SP600125 elevated MMP 9 mRNA expression to a much greater extent at 8 h and 24 h. Accordingly, a marked increase β-Sitosterol in MMP 9 activity was observed in SP600125 treated Raw 264.7 cells at 24 h. The inductive effect of SP600125 on MMP 9 expression was concentration dependent. Both MMP 9 mRNA expression and MMP 9 secre tion were increased by SP600125 in a concentration dependent manner. To confirm whether the inducing effect of SP600125 on MMP 9 expression was mediated through JNK activity, phosphorylation of the JNK substrate c Jun was determined after treatment with SP600125. As shown in Figure 2C, phosphorylation of c Jun decreased from 10 min to 8 h after SP600125 treatment. Next, we determined the level of regulation of MMP 9 expression by SP600125. Raw 264.7 cells were pre treated with actinomycin D 1 h prior to 10 ?M SP600125.
As shown in Figure Dacinostat 2D, actinomycin D successfully inhibited MMP 9 induction by SP600125 at 8 h. This data indicates that SP600125 induced MMP 9 mRNA at transcriptional level. Effects of SP600125 on ERK, p38 MAPK, Akt, and NF ?B As ERK, p38 MAPK, or Akt mediate MMP 9 induction , we tried to determine the effects of SP600125 on the activation of ERK, p38 MAPK, or Akt. As shown in Figure 3A, SP600125 did not elevate the phosphorylation of ERK, p38 MAPK, or Akt. Furthermore, SP600125 did not affect the phosphorylation of p65, which is a component of NF ?B involved in the induction of MMP 9. These results suggest that SP600125 may not induce MMP 9 expression through the activation of ERK, p38 MAPK, Akt, or p65 of NF ?B.
Suppression of MMP 9 expression by JNK1 Next, we performed a knock down experiment to exclude a nonspecific effect of SP600125 and to identify JNK isoform involved in the negative regulation of MMP 9 expression, in light of different potential biological roles of JNK1 and JNK2. JNK1 or JNK2 siRNA successfully suppressed levels of JNK1 or JNK2 protein, respectively . Knock down of JNK1 by JNK1 siRNA increased both MMP 9 mRNA expression and MMP 9 secretion. In addition, JNK1 siRNA increased MMP 9 expression in a concentration dependent manner. In contrast, JNK2 siRNA induced MMP 9 expression to a much lesser degree. However, it may not be conclusive that JNK2 siRNA caused MMP 9 induction, because JNK2 siRNA slightly inhibited JNK1 expression. These data suggest that JNK1 can specifically suppress basal MMP 9 expression in Raw 264.7 cells.
Serum inhibits SP600125 mediated MMP 9 induction The above experiments were performed in the absence of serum to exclude influence of serum. Next, we tried to confirm SP600125 mediated MMP 9 induction in the presence of different concentrations of mouse serum and FBS. Surprisingly, SP600125 mediated induction of MMP 9 was attenuated by 10 mouse serum or FBS. FBS displayed weaker inhibitory effect than did mouse serum. Next, Raw 264.7 cells were treated with different concentrations of mouse serum. Mouse serum inhibited both basal and SP60015 induced MMP 9 expression in a concentrationdependent manner. Both basal and SP600125 induced MMP 9 expression was almost completely abolished by 10 mouse serum. As IFN ??is known to inhibit MMP 9 expression, we investigated whether IFN ??was responsible for MMP 9 suppression in mouse serum.