Effect of celecoxib on BK-induced MMP-9 expression in brain astrocytes
The COX-2/PGE2 system is also critical to brain inflammatory diseases [31]. First, we investigate the effect of COX-2/PGE2 system on BK-induced MMP-9 expression, rat brain astrocytes (RBA) were pretreated with or without a selective inhibitor of COX-2 activity celecoxib (CLC) for 1 h and then incubated with BK for the indicated time intervals. As shown in Fig. 1a, pretreatment with CLC (30 mM) significantly attenuated BK-induced MMP-9 expression determined by zymography. The result suggested that COX-2 might play a regulatory role in BK-induced MMP-9 expression. We further determined whether COX-2 contributes to BK-induced MMP-9 expression via regulating the transcriptional level, analyzed by RT-PCR. The data showed that pretreatment of RBA with different concentrations of CLC (1, 10, and 30 mM) markedly blocked BK-induced MMP-9 mRNA expression in a concentration-dependent manner (Fig. 1b). These results suggested that COX-2 may be a critical element in BK-induced MMP-9 expression in RBA cells. To further confirm the suggestion, we determined whether BK stimulates the downstream product of COX-2, prostaglandin E2 (PGE2), increase and the effect of CLC on the event, the conditioned media were collected and measured PGE2 levels using an EIA kit. The data showed that BK-induced PGE2 biosynthesis was inhibited by pretreatment of cells with CLC (Fig. 1c). Moreover, we found that BK-induced MMP-9 expression was attenuated by knockdown of COX-2 by transfection of RBA cells with the COX-2 siRNA (Fig. 1d). These results demonstrated that COX-2-derived PGE2 production may contribute to BK-induced MMP-9 expression in RBA cells.
BK induces MMP-9 expression via PGE2 receptors
Next, to determine whether PGE2 receptors (E-prostanoid; EP) contribute to BK-induced MMP-9 expression, the EP receptor antagonists were used. The RBA cells were pretreated with the antagonist of EP1 (Sc-19220), EP3 (L798-106), or EP4 (GW627368) and then incubated with BK (10 nM) for the indicated time intervals. The data showed that pretreatment with Sc-19220 (Sc, 3 mM), L798-106 (L789, 3 mM), or GW627368 (GW, 1 mM) attenuated BK-induced MMP-9 expression during the period of observation (Fig. 2), suggesting that BK induces MMP-9 expression via the PGE2-dependent EP receptors (e.g., EP1, EP3, and EP4) in RBA cells. These data indicated that BK-induced MMP-9 expression may be mediated through COX-2-derived PGE2 autocrine in RBA cells.
PGE2 induces de novo MMP-9 expression via EP receptors
Here, to further demonstrate whether BK-induced PGE2 production is important for MMP-9 expression, the RBA cells were directly incubated with PGE2 for the indicated time intervals and concentrations. As shown in Fig. 3a, PGE2 induced MMP-9 expression in a time- and concentration-dependent manner, a significant increase within 4-24 h. Moreover, we also demonstrated that PGE2 induced concentration-dependently MMP-9 mRNA expression by RT-PCR analysis (Fig. 3b). To determine whether PGE2-induced MMP-9 expression is mediated through EP receptors, cells were pretreated with the antagonist of EP1 (Sc), EP3 (L798), or EP4 (GW) and then incubated with PGE2 (10 mM) for the indicated time intervals. The results showed that pretreatment with Sc (3 mM), L798 (3 mM), or GW (1 mM) suppressed PGE2-induced MMP-9 expression during the period of observation (Fig. 3c), indicating that PGE2 could indeed induce de novo MMP-9 expression through the EP receptors, including EP1, EP3, and EP4 in these cells.
Involvement of c-Src in BK- and PGE2-induced MMP-9 expression
To simultaneously investigate the signaling mechanism of BK- and PGE2-induced MMP-9 expression, the pharmacological inhibitors of signaling molecules were used. First, we determined the role of c-Src in BK- and PGE2-induced MMP-9 expression, cells were pretreated with the inhibitor of c-Src (PP1) for 1 h and then incubated with BK or PGE2 for the indicated times. As shown in Fig. 4a and b, pretreatment with PP1 (1 mM) significantly attenuated BK- and PGE2-induced MMP-9 expression, suggesting that c-Src was involved in these responses. To further demonstrate the effect of PP1 on BK- and PGE2-stimulated c-Src phosphorylation, the phosphorylation of c-Src was analyzed by Western blot. The data showed that pretreatment with PP1 blocked BK-stimulated phosphorylation of c-Src (Fig. 4c, left panel). Additionally, PGE2 also stimulate time-dependently c-Src phosphorylation which was blocked by pretreatment of RBA with PP1 (Fig. 4c, right panel). These data suggested that BK induces MMP-9 expression via a PGE2-mediated c-Src phosphorylation cascade in these cells.
