RESUMO
Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.
Assuntos
Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Animais , Apoptose , Divisão Celular , Células Cultivadas , Códon , Cruzamentos Genéticos , Cisteína , DNA Complementar , Feminino , Expressão Gênica , Glomerulonefrite/patologia , Inflamação , Rim/patologia , Fígado/citologia , Fígado/patologia , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Mitose , Mutagênese Sítio-Dirigida , Miocárdio/patologia , Mapeamento por Restrição , Serina , Suínos , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/genéticaRESUMO
Transforming growth factor-beta 1 (TGF-beta 1) is a homodimeric protein stabilized by a single disulfide bridge between Cys77 on the respective monomers and two paired complementary hydrophobic interfaces between the two subunits. A TGF-beta 1 mutant with Cys77 replaced by serine has been expressed in stably transfected Chinese hamster ovary cells and purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirms that the sole interchain disulfide bond in TGF-beta 1 has been eliminated. It is 20% as potent as native TGF-beta 1 in the induction of plasminogen activator inhibitor-1 promoter expression in mink lung epithelial cells (Mv1Lu), although it is less than 1% as potent as native TGF-beta 1 in inhibition of growth in the same cell line. The mutant acts as a full agonist in both bioassays. [Ser77]TGF-beta 1 binds to soluble type II receptors and competes with native TGF-beta 1 in sandwich-enzyme-linked immunosorbent assays; however, in Mv1Lu cells, the mutant shows preferential cross-linking to type I rather than type II receptors. [Ser77]TGF-beta 1 is a useful tool for understanding the different ligand-receptor complexes and numerous biological activities of this multifunctional cytokine.
Assuntos
Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/química , Animais , Bioensaio , Células CHO , Linhagem Celular , Cricetinae , Cisteína/química , Dissulfetos/química , Técnicas In Vitro , Camundongos , Vison , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes , Serina/química , Relação Estrutura-AtividadeRESUMO
We show that 9-cis-retinoic acid (9cRA) is a potent inhibitor of mammary carcinogenesis induced by N-nitroso-N-methylurea in Sprague-Dawley rats. Rats were first treated with a single dose of N-nitroso-N-methylurea (50 mg/kg body weight) and then fed non-toxic levels of 9cRA (120 or 60 mg/kg of diet). 9cRA was highly effective in reducing tumor incidence, average number of tumors per rat, and average tumor burden, as well as extending tumor latency. The combination of 9cRA with low levels of tamoxifen (TAM; fed at either 1.0 or 0.5 mg/kg of diet) was particularly effective; addition of 9cRA to a TAM regimen doubled the number of animals that were tumor-free at autopsy and significantly diminished tumor number and tumor burden. For suppression of carcinogenesis in vivo, 9cRA was much more potent than all-trans-retinoic acid, both as a single agent or in combination with TAM, although both retinoids had equivalent inhibitory effects on DNA synthesis in cultured human breast cancer cell lines. Both 9cRA and all-trans-retinoic acid induce the expression of the adhesion molecule, E-cadherin, in the SK-BR-3 cell line. We suggest that clinical evaluation of the combination of 9cRA and TAM, either for chemoprevention or for adjuvant therapy, should be considered.
Assuntos
Neoplasias Mamárias Experimentais/prevenção & controle , Tamoxifeno/uso terapêutico , Tretinoína/análogos & derivados , Tretinoína/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Artrite/metabolismo , Fibroblastos/metabolismo , Peptidoglicano , Streptococcus pyogenes , Membrana Sinovial/metabolismo , Fatores de Crescimento Transformadores/biossíntese , Animais , Artrite Experimental/etiologia , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Sistema Livre de Células , Feminino , Fibroblastos/classificação , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Fenótipo , Testes de Precipitina , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos Lew , Membrana Sinovial/patologia , Fatores de Crescimento Transformadores/metabolismo , Fatores de Crescimento Transformadores/fisiologiaRESUMO
Exuberant tumor-like synovial cell proliferation with invasion of periarticular bone is a feature of rheumatoid arthritis in humans and of streptococcal cell wall (SCW)-induced arthritis in rats. These histologic observations prompted us to examine synoviocytes from arthritic joints for phenotypic characteristics of transformed cells. The capacity to grow in vitro under anchorage-independent conditions is a characteristic that correlates closely with potential in vivo tumorigenicity. In medium supplemented with 20% serum or in basal media supplemented with platelet-derived growth factor (PDGF), early passage synoviocytes from both SCW-induced and rheumatoid arthritic joints formed colonies in soft agarose. Epidermal growth factor (EGF), interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and transforming growth factor-beta (TGF-beta) did not support growth, although EGF enhanced PDGF-dependent growth. On the other hand, TGF-beta, as well as all-trans-retinoic acid, inhibited colony growth. Early passage normal rat and human synoviocytes also grew under the same conditions, but lung, skin, and late-gestation embryonic fibroblast-like cells did not. Considered in the context of other published data our findings provide cogent evidence that synoviocytes, but not other types of fibroblast-like cells, readily acquire phenotypic characteristics commonly associated with transformed cells. Expression of the transformed phenotype in the inflammatory site is likely regulated by paracrine growth factors, such as PDGF and TGF-beta.
Assuntos
Artrite Experimental/patologia , Artrite/patologia , Inibidores do Crescimento/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Retinoides/farmacologia , Membrana Sinovial/patologia , Fatores de Crescimento Transformadores/farmacologia , Animais , Adesão Celular , Comunicação Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Feminino , Humanos , Ratos , Ratos Endogâmicos Lew , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/fisiologiaRESUMO
The regulatory role of retinoids in growth and differentiation has been examined in vitro and in vivo by light and scanning electron microscopy using retinoid-deficient and control quail embryos between the 5-15 somite stage, as well as 2- and 2.5-day-old embryos. Fertile, retinoid-deficient eggs were obtained from flocks of quail maintained on a retinoid- and carotenoid-deficient diet, supplemented only with small amounts of retinoic acid methyl ester as described by Thompson et al. 1969. As described previously, retinoid deprivation during embryonal development causes abnormalities in organs of epithelial and mesenchymal origin, most dramatically preventing the formation of the extraembryonal circulatory system in the avian embryo. Our in vivo studies show that the basis for the latter defect is the failure of the primitive heart tubes to open at their posterior end, thus preventing the formation of omphalomesenteric veins normally connecting the embryonal with the extraembryonal circulatory system. Early manifestation of the retinoid-deficient defect may result also in formation of a cardia bifida, late manifestation in development of a single dilated ventricle. In contrast, the extraembryonal vascular system of blood islands is well developed. Heart function as shown by the rate of heart beat is reduced in deficient embryos. Our in vitro studies demonstrate similar defects in the development of the circulatory system by culture of normal 24-h embryos on retinoid-deficient agar medium; conversely, normal development is observed upon culture of retinoid-deficient embryos on retinoid-containing agar medium.