RESUMO
We isolated a single-cell-derived cell line from a spinal hamartoma, a which occurred in a newborn boy and was associated with a rudimentary limb. The maternal cells (HHC-7) differentiated into osteoblasts, chondrocytes, adipocytes, and skeletal muscles when they were cultured in differentiation-inducing media specific to each mesenchymal cell. We isolated a single-cell-derived clonal cell line (Clone K) after transfection with SV40 T antigen. These cells expressed CD73 and CD117, while being negative for expression of CD45. Clone K cells cultured in an osteogenic differentiation medium increased ALP activity and expressed mRNAs for Runx2 and osteocalcin. Treatment with rhBMP-2 induced Clone K cells to differentiate into both osteoblasts and chondrocytes. These cells expressed mRNAs for Sox9 and aggrecan in addition to osteogenic markers. Culture in an adipogenic differentiation medium induced Clone K cells to differentiation into adipocytes, which expressed mRNAs for PPARgamma2 and a2P. Clone K cells cultured in a serum-depleted medium generated desmin-positive cells and expressed MyoD1 mRNA. Clone K cells exhibited numerous alpha-smooth muscle actin-positive cells; however, treatment with rhBMP-2 decreased their number. Clone K cells, transplanted with a carrier containing rhBMP-2 into the muscles of SCID mice, generated ectopic endochondral bone formation. In these tissues, several osteoblasts and chondrocytes expressed SV40 T antigen, indicating their Clone K cell origin. Thus, Clone K cells are useful tools for analyzing the characteristics of human multipotential mesenchymal progenitors.
Assuntos
Diferenciação Celular , Linhagem Celular , Hamartoma/patologia , Mesoderma/citologia , Doenças da Coluna Vertebral/patologia , Adipócitos/citologia , Animais , Condrócitos/citologia , Células Clonais , Hamartoma/metabolismo , Hamartoma/cirurgia , Humanos , Recém-Nascido , Masculino , Mesoderma/metabolismo , Mesoderma/transplante , Camundongos , Camundongos SCID , Músculo Esquelético/citologia , Osteoblastos/citologia , Doenças da Coluna Vertebral/metabolismo , Doenças da Coluna Vertebral/cirurgia , TransfecçãoRESUMO
We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Separação Celular , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Crânio/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Proteína Morfogenética Óssea 2 , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Separação Celular/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Crânio/citologia , Crânio/metabolismoRESUMO
We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Calo Ósseo/fisiologia , Fibroblastos/transplante , Terapia Genética/métodos , Proteínas de Neoplasias , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Proteína Morfogenética Óssea 2 , Calo Ósseo/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core , Fibroblastos/fisiologia , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteogênese/fisiologia , Pele/citologia , Crânio/citologia , Crânio/lesões , Crânio/fisiologia , Transdução Genética/métodosRESUMO
SOX9 is a transcription factor that is essential for chondrogenesis and testis differentiation, and haploinsufficiency of SOX9 causes campomelic dysplasia, severe skeletal malformation syndrome with variably penetrant XY sex reversal. Here we demonstrate that in several cell lines that express SOX9, 30-bp element in the first intron of human SOX9 gene act as a potential enhancer in the ATDC5 chondroprogenitor cell line, despite the apparent absence of cell-specific regulatory elements within a 5.5-kb promoter region. Deletion and site-specific mutational analyses reveal that the last 12 bp of the 30-bp element are critical for transcriptional activity, while 5'-half sequences are necessary for full transactivation. Gel retardation assays indicate the possible involvement of several binding factors along the length of this element. These results suggest that functionally interdependent elements in the 30-bp enhancer region of the first intron account for basal expression levels of Sox9 in ATDC5.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Proteínas de Grupo de Alta Mobilidade/genética , Íntrons/fisiologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Células 3T3 , Animais , Sequência de Bases , DNA/análise , Análise Mutacional de DNA , Humanos , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Ratos , Fatores de Transcrição SOX9 , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
