selleck catalog To examine whether ROS participated in TGF b1 induced MMP 9 expression, cells were pretreated with N acetyl cysteine for 1 h and then incubated with TGF b1 for 16 h. Our results show that pretreatment with NAC reduced TGF b1 induced MMP 9 expression and its mRNA accumulation, implying that ROS may con tribute to induction of MMP 9 by TGF b1 in RBA 1 cells. To determine Inhibitors,Modulators,Libraries whether generation of ROS was involved in TGF b1 induced MMP 9 expression in RBA 1 cells, a fluorescent probe DCF DA was used to determine the generation of ROS in these cells. RBA 1 cells were labeled with DCF DA, incubated with TGF b1 for the indicated time intervals, and the fluorescence intensity was measured at 485 nm excitation and 530 nm emission.

The data reveal that TGF b1 stimulated intracellular ROS genera tion in a time dependent manner with a maximal response within 10 min and sustained over 60 min. Furthermore, TGF b1 stimulated ROS gen eration was markedly attenuated by pretreatment Inhibitors,Modulators,Libraries with NAC, demonstrating that NAC is an efficient ROS scavenger. Next, to determine whether TGF b1 induced MAPK phosphorylation occurs via a ROS dependent pathway, we pretreated cells with NAC for 1 h and then incubated them with TGF b1 for 10 min or Inhibitors,Modulators,Libraries 4 h. These results show that pretreat ment with NAC significantly reduced TGF b1 stimulated phosphorylation of ERK12 and JNK12 in RBA 1 cells. In addition, the role of ROS in TGF b1 induced cell migration was assessed by a cell migration assay. The imaging data show that TGF b1 induced cell migration Inhibitors,Modulators,Libraries is attenuated by pretreatment with NAC.

Furthermore, to demonstrate the direct role of ROS in MMP Inhibitors,Modulators,Libraries 9 up regulation, cells were directly exposed to various concentrations of H2O2 or to combination of 1 mM of H2O2 and 15 ngml of TGF b1 for 24 h. The data show that expo sure of cells selleck chem Oligomycin A to H2O2 concentration dependently induced MMP 9 expression which was blocked by pretreatment with NAC, suggesting that ROS play a critical role in up regulation of MMP 9 in RBA 1 cells. These results suggest that ROS dependent ERK12 and JNK12 cascades may contribute to TGF b1 induced MMP 9 expression and cell migration in RBA 1 cells. NFB is required for TGF b1 induced MMP 9 expression and cell migration in RBA 1 cells Recent findings have suggested that NFB is a funda mental transcription factor for induction of several genes such as MMP 9 in astrocytes. Moreover, as shown in Figures 1C and 1D, we found that TGF b1 induces MMP 9 expression at the transcriptional level. The MMP 9 gene promoter with potential binding ele ments is required for recognition of transcription factors including NFB. On the other hand, the NFB family is considered to be an essential regulator of both cellular and inflammatory activities.