MMP-9 expression and activation play a critical role in tissue remodeling associated with the pathogenesis of brain diseases . Reduction of MMP activity by pharmacological inhibitors or gene knock-out strategies protects the brain from BBB disruption, cell death, and advanced neuroinflammation [2, 29]. Moreover, BK and related peptides are simultaneously produced and released following brain injury . The role of BK implicated in astrocytic functions is not completely understood. Thus, we investigated the molecular mechanisms underlying BK-induced MMP-9 expression in cultured RBA-1 cells and an animal model. Our results suggest that in brain astrocytes, activation of Ca2+/PKC-α-dependent Nox2/ROS signal leading to induction of AP-1 (c-Fos/c-Jun) is essential for BK-induced MMP-9 gene expression and enhancing cell migration. The findings suggest that BK-induced MMP-9 expression and cell migration may contribute to increase BBB permeability and recruit immune cells, leading to brain inflammation and edema. In addition, astrocytic migration may be involved in brain inflammation and remodeling during brain injuries such as brain wound healing, tissue remodeling, and glial scar formation [2, 3, 19, 20].
Imbalance in the level of ROS has been shown to play a causative role in numerous pathologies of degenerative diseases [23, 31]. ROS concentration-dependently exert a key role in the normal physiological functions and the inflammatory responses . In the brain, ROS also extend to the control of vascular tone which is tightly modulated by metabolic activity within neurons . Moreover, increasing ROS generation by diverse stimuli can regulate the expression of inflammatory mediators in pathogenesis of brain disorders [24, 31]. Recently, the cellular damage in neurodegenerative disorders such as Alzheimer’s disease is attributed to oxidative stress [7, 10, 11]. BK-induced ROS generation has been reported in cerebral arterioles and renal diseases . In this study, our data demonstrated that in both in vitro and in vivo studies, BK induces MMP-9 expression via ROS-dependent pathways in brain astrocytes. Moreover, we found that BK-induced MMP-9 expression is mediated through Nox-dependent ROS generation, since pretreatment with ROS scavenger NAC or Nox activity inhibitor DPI attenuated BK-induced responses. The involvement of Nox-dependent ROS generation in BK-induced responses was further confirmed by NAC or DPI in RBA-1 cells. ROS exert as a major signaling factor which mediates microglial activation induced by inflammatory mediators, including LPS . Herein we are the first group to establish that Nox-dependent intracellular redox signal (ROS generation) contributes to MMP-9 expression induced by BK in brain astrocytes.
Moreover, Nox is considered to be a major source of ROS in several physiological and pathological processes [8, 24]. To date, there are five isoforms of Nox have been discovered, including Nox1-5 . Nox1, Nox2, and Nox4 have been shown to be expressed and are crucial for ROS generation in brain cells [8, 32]. First, our data demonstrated that Nox activity is involved in BK-induced responses by a Nox inhibitor DPI (Figure 2A-C). As previous reports, we also found that RBA-1 cells express Nox1, Nox2, and Nox4 (Figure 2D). Next, BK-induced MMP-9 expression is predominantly mediated through activation of Nox2, confirmed by respective Nox siRNAs (Figure 2E). The involvement of Nox in BK-induced responses is mediated through phosphorylation and translocation of p47phox (a Nox2 component) which was attenuated by a p47phox inhibitor apocynin or p47phox siRNA (Figure 3). These results are consistent with previous studies showing that Nox is expressed in astrocytes and contributes to ROS generation [16, 33] and Nox is involved in LPS-induced MMPs expression in Raw264.7 . Additionally, we also demonstrated that BK-stimulated Nox2-dependent ROS signal and MMP-9 expression is mediated through Ca2+-dependent PKC-α activation, confirmed by an intracellular Ca2+ chelator (BAPTA/AM), the inhibitor of ER Ca2+-ATPase (TG), pan-PKC (GF109203X), or PKC-α (Gö6976) and PKC-α siRNA (Figure 4, 5). These data demonstrated that BK stimulates Ca2+-dependent PKC-α activation linking to Nox2/ROS generation and MMP-9 expression in RBA-1 cells. It is consistent with previous studies indicating that overexpression of PKC-α can increase phosphorylation of p47phox and induce both its translocation and Nox activation in human neutrophils .