The thyroid gland is one of the most sensitive organs in ionizing radiation (IR)-induced carcinogenesis. To determine, therefore, the specific cascade of IR-induced signal transduction in human thyroid cells, we investigated the functional role of protein kinase C (PKC), especially its interlocking activation of c-Jun NH(2)-terminal kinase (JNK) pathway. In the present study, using adenovirus expression vectors for diverse dominant-negative (DN) types of PKC isoforms (alpha, beta2, delta, epsilon and zeta) expressed in primary cultured human thyroid cells, only DN/PKC delta suppressed IR-induced JNK activation. In addition, Rottlerin, a PKC delta specific inhibitor, inhibited IR-induced JNK activation. IR-induced activation of transcription factor AP-1, downstream target of JNK, was also attenuated by DN/PKC delta. To examine the involvement of upstream kinases of JNK, we performed immune-complex kinase assays of mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. IR activated MKK7 but not MKK4, and this activation was inhibited by Rottlerin. Furthermore, IR-induced JNK activation was suppressed by overexpression of kinase-deficient MKK7. Our results indicate that IR selectively activates the cascade of PKC delta-MKK7-JNK-AP-1 in human thyroid cells, suggesting a not apoptotic but radio-resistant role of PKC delta in human thyroid cells following IR.
Assuntos
Isoenzimas/metabolismo , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Glândula Tireoide/efeitos da radiação , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 7 , Fosforilação , Proteína Quinase C-delta , Radiação Ionizante , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/metabolismo , Tirosina/metabolismo , Raios Ultravioleta , Raios XRESUMO
We have recently demonstrated that Tie-1 and Tie-2 are expressed in synovial cells from rheumatoid arthritis (RA). To elucidate the possible involvement of Tie receptors in synovial proliferation, we analyzed their expression by immunostaining in five cases of giant cell tumor of tendon sheath (GCTTS), which represents a proliferating lesion of synovial cells. Strong immunoreactivity for both Tie-1 and Tie-2, regardless of the individual patient's profile, was observed in all cases of GCTTS. Six sets of double immunohistochemical stainings for Tie-1/Tie-2 and fibronectin, CD68, or CD34 were carried out to determine the phenotype of Tie-1 and Tie-2-positive tumor components. In these studies, both Tie-1 and Tie-2 immunoreactivity were widely observed in the fibronectin-positive fibroblastic and the CD68-positive histiocytic mononuclear cells, as well as in the osteoclast-like giant cells. In tumor vasculature, Tie receptors were expressed in the CD34-positive endothelial cells possessing proliferating cell nuclear antigen (PCNA) immunoreactivity. We also evaluated the correlation of Tie-1/Tie-2 expression and proliferating cells in GCTTS by using double staining of Tie-1/Tie-2 together with PCNA. Overexpression of PCNA immunoreactivity was frequently found in Tie receptors-positive cells with no obvious differences in the expression pattern of Tie-1 and Tie-2. These findings suggest the possible involvement of Tie receptors in the pathogenesis of GCTTS other than solely via their involvement in angiogenesis and subsequent vascularization. It was demonstrated that Tie-2 immunoreactivity was restricted to the fibroblastic, but not histiocytic, phenotype in RA synovium, suggesting different regulatory control of Tie-2 expression in GCTTS and RA synovium. Overexpression of Tie receptors in GCTTS may imply a biological role for these receptors in synovial proliferation.