PGE2 induces MMP-9 expression through EP receptor-mediated ERK1/2 activation
Activation of MAPKs by BK could modulate cellular functions of brain cells [22]. Moreover, the ERK1/2 is involved in BK-induced MMP-9 expression in brain astrocytes [21]. Thus, to determine whether ERK1/2 also participated in PGE2-induced MMP-9 expression, cells were pretreated with or without U0126 (1 mM) for 1 h and then incubated with PGE2 for the indicated time intervals. As shown in Fig. 5a, PGE2-induced MMP-9 expression was attenuated by pretreatment with U0126, suggesting that ERK1/2 may be involved in PGE2-induced MMP-9 expression. We further demonstrated that PGE2 stimulated time-dependently ERK1/2 phosphorylation by Western blot (Fig. 5b). These results suggested that PGE2-induced MMP-9 expression is mediated through ERK1/2 pathway in RBA cells. Next, to determine whether PGE2-stimulated ERK1/2 phosphorylation is mediated through EP receptor-dependent pathway, cells were pretreated with the antagonist of EP1 (Sc), EP3 (L798), or EP4 (GW) and then incubated with PGE2 (10 mM) for the indicated time intervals. The results showed that pretreatment with Sc (3 mM), L798 (3 mM), or GW (1 mM) significantly blocked PGE2-stimulated ERK1/2 phosphorylation during the period of observation (Fig. 5c), suggesting that PGE2 stimulated EP receptor (e.g., EP1, EP3, and EP4)-dependent ERK1/2 phosphorylation in these cells. These results demonstrated that PGE2-induces MMP-9 expression is mediated through EP receptor-dependent ERK1/2 activation in RBA-1 cells.
Jak2/STAT3 cascade is required for BK-induced PGE2 autocrine linking to MMP-9 expression
The Jak/STAT3 cascade is activated upon ligand binding to certain G protein-coupled receptors (GPCRs) including the BK in various cell types like endothelial cells [32]. Moreover, activation of Jak/STAT3 signaling pathway has been shown to regulate MMP-9 expression in tumor invasion and metastasis [33-35]. To examine whether the Jak/STAT3 signaling pathway is also involved in BK-induced COX-2/PGE2-mediated MMP-9 expression, the inhibitors of Jak2 (AG490) and STAT3 (CBE: cucurbitacin E) were used. As shown in Fig. 6a, cells were pretreated with AG490 (1 mM) or CBE (0.1 mM) and then incubated with BK (10 nM) or PGE2 (10 mM) for the indicated time intervals. The data showed that pretreatment with AG490 (1 mM) or CBE (0.1 mM) both markedly attenuated BK-induced MMP-9 expression. Similarly, pretreatment of cells with AG490 or CBE both also significantly attenuated PGE2-induced MMP-9 expression (Fig. 6b). These results suggested that the Jak2/STAT3 cascade was involved in BK- or PGE2-induced MMP-9 expression in RBA cells. To further determine whether activation of Jak2/STAT3 cascade in BK-induced responses mediated through phosphorylation of Jak2/STAT3 cascade, as shown in Fig. 6c, BK time-dependently stimulated phosphorylation of Jak2/STAT3 cascade determined by Western blot. A significant response was obtained within 1-3 min. Moreover, pretreatment with the inhibitor of Jak2 (AG) significantly inhibited BK-stimulated phosphorylation of Jak2/STAT3 cascade. We further demonstrate the role of COX-2 in BK-stimulated phosphorylation of Jak2/STAT3 cascade, cells were pretreated with CLC and then incubated with BK for 3 min. The data showed that pretreatment with CLC (30 mM) significantly blocked BK-stimulated phosphorylation of Jak2/STAT3 cascade (Fig. 6c), suggesting that BK-stimulated phosphorylation of Jak2/STAT3 cascade is mediated through COX-2/PGE2 system. Subsequently, to confirm the role of COX-2/PGE2 system in activation of Jak2/STAT3 cascade, cells were directly incubated with PGE2. As shown in Fig. 6d, PGE2 stimulated phosphorylation of Jak2/STAT3 cascade at 3 min determined by Western blot. Pretreatment with AG also significantly blocked this PGE2 response. To demonstrate the effect of the signaling molecules, including ERK1/2 and c-Src in PGE2-stimulated phosphorylation of Jak2/STAT3 cascade, cells were pretreated with U0126 or PP1 and then incubated with PGE2 for 3 min. The data showed that pretreatment with PP1 markedly blocked PGE2-stimulated phosphorylation of Jak2 and STAT3. Moreover, pretreatment with U0126 inhibited STAT3 phosphorylation, but not Jak2, indicating that PGE2-stimulated STAT3 phosphorylation was mediated through c-Src/Jak2/ERK1/2 pathway. The results suggested that BK-stimulated activation of Jak2/STAT3 cascade via COX-2/PGE2 system is required for MMP-9 up-regulation in RBA cells.
Effect of COX-2/PGE2 system on BK-induced MMP-9-dependent astrocytic function changes
Ultimately, to demonstrate the effect of COX-2/PGE2 system on BK-induced MMP-9-dependent astrocytic function changes, we evaluated the cell migration of RBA cells. The images of cell migration induced by BK (10 nM) were observed and taken at 48 h and the number of migratory cells was counted and the statistical data were presented in Fig. 7a. The data showed that pretreatment with CLC (30 mM) significantly blocked BK-induced cell migration, suggesting that the COX-2/PGE2 system may be involved in BK-induced astrocytic migration. Moreover, cells were directly incubated with PGE2 (30 mM) for 48 h and the images of cell migration were observed and taken at 48 h (Fig. 7a, insert panel). The data showed that pretreatment with PP1 (1 mM), AG (1 mM), U0 (1 mM), or CBE (0.1 mM) all significantly reduced PGE2-induced cell migration, suggesting that PGE2 could induce astrocytic migration via c-Src/Jak2-ERK1/2-STAT3 cascade. These results demonstrated that COX-2-derived PGE2 participated in BK-induced astrocytic migration through activation of c-Src/Jak2-ERK1/2-STAT3 pathway.