The excessive increase of oxidative stress during injuries not only causes oxidative damage to cellular macromolecules, but also modulates the pattern of gene expression through functional alterations of transcription factors. The transcription factor AP-1 is well known to be modulated during oxidative stress associated with inflammatory diseases . Moreover, BK-induced gene expression has been shown to be mediated through one of transcription factors such as AP-1 . However, the mechanistic connection between the MMP-9 expression and the ROS-dependent pathway induced by BK has not been established in RBA-1 cells. In this study, we demonstrated that AP-1 is essential for BK-induced MMP-9 expression which was inhibited by an AP-1 inhibitor TSIIA (Figure 6A). The involvement of AP-1 in BK-induced responses was further confirmed by determining induction of AP-1 (c-Fos/c-Jun), including phosphorylation of c-Jun, accumulation of c-Fos in nucleus, and AP-1 promoter reporter activity. These responses were attenuated by the Ca2+ chelator (BAPTA/AM), the inhibitor of PKC-α (Gö6976), p47phox (apocynin), Nox (DPI), or a ROS scavenger (NAC), suggesting that the Ca2+/PKC-α/p47phox/Nox/ROS cascade is involved in activation of AP-1 which contributes to BK-induced MMP-9 expression in RBA-1 cells. Moreover, the role of AP-1 in BK-induced MMP-9 expression was also confirmed by either c-Fos or c-Jun siRNA to inhibit BK-induced MMP-9 expression. These results are consistent with the involvement of AP-1 in MMP-9 expression induced by ox-LDL and LTA in RBA-1 cells [21, 22].
To confirm AP-1 indeed binds to the promoter region of MMP-9 gene, the binding activity of AP-1 was determined by a ChIP-PCR assay. In this study, BK stimulated AP-1 (c-Fos and c-Jun) recruitment and activation via Ca2+/PKC-α-dependent Nox/ROS cascade and enhancing MMP-9 promoter activity. We further confirmed that AP-1 binding site (-503 to -497) within MMP-9 promoter is required for BK-induced MMP-9 transcriptional activity by using an AP-1-mutated MMP-9 promoter construct. These results demonstrated that activation of AP-1 (either c-Fos or c-Jun) is essential for BK-induced MMP-9 gene expression which is mediated through a Ca2+/PKC-α-dependent Nox/ROS signal in RBA-1 cells. It is consistent with previous studies showing that AP-1 is involved in MMP-9 expression in various cell types [22, 37].
Several reports have shown the multiple effects of BK and MMP-9 on brain glial cells [30, 38]. In brain injury, up-regulation of BK, ROS, and MMP-9 may increase BBB permeability, recruit immune cells infiltrating through BBB into the tissues, and subsequently result in brain inflammation and edema . In this study, we suggested that BK-induced Nox2-mediated ROS signal, MMP-9 expression, and astrocytic migration might be involved in brain inflammation and remodeling during brain injuries. Both ROS  and MMP-9  have been reported to play a critical role in cell motility in several cell types. Herein we demonstrated that BK induces MMP-9 expression and cell migration via PKC-α-dependent Nox/ROS signaling pathway in RBA-1 cells. The results are consistent with recent reports indicating that the ROS-dependent MMP-9 expression is essential for cell migration by various stimuli such as TGF-β1 or LPS [20, 34]. Hence, we suggest that ROS-mediated MMP-9 up-regulation by BK is associated with cell migration in RBA-1 cells. Taken together, BK, ROS, and MMP-9 may have multiple effects on different types of brain glial cells, inducing inflammatory and remodeling roles.