Assuntos
Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Membrana Sinovial/metabolismo , Sinovite Pigmentada Vilonodular/metabolismo , Tendões/metabolismo , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Divisão Celular , Feminino , Fibronectinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptor de TIE-1 , Receptor TIE-2 , Receptores de TIE , Membrana Sinovial/patologia , Sinovite Pigmentada Vilonodular/patologia , Tendões/patologiaRESUMO
Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)
Assuntos
Receptores de Ativinas Tipo I , Proteínas de Ligação a DNA/fisiologia , Produtos do Gene tax/fisiologia , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transativadores/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Proteínas de Xenopus , Animais , Sítios de Ligação , Células COS , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Chlorocebus aethiops , Genes pX , Humanos , Neoplasias Hepáticas/patologia , Pulmão , Substâncias Macromoleculares , Vison , Fatores de Crescimento Neural , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Sequências Reguladoras de Ácido Nucleico , Proteínas Smad , Proteína Smad2 , Proteína Smad3 , Proteína Smad4 , Transativadores/genética , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Xenopus laevisRESUMO
Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Receptores de Ativinas , Ativinas , Animais , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Quimera/genética , Quimera/imunologia , Quimera/metabolismo , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Marcação de Genes , Genes Reporter/efeitos dos fármacos , Inibinas/farmacologia , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Fosfoproteínas/genética , Fosforilação , Testes de Precipitina , Proteína Smad2 , Proteína Smad3 , Transativadores/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
To investigate the relationship of chondrocyte apoptosis and cartilage destruction, we performed in situ nick end labeling (ISNEL), electron microscopy, and immunohistochemistry against apoptosis-related proteins, p53 and c-myc, in the articular cartilages of patients with rheumatoid arthritis (RA; n = 12) and osteoarthritis (OA; n = 12), and in control articular cartilages from patients with femoral neck fracture (n = 8). The distribution of stained chondrocytes was evaluated semiquantitatively in relation to the degree of cartilage destruction. ISNEL-positive chondrocytes with apoptotic morphological features were identified in a relatively early phase of cartilage destruction, and correlated positively and significantly in a number with the degree of cartilage degeneration. Comparison of RA and OA revealed a significantly greater number of ISNEL-positive chondrocytes in RA cartilage. In contrast, the specimens of normal subjects contained few cells with apoptotic changes. Similarly to the distribution of ISNEL staining, the expression of p53 and c-myc proteins was observed in chondrocytes within the degraded lesions, and showed a positive correlation with the number of ISNEL-stained cells. These results suggest that the degree of chondrocyte apoptosis is closely related to cartilage destruction and that chondrocytes in RA more readily undergo apoptosis than those in OA. The expression of p53 and c-myc proteins in ISNEL-positive areas may reflect the involvement of these proteins in the apoptotic process in articular chondrocytes in inflammatory arthritis.
Assuntos
Apoptose/genética , Artrite Reumatoide/genética , Cartilagem Articular/patologia , Condrócitos/patologia , Osteoartrite/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/patologia , Feminino , Expressão Gênica/fisiologia , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Osteoartrite/patologiaRESUMO
Intraarticular injection of dexamethasone (DEX) accelerates cartilage degradation due to the suppression of chondrocyte proliferation and extracellular matrix formation. The present study first demonstrated the interaction between DEX and TGF beta, a potent growth factor for cultured rat articular chondrocytes (CRAC), and then investigated the molecular mechanism by which DEX counteracts TGF beta-induced chondrocyte proliferation and differentiation through the regulation of AP-1 activity. DEX reduced serum-deprived and TGF beta-stimulated cell growth and [(3)H]-thymidine incorporation of CRAC. DEX also inhibited the expression of (alpha)1 type II collagen with concomitant suppression of the promoter activity. Transfection studies using a reporter vector with AP-1 responsive elements showed that DEX reduced TGF beta-activated but not basal luciferase activities. Activation of 3TP-luc, another AP-1 responsive element containing reporter was also blocked by DEX. GAL4-Elk1 studies revealed that DEX suppressed TGF beta-induced ERK activation which led to c-fos gene expression followed by increase in AP-1 complex formation, whereas the Smad pathway was not involved in DEX-dependent negative regulation of AP-1 in a reporter assay that requires FAST1-Smad2 for the activation. DEX also eliminated TGF beta-induced c-fos mRNA expression and ERK activation in Northern analysis and in vitro kinase assay, respectively. Further, DNA synthesis and transactivation of type II collagen by TGF beta were inhibited by PD98059, an inhibitor of MEK. Our results indicate that DEX suppressed TGF beta-induced chondrocyte proliferation and type II collagen expression, probably through selective inhibition of ERK integrated AP-1 activation.
