The purified DNA was together sub jected to PCR amplification using the primers specific for the region containing the distal AP 1 binding site present in the MMP 9 promoter region, sense primer PCR fragments were analyzed on 2% agarose in 1 TAE gel containing ethidium bro mide and the size was compared to a molecu lar weight marker. Inhibitors,Modulators,Libraries Statistical Inhibitors,Modulators,Libraries analysis of data Concentration effect curves were fitted and EC50 values were estimated using a GraphPad Prism Program. Data were expressed as mean ? S. E. M. and analyzed by one way ANOVA fol lowed with Tukeys post hoc test. P 0. 05 was consid ered significant. Results AP 1 is involved in JEV induced proMMP 9 expression The promoter region of MMP 9 possesses an AP 1 binding site that is regulated by several external sti muli in different cell types.
Therefore, we first determined whether JEV induced MMP 9 expression was mediated through AP 1 in RBA 1 cells. As shown by the gelatin zymographic experiments in Figure 1A, pretreatment with an inhibitor of AP 1 attenuated JEV induced MMP 9 expression in a con centration dependent manner. Within the AP 1 sub family, c Jun is an important transcriptional activator and Inhibitors,Modulators,Libraries c Fos transactivates MMPs by binding directly to promoter AP 1 motifs. Thus, we used the siRNA transfection technique to verify whether c Jun and c Fos were required for MMP 9 expression induced by JEV. As shown in Figure 1B, transfection with either c Jun or c Fos siRNA down regulated total c Jun or c Fos protein expression and signifi cantly reduced JEV induced MMP 9 expression in RBA cells.
Next, we found that the action of AP 1 in Inhibitors,Modulators,Libraries regulating MMP 9 expression occurred at the transcriptional level in RBA 1 cells, since pretreatment with tanshinone sig nificantly attenuated JEV induced MMP 9 mRNA accu mulation. To ensure the transcriptional regulation of MMP 9 gene in this context, RBA 1 cells were transfected with a luciferase reporter vector con taining an exogenous MMP 9 promoter, and the cells were then stimulated with JEV for 6 h. As shown in Fig ure 1D, JEV infection stimulated MMP 9 promoter activity, which was attenuated by pretreatment with tan shinone in RBA cells. To further confirm the role of AP 1 in JEV mediated MMP 9 promoter induction, a point mutated AP 1 MMP 9 promoter construct was used. As shown in Figure 1E, JEV stimulated MMP 9 promoter activity was prominently lost in RBA 1 cells transfected with the point mutated AP 1 MMP 9 pro moter.
Inhibitors,Modulators,Libraries These results suggest that AP 1 is required for JEV induced MMP 9 expression in RBA 1 cells. AP 1 expression is mediated via c Src, PDGFR, and PI3K Akt by JEV infection The regulation of AP 1 activity depends on changes things in c Jun and c Fos gene transcription and mRNA accumu lation. In addition, we demonstrated that transfec tion of c Jun or c Fos siRNA diminished JEV induced MMP 9 expression.
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