Hui-Hsin Wang

Transforming growth factor-β1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-κB pathways

BackgroundTransforming growth factor-β (TGF-β) and matrix metalloproteinases (MMPs) are the multifunctional factors during diverse physiological and pathological processes including development, wound healing, proliferation, and cancer metastasis. Both TGF-β and MMPs have been shown to play crucial roles in brain pathological changes. Thus, we investigated the molecular mechanisms underlying TGF-β1-induced MMP-9 expression in brain astrocytes.MethodsRat brain astrocytes (RBA-1) were used. MMP-9 expression was analyzed by gelatin zymography and RT-PCR. The involvement of signaling molecules including MAPKs and NF-κB in the responses was investigated using pharmacological inhibitors and dominant negative mutants, determined by western blot and gene promoter assay. The functional activity of MMP-9 was evaluated by cell migration assay.ResultsHere we report that TGF-β1 induces MMP-9 expression and enzymatic activity via a TGF-β receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. ROS production leads to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activation of the NF-κB transcription factor. Activated NF-κB turns on transcription of the MMP-9 gene. The rat MMP-9 promoter, containing a NF-κB cis-binding site, was identified as a crucial domain linking to TGF-β1 action.ConclusionsCollectively, in RBA-1 cells, activation of ERK1/2- and JNK-NF-κB cascades by a ROS-dependent manner is essential for MMP-9 up-regulation/activation and cell migration induced by TGF-β1. These findings indicate a new regulatory pathway of TGF-β1 in regulating expression of MMP-9 in brain astrocytes, which is involved in physiological and pathological tissue remodeling of central nervous system.

Oxidized low-density lipoprotein induces matrix metalloproteinase-9 expression via a p42/p44 and JNK-dependent AP-1 pathway in brain astrocytes

Upregulation of matrix metalloproteinases (MMPs), especially MMP‐9, by oxidized low‐density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL‐induced MMP‐9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP‐9 via a MAPK‐dependent AP‐1 activation in rat brain astrocyte (RBA)‐1 cells. Results revealed by gelatin zymography, RT‐PCR, and Western blotting analyses showed that oxLDL‐induced proMMP‐9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA‐1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP‐1 (i.e., c‐Fos/c‐Jun) is crucial for oxLDL‐induced proMMP‐9 expression which was attenuated by pretreatment with AP‐1 inhibitor (curcumin). The regulation of MMP‐9 gene transcription by AP‐1 was confirmed by oxLDL‐stimulated MMP‐9 luciferase activity which was totally lost in cells transfected with the AP‐1 binding site‐mutated MMP‐9 promoter construct (mt‐AP1‐MMP‐9). These results suggested that oxLDL‐induced proMMP‐9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP‐1 activation. Understanding the regulatory mechanisms underlying oxLDL‐induced MMP‐9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases.

Nitric oxide production by endothelin-1 enhances astrocytic migration via the tyrosine nitration of matrix metalloproteinase-9.

The deleterious effects of endothelin‐1 (ET‐1) in the central nervous system (CNS) include disturbance of water homeostasis and blood–brain barrier (BBB) integrity. In the CNS, ischemic injury elicits ET‐1 release from astrocytes, behaving through G‐protein coupled ET receptors. These considerations raise the question of whether ET‐1 influences cellular functions of astrocytes, the major cell type that provides structural and functional support for neurons. Uncontrolled nitric oxide (NO) production has been implicated in sterile brain insults, neuroinflammation, and neurodegenerative diseases, which involve astrocyte activation and neuronal death. However, the detailed mechanisms of ET‐1 action related to NO release on rat brain astrocytes (RBA‐1) remain unknown. In this study, we demonstrate that exposure of astrocytes to ET‐1 results in the inducible nitric oxide synthase (iNOS) up‐regulation, NO production, and matrix metalloproteinase‐9 (MMP‐9) activation in astrocytes. The data obtained with Western blot, reverse transcription‐PCR (RT‐PCR), and immunofluorescent staining analyses showed that ET‐1‐induced iNOS expression and NO production were mediated through an ETB‐dependent transcriptional activation. Engagement of Gi/o‐ and Gq‐coupled ETB receptors by ET‐1 led to activation of c‐Src‐dependent phosphoinositide 3‐kinase (PI3K)/Akt and p42/p44 mitogen‐activated protein kinase (MAPK) and then activated transcription factor nuclear factor‐κB (NF‐κB). The activated NF‐κB was translocated into nucleus and thereby promoted iNOS gene transcription. Ultimately, NO production stimulated by ET‐1 enhanced the migration of astrocytes through the tyrosine nitration of MMP‐9. Taken together, these results suggested that in astrocytes, activation of NF‐κB by ETB‐dependent c‐Src, PI3K/Akt, and p42/p44 MAPK signalings is necessary for ET‐1‐induced iNOS gene up‐regulation. J. Cell. Physiol. 226: 2244–2256, 2011.