Assuntos
Anti-Inflamatórios/farmacologia , Condrócitos/efeitos dos fármacos , Colágeno/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Cartilagem Articular/citologia , Cartilagem Articular/efeitos dos fármacos , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fator de Transcrição AP-1/metabolismoRESUMO
OBJECTIVE: TGFbeta is a potent stimulator of cell growth in cultured rat articular chondrocytes (CRAC). The stimulatory effect is mediated through the immediate induction of c-fos gene by activating ERK of MAPK. The present study was undertaken to investigate the upstream regulators involved in TGFbeta-induced ERK activation in CRAC and to compare the results with the events in HepG2 cells. RESULTS: In vitro kinase and trans-reporting assays showed that TGFbeta preferentially activated ERK and JNK pathways in CRAC and HepG2, respectively. ERK activation in CRAC was selectively inhibited by PD98059, a MEK inhibitor. Overexpression of wild or active forms of MEKK1, the upstream activator of ERK and JNK, decreased the TGFbeta-induced 3TP-luciferase activity in CRAC. In contrast, in HepG2 dominant negative form of MEKK1 or SEK1 ligand-dependent reporter activity was diminished. Transfection of TAK1, another MAPKKK, also positively and negatively regulated 3TP transcriptional activity of HepG2 and CRAC, respectively. Activation of PKA by 8-bromo-cyclic AMP or forskolin, and inhibition of PKC by calphostin C, resulted in a significant decrease in 3TP activity as well as in vitro ERK kinase activity in CRAC. CONCLUSIONS: The results indicate that TGFbeta transduces a predominant signal pathway through MEK-ERK-Elk1, independent of MEKK1 or TAK1 pathway in CRAC. However, in HepG2, activation of MEKK1 and TAK1 is essential for TGFbeta-induced signal transmission. The results also demonstrated that in CRAC, MEK-ERK pathway activated by TGFbeta is negatively regulated by PKA cascade but transactivated by PKC.
Assuntos
Comunicação Celular/fisiologia , Condrócitos/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteína Quinase C/fisiologia , Fator de Crescimento Transformador beta/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Cartilagem Articular/citologia , Células Cultivadas/citologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Masculino , Proteína Quinase C/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas/citologia , Regulação para CimaRESUMO
OBJECTIVE: To investigate the involvement of Tie-1 and Tie-2, receptor tyrosine kinases required for angiogenesis, in synovial proliferation and angiogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissues from 10 patients with RA and three control subjects were analysed by double immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Expression of Tie-1 and Tie-2 was seen in all synovia, but predominantly in papillary projected portions. In synovial lining cells, Tie-2 was expressed mainly in the basal layer and frequently colocalised with vimentin and proliferating cell nuclear antigen (PCNA), whereas Tie-1 was also expressed in the superficial layer. In stromal cells, Tie-2 immunoreactivity was restricted to vimentin positive fibroblast-but not macrophage derived cells, whereas Tie-1 expression was not dependent on the phenotype. Tie receptors were also highly expressed in the endothelium and surrounding pericytes of capillaries scattered over the papillary proliferated synovium without notable difference in the expression of the two receptors. Furthermore, Tie positive vessels often overexpressed PCNA. In normal synovia, expression of Tie receptors was restricted to the capillary endothelium. RT-PCR confirmed the expression of Tie-1 and Tie-2 in RA synovial tissues and also in the cultured synoviocytes. CONCLUSION: The results suggest the possible involvement of overexpressed Tie-1 and Tie-2 in synovial lining and stromal cells in the pathophysiology of RA synovitis, probably through distinct mechanisms. Furthermore, expression of Tie receptors in actively growing vasculature may reflect the direct involvement of these receptors in angiogenesis and subsequent vascularisation.
