| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 92 === Bradykinin (BK), an inflammatory mediator, has been detected producing in brain trauma and diseases. BK has been shown to increase the expression of proteins such as matrix metalloproteinases (MMPs) on brain cells and contributes to the pathophysiology of inflammatory responses. However, the mechanisms regulating MMP-9 expression by BK in rat brain astrocytes-1 (RBA-1) remain unclear.
First section, we investigated the mechanisms underlying BK-induced MMP-9 expression in RBA-1 cells using zymographic, Western blotting, and RT-PCR analyses. BK increased expression of MMP-9 mRNA and protein in a time- and concentration-dependent manner. BK-induced MMP-9 mRNA and protein expression was inhibited by MEK1/2 inhibitor PD98059, PI3-K inhibitor LY294002, and NF-κB inhibitor helenalin. In accordance with these findings, BK-induced phosphorylation of p42/p44 MAPK and Akt and activation of NF-κB was attenuated by prior treatment with PD98059, LY294002, and helenalin, respectively. The effects of BK on MMP-9 expression and p42/p44 MAPK and Akt phosphorylation were inhibited by B2 BK receptor antagonist Hoe140, indicating the involvement of B2 BK receptors revealed by [3H]-BK binding assay. Furthermore, BK-stimulated translocation of NF-κB into the nucleus was revealed by Western blotting and immnofluorescence staining and blocked by Hoe140, PD98059, LY294002, and helenalin. Taken together, these results suggest that in RBA cells, activation of p42/p44 MAPK and PI3-K/Akt cascades mediated through NF-κB pathway are essential for BK-induced MMP-9 gene expression.
Previous studies indicated that p42/p44 MAPK is important to regulate several cell functions. Therefore, we further investigated the detailed mechanisms of p42/p44 MAPK phosphorylation stimulated by BK in RBA-1 cells. Here we show that BK-stimulated p42/p44 MAPK activation was attenuated by pretreatment with B2 BK receptor antagonist Hoe140, either pertussis toxin (PTX) or cholera toxin (CTX) for 24 h, phosphatidylinositide-phospholipase C (PI-PLC) inhibitor U73122, phosphatidyl-choline (PC)-PLC inhibitor D609, removal of Ca2+ by addition of BAPTA/AM plus EGTA and depletion of endoplasmic reticulum (ER) Ca2+ stores by thapsigargin (TG), protein kinase C (PKC) inhibitors staurosporine and GF109203X, down-regulation of PKC by pretreatment with PMA for 24 h, transfection of dominant-negative Ras and Raf1, and inhibitors of MEK1/2 PD98059 and U0126. Furthermore, we found that BK-induced intracellular Ca2+ increasing was mediated through B2 BK receptors and TG-sensitive Ca2+ stores in RBA-1 by a fluorescent assay using Fura-2/AM as an indicator. Moreover, our results demonstrated that BK-induced translocation of PKC including PKC-α, -βI, -γ, -ε, -δ, -μ and -ξ isoforms. In addition, BK-induced p42/p44 MAPK activation was inhibited by pretreatment with PKC- specific inhibitor rottlerin. Moreover, we show that BK-stimulated several protein tyrosine kinases phosphorylation such as proline-rich tyrosine kinase (PYK2), Src family kinases, and SHC which were complexed each other by using anti-phospho-tyrosine antibody and immuoprecipitation. In addition, BK stimulated activation of JNK/c-Jun cascade and translocation of p42/p44 MAPK into nucleus. Taken together, these results conclude that BK-stimulated p42/p44 MAPK activation via a Ras- and Raf1-dependent pathway mediated through a PTX-sensitive G protein-coupled BK B2 receptor that was involved in the activation of PI-PLC, PC-PLC, Ca2+, PKC (PKC-δ) and PYK2/c-Src/SHC complex in RBA-1 cells.
Astrocytes play a critical role in normal brain physiology and pathophysiology. Several studies reported that cellular functions of astrocytes were regulated by various stimuli, including proliferation, migration and gene expression. These cellular functions regulated by BK were investigated in RBA-1 cells. On the basis of [3H]-thymidine incorporation and promoter assay, the results demonstrated that BK-induced transcriptional activation of NF-B and AP-1, but had no effect on cell proliferation. We also demonstrated that BK induced expression of inducible nitric oxide synthase (iNOS), cPLA2 and cyclooxygenase 2 (COX-2) mRNA and protein in a time-dependent manner. Moreover, prostaglandin E2 (PGE2) was detected in culture media in RBA-1 cells exposed to BK, revealing up-regulation of COX-2 in these cells. We found that BK-induced PYK2 phosphorylation, focal adhesion kinase (FAK) dephosphorylation, cell migration and morphological change. BK also stimulated expression of a neural marker, neuron-specific enolase (NSE), and down-regulation of an astrocytic marker, glial fibrillary acidic protein (GFAP). These results suggest that BK induced neural differentiation of RBA-1 cells.
This study may provide insights into the regulation of MMP-9 expression in CNS, which may occur in vivo in pathological situations such as CNS inflammation and brain astrocytoma. Furthermore, BK-induced some gene expression and cell differentiation in RBA-1 cells that might provide a new therapeutic strategy of brain diseases.
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