Sphingosine 1‐phosphate induces EGFR expression via Akt/NF‐κB and ERK/AP‐1 pathways in rat vascular smooth muscle cells

Sphingosine 1‐phosphate (S1P) has been shown to regulate expression of several genes in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating epidermal growth factor receptor (EGFR) expression by S1P in aortic VSMCs remain unclear. Western blotting and RT‐PCR analyses showed that S1P induced EGFR mRNA and protein expression in a time‐ and concentration‐dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and phosphatidylinositide 3‐kinase (PI3K; wortmannin), and transfection with dominant negative mutants of ERK and Akt, respectively. These results suggested that S1P‐induced EGFR expression was mediated through p42/p44 MAPK and PI3K/Akt pathways in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt which was attenuated by U0126 and wortmannin, respectively. Furthermore, S1P‐induced EGFR upregulation was blocked by a selective NF‐κB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P‐induced activation of NF‐κB was blocked by wortmannin, but not by U0126, suggesting that activation of NF‐κB was mediated through PI3K/Akt. Moreover, S1P‐induced EGFR expression was inhibited by an AP‐1 inhibitor curcumin and tanshinone IIA. S1P‐stimulated AP‐1 subunits (c‐Jun and c‐Fos mRNA) expression was attenuated by U0126 and wortmannin, suggesting that MEK and PI3K/ERK cascade linking to AP‐1 was involved in EGFR expression. Upregulation of EGFR by S1P may exert a phenotype modulation of VSMCs. This hypothesis was supported by pretreatment with AG1478 or transfection with shRNA of EGFR that attenuated EGF‐stimulated proliferation of VSMCs pretreated with S1P, determined by XTT assay. These results demonstrated that in VSMCs, activation of Akt/NF‐κB and ERK/AP‐1 pathways independently regulated S1P‐induced EGFR expression in VSMCs. Understanding the mechanisms involved in S1P‐induced EGFR expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis. J. Cell. Biochem. 103: 1732–1746, 2008.

Reactive Oxygen Species-Dependent c-Fos/Activator Protein 1 Induction Upregulates Heme Oxygenase-1 Expression by Bradykinin in Brain Astrocytes.

Heme oxygenase-1 (HO-1) plays a crucial role in tissue pathological changes such as brain injuries. Our previous studies have demonstrated that bradykinin (BK) induces the expression of several inflammatory proteins, including matrix metalloproteinase-9 and COX-2, via mitogen-activated protein kinases and nuclear factor-κB (NF-κB) in rat brain astrocytes (RBA-1). However, the molecular mechanisms underlying BK-induced HO-1 expression in RBA-1 cells remain poorly defined. Here we demonstrated that BK induced HO-1 expression and enzymatic activity via a B(2) BK receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. NADPH oxidase (Nox)-dependent ROS generation led to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activated the downstream molecules NF-κB and c-Jun, respectively. The c-Fos, an activator protein 1 (AP-1) subunit, was upregulated by activation of NF-κB and c-Jun, which bound to HO-1 promoter and thereby turned on transcription of HO-1 gene. The rat HO-1 promoter containing a putative AP-1 cis-binding site was identified as a crucial domain linking to BK action. Taken together, these results suggested that in RBA-1 cells, activation of ERK/NF-κB and JNK/c-Jun cascades by a Nox/ROS-dependent event enhancing c-Fos/AP-1 activity is essential for HO-1 upregulation and activation induced by BK. Moreover, ROS-dependent NF-E2-related factor 2 activation also contributes to HO-1 induction by BK in astrocytes.

