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transforming growth factor beta (TGF-beta) is an important regulator of apoptosis in some cell types, but the underlying molecular mechanisms are largely unknown. TGF-beta signals through type I and type II receptors and downstream effector proteins, termed Smads. TGF-beta induces the phosphorylation of Smad2 and Smad3 (receptor-activated Smads) which associate with Smad4 and translocate to the nucleus, where they regulate gene transcription [1]. Smad7 protein is induced by TGF-beta1 and has been classified as an inhibitory Smad. Smad7 prevents phosphorylation of receptor-activated Smads, thereby inhibiting TGF-beta-induced signaling responses [1]. Smad7 expression is increased in rat prostatic epithelial cells undergoing apoptosis as a result of castration [2]. Here we have shown that TGF-beta1 treatment or ectopic expression of Smad7 in human prostatic carcinoma cells (PC-3U) induces apoptosis. Furthermore, TGF-beta1-induced apoptosis was prevented by inhibition of Smad7 expression, by antisense mRNA in stably transfected cell lines or upon transient transfection with antisense oligonucleotides in several investigated cell lines. These findings provide evidence for a new effector function for Smad7 in TGF-beta1 signaling.
Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Masculino , Neoplasias da Próstata , Proteína Smad7 , Transativadores/genética , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais CultivadasRESUMO
Primary and secondary forms of focal segmental glomerulosclerosis (FSGS) are characterized by depletion of podocytes and constitute a central manifestation of chronic progressive glomerular diseases. Here we report that podocytes undergo apoptosis at early stages in the course of progressive glomerulosclerosis in TGF-beta1 transgenic mice. Apoptosis is associated with progressive depletion of podocytes and precedes mesangial expansion. Smad7 protein expression is strongly induced specifically in damaged podocytes of transgenic mice and in cultured murine podocytes treated with TGF-beta. TGF-beta1 and Smad7 each induce apoptosis in podocytes, and their coexpression has an additive effect. Activation of p38 MAP kinase and caspase-3 is required for TGF-beta-mediated apoptosis, but not for apoptosis induced by Smad7. Unlike TGF-beta, Smad7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NF-kappaB. Our results suggest a novel functional role for Smad7 as amplifier of TGF-beta-induced apoptosis in podocytes and a new pathomechanism for podocyte depletion in progressive glomerulosclerosis.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Transativadores/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Mesângio Glomerular/patologia , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína Smad7 , Fator de Crescimento Transformador beta/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We have recently cloned the cDNA for transforming growth factor type beta 3 (TGF-beta 3), a new member of the TGF-beta gene family. We examined the biological effects of recombinant TGF-beta 3 protein in osteoblast-enriched bone cell cultures. In this report we demonstrate that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity. In a direct comparison between TGF-beta 3 and TGF-beta 1, TGF-beta 3 appeared to be three- to fivefold more potent than TGF-beta 1. Our cross-linking experiments with iodinated TGF-beta showed that in osteoblast-enriched bone cell cultures, both TGF-beta 3 and TGF-beta 1 associated with the same three cell surface binding sites. Scatchard analysis of receptor competition studies indicated the presence of high-affinity binding sites for TGF-beta 3 in the picomolar range. TGF-beta 3 showed an approximately fourfold-higher apparent affinity than TGF-beta 1 in overall binding.
Assuntos
Osso e Ossos/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Crescimento Transformadores/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Colágeno/biossíntese , Replicação do DNA/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Cinética , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Biossíntese de Proteínas , Ratos , Ratos Endogâmicos , Receptores de Fatores de Crescimento Transformadores beta , Proteínas Recombinantes/metabolismo , Transfecção , Fatores de Crescimento Transformadores/metabolismoRESUMO
TGF-ß signaling in liver cells has variant roles in the dynamics of liver diseases, including hepatocellular carcinoma (HCC). We previously found a correlation of high levels of the important endogenous negative TGF-ß signaling regulator SMAD7 with better clinical outcome in HCC patients. However, the underlying tumor-suppressive molecular mechanisms are still unclear. Here, we show that conditional (TTR-Cre) hepatocyte-specific SMAD7 knockout (KO) mice develop more tumors than wild-type and corresponding SMAD7 transgenic mice 9 months after diethylnitrosamine (DEN) challenge, verifying SMAD7 as a tumor suppressor in HCC. In line with our findings in patients, Smad7 levels in both tumor tissue as well as surrounding tissue show a significant inverse correlation with tumor numbers. SMAD7 KO mice presented with increased pSMAD2/3 levels and decreased apoptosis in the tumor tissue. Higher tumor incidence was accompanied by reduced P21 and upregulated c-MYC expression in the tumors. Activation of signal transducer and activator of transcription factor 3 signaling was found in Smad7-deficient mouse tumors and in patients with low tumoral SMAD7 expression as compared with surrounding tissue. Together, our results provide new mechanistic insights into the tumor-suppressive functions of SMAD7 in hepatocarcinogenesis.