SMAD3 Mutant Carrying Mutation P197Nâ†'I Failed To Inhibit The Expression Of MMP-2 In Smad3 Deficient Fibroblasts

Transforming growth factor β (TGF-β) is a superfamily of polypeptide signal factors. They play a central role in the regulation of cell proliferation, differentiation, adherence, migration and apoptosis during vertebrate development. Smads family is important to transduce TGF-β signals from the cell membrane to the nucleus. Mutations in Smads family showed that they are critical for regulating physiological and pathological processes, such as embryonic development, bone formation and cancer progression. In this study, we designed our experiments from two facets, what we has done makes good foundation for further studying on Smads gene function.Smad3 is one of the important receptor-activated Smads.Targeted disruption of Smad3 has revealed that Smad3 is involved in physiological and pathological procedures of maintaining the immune system of the body, wound healing and formation and maintenance of the cartilage and bone. Our previous studies have shown that the mis-sense mutation of Smad3 (P197N-I) could be associated with the pathogenesis of human osteoarthritis. In this study, an expression vector carrying this Smad3 mutation was constructed. The Smad3 deficient fibroblasts were transfected with wild type and mutant Smad3 expression constructs, the expression level and activities of MMP-2 was detected. The results showed that the activities of MMP-2 of fibroblasts transfected with wild type Smad3 were significantly decreased, while Smad3 mutant failed to down-regulate the expression of MMP-2. All these data suggests that the osteoarthritis related SmadB mutant could loss the ability to inhibit the expression of MMP-2 in fibroblasts.Wangjian generated the osteoblast specific Cre transgenic mice. The transgenic vector was constructed with the Cre gene directed by OG2, and the transgenic mice were got by microinjection. There is a problem is that the microinjected foreign gene integrated in the genome as multiple copies and randomly, resulting in the unpredictability of the foreign gene expression level. In this study, to check the Cre mediated recombination in multiple tissues, we mated the osteoblast specific Cre transgenic mice with the Smad4 co/co mice. The Southern-Blot showed that the Smad4 gene was knocked out only in bone (calvaria and leg bone). The results proved that the Cre recombinase was expressed specially in bone and had mediated the recombinant between the LoxP sites.