Assuntos
Artrite Reumatoide/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Membrana Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Divisão Celular/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Membrana Sinovial/citologia , Vimentina/metabolismoRESUMO
Structural change of Cytochrome c peroxidase (CcP) due to interaction with lysine peptides (Lysptds) has been studied by absorption spectra and measurements on electron transfer between cytochrome c (cyt c) and CcP in the presence of Lysptd. Peaks were observed in the difference absorption spectrum of CcP between in the presence and absence of Lysptds, demonstrating a structural perturbation of CcP, at least at its heme site, on interaction with Lysptd. The interaction between CcP and Lysptd was electrostatic, since no significant peak was detected in the difference absorption spectrum when 100 mM of NaCl was added to the solution. Lysptds competitively inhibited electron transfer from cyt c to CcP, which indicated that they interacted with CcP at the same site as cyt c and would be models of the CcP interacting site of cyt c.
Assuntos
Grupo dos Citocromos c/metabolismo , Citocromo-c Peroxidase/química , Polilisina/farmacologia , Citocromo-c Peroxidase/efeitos dos fármacos , Citocromo-c Peroxidase/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Conformação Proteica , Análise Espectral , Leveduras/enzimologiaRESUMO
Transforming growth factor-beta1 (TGF-beta1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-beta1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-beta1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-beta1, we performed in vitro kinase assays using specific substrates. TGF-beta1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5 min, which decreased to basal levels within 240 min after TGF-beta1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240 min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-beta1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-beta1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-beta1-induced cell growth by 40%. These results indicate that TGF-beta1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-beta1.
Assuntos
Condrócitos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Condrócitos/citologia , Dimetil Sulfóxido/farmacologia , Ativação Enzimática , Masculino , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Ratos , Ratos Sprague-Dawley , Solventes/farmacologiaRESUMO
To examine whether synthetic vitamin D3 analog, 22-oxa-1,25(OH)2D3 (OCT) has an inhibitory effect on the growth of thyroid carcinoma, we tested the in vitro and in vivo effects of OCT on the growth of a well-differentiated thyroid cancer cell line, NPA. OCT bound to its receptor at the same rate as 1,25(OH)2D3, and inhibited the proliferation of NPA cells in vitro in a dose-dependent manner, similar to that observed with 1,25 (OH)2D3. Northern blot analysis showed that steady-state and fetal bovine serum-stimulated levels of c-myc mRNA were suppressed after 0.5-4 hour treatment with OCT. Transfection studies with the deletion mutants of the 5'-up-stream flanking region of c-myc/chloramphenicol acetyltransferase chimera genes indicated the presence of an OCT responsive element between -410 and -106. Next, we examined OCT effects in implanted NPA tumor cells in nude mice. OCT showed no remarkable hypercalcemic effect compared to 1, 25 (OH2)D3, but OCT and 1, 25 (OH2)D3, had no significant inhibitory effect in vivo after either intra-tumor or intra-peritoneum injection. Our results demonstrate that OCT inhibits the proliferation of well-differentiated thyroid cancer in an in vitro system associated with the suppression of c-myc mRNA, but this inhibitory effect was not reproducible in in vivo model.
