RESUMO
SMAD3 is one of the receptor-activated SMADs which are important in TGF-beta signal transduction. Smad3-null mice show accelerated cutaneous wound healing compared with wild-type mice. In this work, we investigated the functions and the mechanism of Smad3-mediated TGF-beta signal on matrix metaloproteinase-2 (MMP-2) in mouse fibroblast. We found that MMP-2 at wound bed was expressed earlier in Smad3-null mice than that in wild type and heterozygotes. In the sera of wounding mice, the activity of MMP-2 was also remarkably higher in Smad3-null mice than that in the other two. The embryonic fibroblasts were separated from Smad3 knockout mice to test the function of Smad3 in modulating the expression of MMP-2. The results showed that the expression and activity of MMP-2 in Smad3-null fibroblasts were higher than those in wild type cells. TGF-beta1 could increase the MMP-2 activities in both Smad3 mutant and wild type fibroblasts. The expression and activity of MMP-2 were inhibited by over expression of SMAD3 in Smad3-null fibroblasts, while the expression and activity of MMP-2 were increased by over expression of anti-sense Smad3 in wild type cells. All these results showed that SMAD3 inhibited the expression of MMP-2 in mouse embryonic fibroblasts.
Assuntos
Fibroblastos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Proteína Smad3/metabolismo , Animais , Northern Blotting , Western Blotting , Células Cultivadas , DNA Antissenso/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genótipo , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Knockout , Proteína Smad3/genética , Proteína Smad3/fisiologia , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
Osteoarthritis (OA) is the most common joint disease worldwide. Recent studies have shown that targeted disruption of Smad3 in mouse results in OA. To reveal the possible association between the Smad3 gene mutation and human OA, we employed polymerase chain reaction-single strand conformation polymorphism and sequencing to screen mutations in all nine exons of the Smad3 gene in 32 patients with knee OA and 50 patients with only bone fracture. A missense mutation of the Smad3 gene was found in one patient. The single base mutation located in the linker region of the SMAD3 protein was A --> T change in the position 2 of codon 197 and resulted in an asparagine to isoleucine amino-acid substitution. The expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 in sera of the patient carrying the mutation were higher than other OA patients and controls. This is the first report showing that the Smad3 gene mutations could be associated with the pathogenesis of human OA.
Assuntos
Proteínas de Ligação a DNA/genética , Expressão Gênica , Osteoartrite/genética , Transativadores/genética , Análise Mutacional de DNA , Eletroforese , Humanos , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/genética , Mutação de Sentido Incorreto/genética , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA , Proteína Smad3RESUMO
A keratinocyte-specific Cre transgenic construct (pK5-Cre) containing the keratin 5 promoter, Cre recombinase gene and polyA of human growth hormone gene was generated. The 4.2 kb DNA fragment of K5-Cre-hGH was introduced into 720 fertilized zygotes by microinjection. 695 injected eggs were implanted into the oviduct of 29 female mice respectively, from which 48 off-spring were obtained. Twelve mice carrying the transgene were identified by genotyping, and the integration efficiency is 25%. The K5-Cre transgenic mice were crossed with Smad4 conditional gene targeting mice to check the tissue-specific expression of the Cre recombinase and the Cre mediated recombination in multiple tissues. The results showed that the Cre recombinase was expressed in skin tissue only and successfully mediated the recombination between the loxP sites in vivo.
