Transforming growth factor-β1 stimulates matrix metalloproteinase-9 production through ERK activation pathway and upregulation of Ets-1 protein

Objective To investigate the molecular mechanism of transforming growth factor β1 (TGF β1) in regulating the production of matrix metalloproteinase 9 (MMP 9) protein. Methods Mouse immortal podocyte cells were cultured. Different concentrations (1, 2, and 5 ng/l) of TGF β1 were added into the culture mediom. Cell culture without TGF β1 stimulation was used as control group. The activity of MMP 9 in the supernatant of the culture medion was assayed by gelatin zymography, expression of MMP 9 mRNA was assessed by RT PCR; the activation of ERK pathway and the level of a transcriptional factor Ets 1 protein was analyzed by Western blotting. PD98059, a specific inhibitor of ERK1/2 activation, was added into the culture fluid of the podocytes for 30 minutes, than 2ng/ml TGF β1 was added. The above mentioned tested were conducted to observe the influence of the inhibitor of ERK1/2 activation. Results The MMP 9 activity was very week in the supernatant of culture fluid of the control group and was increased in the TGF β1 groups dose dependently. After the podocytes were co incubated with 1 ng/ml TGF β1 for 24 hours, the MMP 9 activity was 26 86 times that of the control group ( P 0 01). Since the 12 th hour after co incubation with 2ng/ml TGF β1 the MMP 9 activity in the supernatant of culture fluid began to be significantly increased and remained at high level till the 48 th hour. RT PCR showed that low level MMP 9 mRNA expression in the control group. After stimulation of 2 ng/ml TGF β1 for 6 hours the MMP 9 mRNA expression was 2 71 times that of the control group ( P 0 01) and the high level expression lasted 24 hours. Western blotting showed low level Ets 1 protein in the control group. At the time point of 12 th hour after stimulation of TGF β1 the Ets 1 protein expression was increased in all the three TGF β1 groups. After stimulation with 2ng/ml TGF β1 for 4 hours the Ets 1 protein expression was 2 71 times that of the control group ( P 0 01). After pretreatment of the podocyte with PD98059 for 30 minutes, the added 2 ng/ml TGF β1 failed to increase the MMP 9 activity and up regulate the MMP 9 mRNA expression. Conclusion TGF β1 stimulates the production of MMP 9 by activation of cytoplasmic ERK signaling pathway and upregulation of Ets 1 expression.