Assuntos
Antineoplásicos/farmacologia , Calcitriol/análogos & derivados , Carcinoma Papilar/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Northern Blotting , Calcitriol/farmacologia , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Contagem de Células/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , Genes myc , Humanos , Camundongos , Camundongos Nus , Receptores de Calcitriol/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
We have previously reported that type I transforming growth factor beta (TGF-beta1) is a potent stimulator of cell growth in articular chondrocytes. In this study, we examined the mechanism of TGF-beta1 induced cellular proliferation by using cultured rat articular chondrocytes (CRAC). A time-course study of [3H]thymidine incorporation upon TGF-beta1 (1 ng/mL) or 10% fetal bovine serum stimulation revealed that TGF-beta1 directly stimulates DNA synthesis in CRAC. Pretreatment with H7, an inhibitor for protein kinase C (PKC), completely blocks TGF-beta1-induced proliferation. Since TGF-beta1 has been shown to transduce signals through MAP kinase cascades, we investigated the induction of several protooncogenes by Northern blotting. TGF-beta1 addition causes an immediate and transient induction of c-fos but not myc or jun mRNA. Furthermore, this c-fos expression is not inhibited by cycloheximide, but is completely abolished by pretreatment with TPA, so that the c-fos gene is a direct target of TGF-beta1 signalling and PKC is involved in this c-fos induction. To refine our understanding of TGF-beta1 regulation of the c-fos promoter region, we performed chloramphenicol acetyltransferase (CAT) assays. A serial deletion analysis of the c-fos promoter region reveals a TGF-beta1 responsive element in a region between -403 and -329 bp upstream of the transcription initiation site. We attempted gel shift assays on this response element with CRAC nuclear extracts. Although this region contains a sis-inducible binding element, we fail to detect specific DNA-protein complexes. Our results, however, suggest that TGF-beta1 acts as a primary mitogen in CRAC and this mitogenic activity requires PKC activation. Finally, the subsequent induction of c-fos expression occurs through an as yet unidentified transactivation mechanism.
Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Condrócitos/fisiologia , Regulação da Expressão Gênica , Genes fos , Mitógenos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Condrócitos/efeitos dos fármacos , DNA/biossíntese , Eletroforese , Expressão Gênica , Ratos , Ratos Sprague-Dawley , Ativação TranscricionalRESUMO
Smads are central mediators of signal transduction for the TGFbeta superfamily. However, the precise functions of Smad-mediated signaling pathways in early development are unclear. Here we demonstrate a requirement for Smad2 signaling in dorsoanterior axis formation during Xenopus development. Using two point mutations of Smad2 previously identified in colorectal carcinomas, we show that Smad2 ushers Smad4 to the nucleus to form a transcriptional activation complex with the nuclear DNA-binding protein FAST-1 and that the mutant proteins interact normally with FAST-1 but fail to recruit Smad4 into the nucleus. This mechanism of inhibition specifically restricts the dominant-negative activity of these mutants to the activin/Vg1 signaling pathway without inhibiting BMPs. Furthermore, expression of these mutants in Xenopus animal caps inhibits but does not abolish activin and Vg1 induction of mesoderm and in the embryo results in a truncated dorsoanterior axis. These studies define a mechanism through which mutations in Smad2 may block TGFbeta-dependent signaling and suggest a critical role for inductive signaling mediated by the Smad2 pathway in Xenopus organizer function.
Assuntos
Proteínas de Ligação a DNA/genética , Inibinas/genética , Transdução de Sinais/genética , Transativadores/genética , Proteínas de Xenopus , Xenopus/embriologia , Receptores de Ativinas Tipo I , Ativinas , Animais , Células COS , Desenvolvimento Embrionário , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese , Mutação , Fatores de Crescimento Neural , Fosforilação , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento/genética , Proteínas Smad , Proteína Smad2 , Proteína Smad4 , Fatores de Transcrição/genética , Ativação Transcricional/genética , Transfecção , Fator de Crescimento Transformador beta/genéticaRESUMO
Smad proteins are known to transduce signalling of TGF-beta receptor superfamily. We report here the entire sequences of rat Smad2 and Smad4 which have not been identified yet. Entire sequences were identified by degenerated polymerase chain reaction and following phage library screening and 5' RACE. The predicted amino acid sequences of rat Smad2 and Smad4 are highly conserved among rat, human and mouse. We also mapped these Smads to chromosome 18q.12.3. Unlike endothelial cells, TGF-beta1 stimulates articular chondrocyte proliferation as well as extracellular matrix production, and acts as a repairing agent against cartilage destruction. Since both Smad2 and Smad4 are essential factors for TGF-beta signalling, we examined their expression and regulation in cultured articular chondrocytes. Northern blot analysis showed that TGF-beta1 significantly increased the mRNA level of Smad2 but not of Smad4 in a dose- and time-dependent manner, suggesting that the augmentation of TGF-beta1 action is caused by increasing the expression of the downstream signalling molecule.
