These activities indicate the important roles of MMP 9 in tissue destruction and also in tissue remodeling and inflammation. MMP 9 regulation involves transcriptional regulation, post translational cleavage, and antagonism by physiological inhibitors. In Cuscutin transcriptional regulation, MMP 9 expression is controlled by transcriptional factors including AP 1 and NF ?B, which bind to the corresponding binding sites in the MMP 9 promoter region. In various kinds of cells, different stimuli induce MMP 9 expression through activation of the MEK ERK or phosphoinositide 3 kinase Akt signaling pathways, which subsequently activate AP 1 and NF ?B. Also, p38 MAPK up regulates MMP 9 expression in Raw 264.7 cells stimulated with LPS and CpG oligodeoxynucleotide. In comparison with ERK and p38 MAPK, however, the regulatory role of JNK in MMP 9 expression remains contentious.
Inhibition of JNK activity by the JNK inhibitor PCI-24781 SP600125 decreases MMP 9 expression in ovarian carcinoma cells stimulated with PMA, astrocytes stimulated with IL 1??? and cardiac fibroblasts stimulated with IL 1??? Similarly, knockdown of JNK2 by siRNA also inhibits MMP 9 expression induced by TNF ??in A549 cells. In contrast, SP600125 does not suppress MMP 9 expression in Raw 264.7 cells stimulated with LPS and does not inhibit MMP 9 expression induced by PMA in rat astrocytes, even though PMA activates JNK. In this study, we demonstrate that JNK1, but not JNK2, suppresses LPS induced MMP 9 expression in Raw 264.7 cells in the absence of serum. Additionally, we reveal that the presence of the inhibitory factor in serum that represses MMP 9 expression induced by JNK inhibition. Finally, we report the presence of a similar autocrine inhibitory activity in the conditioned media of Raw 264.
7 cells. Results SP600125 augments MMP 9 induction in Raw 264.7 cells stimulated by LPS or TNF ??We first checked the effect of the JNK inhibitor SP600125 on induction of MMP 9 in comparison with other genes already known to be up regulated through JNK by LPS stimulation. Before stimulation, Raw 264.7 cells were deprived of serum overnight with DMEM containing 0.02 BSA. Then, 10 ?M SP600125 was added to the cells 1 h prior to stimulation with 100 ng ml LPS. Total RNA or culture medium was collected after 8 h for MMP 9 RT PCR or after 24 h for MMP 9 gelatin zymography. As shown in Figure 1A, LPS induced the expression of TNF ?? IL 6, and COX 2 mRNA in Raw 264.7 cells. These inductions were abrogated by 10 ?M SP600125.
These observations indicate that induction of TNF ?? IL 6, or COX 2 mRNA by LPS is up regulated by JNK in Raw 264.7 cells. Similarly, LPS and TNF ??increased both MMP 9 mRNA expression and MMP 9 secretion. However, in contrast to TNF ?? IL 6, and COX 2 expression, both MMP 9 expression and MMP 9 secretion was augmented by 10 ?M SP600125. Interestingly, SP600125 elevated basal levels of MMP 9 mRNA expression and MMP 9 secretion without stimulation of LPS or TNF ?? SP600125 induces basal expression of MMP 9 in a time and concentration dependent manner As SP600125 increased the basal expression of MMP 9 even without LPS or TNF ??? we next characterized the effect of SP600125 on basal MMP 9 expression. In the absence of SP600125, both MMP 9 mRNA expression and MMP 9 secretion gradually increased according to the time of incubation in Raw 264.7 cells cultured in 0.02 BSA containing medium.