Assuntos
Cartilagem Articular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Expressão Gênica , Transativadores/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cartilagem Articular/química , Condrócitos/química , Condrócitos/metabolismo , DNA Complementar/química , DNA Complementar/genética , Proteínas de Ligação a DNA/química , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Proteína Smad2 , Proteína Smad4 , Transativadores/químicaRESUMO
Smads transmit signals from transmembrane ser/thr kinase receptors to the nucleus. We now identify SARA (for Smad anchor for receptor activation), a FYVE domain protein that interacts directly with Smad2 and Smad3. SARA functions to recruit Smad2 to the TGFbeta receptor by controlling the subcellular localization of Smad2 and by interacting with the TGFbeta receptor complex. Phosphorylation of Smad2 induces dissociation from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Furthermore, mutations in SARA that cause mislocalization of Smad2 inhibit TGFbeta-dependent transcriptional responses, indicating that the regulation of Smad localization is important for TGFbeta signaling. These results thus define SARA as a component of the TGFbeta pathway that brings the Smad substrate to the receptor.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Serina Endopeptidases , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus , Dedos de Zinco , Células 3T3 , Adulto , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Proteínas de Transporte/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fatores de Crescimento Neural , Fosforilação , Proteínas Serina-Treonina Quinases , Coelhos , Receptor do Fator de Crescimento Transformador beta Tipo II , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteínas Smad , Proteína Smad2 , Proteína Smad3 , Proteína Smad4 , Frações Subcelulares , Células Tumorais Cultivadas , XenopusRESUMO
Four classes of antiviral compounds were evaluated for inhibitory activity against two variants of human herpesvirus 6 (HHV-6A and -6B) and human herpesvirus 7 (HHV-7). These included: (1) a pyrophosphate analog, phosphonoformic acid (PFA); (2) beta-guanine analogs, 9-(2-hydroxyethoxymethyl)guanine (acyclovir or ACV), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir or GCV) and 9-(4-hydroxy-3-hydroxy-3-hydroxymethylbutylyl)guanine (penciclovir or PCV); (3) acyclic nucleoside phosphonates, (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine [cidofovir or (S)-HPMPC] and its cyclic derivative (S)-cyclic-HPMPC (cHPMPC), 9-[[2-hydroxy-1-phosphonomethoxy)ethoxy]methyl]guanine (HPMEMG) and 9-[(2-phosphonylmethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and the seven other related compounds; and (4) a series of benzimidazole ribonucleosides, including 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB). End-point inhibitory concentration (EPC) and 50% effective inhibitory concentration (EC50) values were determined by a dot-blot antigen detection method in cord blood mononuclear cells infected with HHV-6A, HHV-6B or HHV-7 at a multiplicity of infection of 0.004 CCID50/cell. (S)-HPMPC and cHPMPC had an EC50 value of approximately 0.3 microg/ml for HHV-6A, 1.2 microg/ml for HHV-6B and 3.0 microg/ml for HHV-7. These compounds were the most active of those tested against each virus. The EC50 value of GCV for HHV-6A was 0.65 microg/ml, 1.33 microg/ml for HHV-6B, and >7 microg/ml for HHV-7. The EC50 values of ACV and PCV were approximately 6-8 microg/ml for HHV-6A, 16-24 microg/ml for HHV-6B and 121-128 microg/ml for HHV-7. These drugs were the least active. The sensitivity of HHV-7 to the guanine analogs was different from HHV-6, suggesting a difference in selectivity of specific viral enzymes.