Thrombin Induces EGF Receptor Expression and Cell Proliferation via a PKC(δ)/c-Src-Dependent Pathway in Vascular Smooth Muscle Cells

Objection—Thrombin upregulates expression of several proteins in vascular smooth muscle cells (VSMCs) which may contribute to atherosclerosis. Here, we investigated the mechanisms underlying thrombin-induced EGF receptor (EGFR) expression and its effect on VSMCs. Methods and Results—Normal rat VSMCs were used. First, Western blotting and RT-PCR analyses showed that thrombin induces the expression of EGFR at transcription and translation levels in VSMCs. Second, pharmacological inhibitors, dominant negative mutants, and short hairpin RNA interference (shRNA) technology enabled us to demonstrate that thrombin-induced EGFR expression is mediated through PKC(&dgr;)/c-Src–dependent transactivation of EGFR linking to PI3K/Akt and ERK1/2. We further investigated whether the transcription factors AP-1 and NF-&kgr;B are involved in this response by a promoter assay. Finally, data obtained by using EGFR shRNA technology and XTT assay demonstrated that thrombin-enhanced VSMC proliferation was mediated through upregulation of EGFR. Conclusions—Our results demonstrate that thrombin-enhanced VSMC proliferation was mediated through upregulation of EGFR via a PKC(&dgr;)/c-Src–dependent transactivation of EGFR, PI3K-Akt, and ERK, and AP-1/NF-&kgr;B pathway.

Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells: role of ERK1/2, p38 MAPK, and NF-κB

AIMS Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in inflammatory diseases such as atherosclerosis and angiogenesis. As extracellular nucleotides such as ATP have been shown to act via activation of P(2) purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via upregulated expression of inflammatory proteins, such as cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) in VSMCs. METHODS AND RESULTS Western blotting, promoter assay, RT-PCR, and PGE2 immunoassay revealed that ATPgammaS induced expression of COX-2 and prostaglandin (PGE2) synthesis through the activation of p42/p44 MAPK (mitogen-activated protein kinase), p38 MAPK, and nuclear factor-kappaB (NF-kappaB). These responses were attenuated by inhibitors of MAPK/ERK kinase (MEK1/2; U0126), p38 MAPK (SB202190), and NF-kappaB (helenalin), or by tranfection with dominant negative mutants of p42, p38, IkappaB kinase (IKK)alpha, and IKKbeta. Furthermore, the ATPgammaS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaBalpha was blocked by U0126 and helenalin. In addition, the ATPgammaS-stimulated cPLA2 expression was inhibited by U0126, SB202190, helenalin, celecoxib (a selective COX-2 inhibitor), and PGE2 receptor antagonists (AH6809, GW627368X, and SC-19220). However, the inhibitory effect of celecoxib on cPLA2 expression was reversed by addition of exogenous PGE2. CONCLUSION Our results suggest that in VSMCs, activation of p42/p44 MAPK, p38 MAPK, and NF-kappaB is essential for ATPgammaS-induced COX-2 expression and PGE2 synthesis. Newly synthesized PGE2 was observed to act as an autocrine signal contributing to cPLA2 expression, which may be implicated in inflammatory responses. Collectively, our findings provide insights into the correlation between COX-2 and cPLA2 expression in ATPgammaS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vessel disease-related vascular inflammation.

Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

BackgroundLipoteichoic acid (LTA) is a component of Gram-positive bacterial cell walls, which has been found to be elevated in cerebrospinal fluid of patients suffering from meningitis. Moreover, matrix metalloproteinases (MMPs), MMP-9 especially, have been observed in patients with brain inflammatory diseases and may contribute to brain disease pathology. However, the molecular mechanisms underlying LTA-induced MMP-9 expression in brain astrocytes remain unclear.ObjectiveThe goal of this study was to examine whether LTA-induced cell migration is mediated by calcium/calmodulin (CaM)/CaM kinase II (CaMKII)-dependent transactivation of the PDGFR pathway in rat brain astrocytes (RBA-1 cells).MethodsExpression and activity of MMP-9 induced by LTA was evaluated by zymographic, western blotting, and RT-PCR analyses. MMP-9 regulatory signaling pathways were investigated by treatment with pharmacological inhibitors or using dominant negative mutants or short hairpin RNA (shRNA) transfection, and chromatin immunoprecipitation (ChIP)-PCR and promoter activity reporter assays. Finally, we determined the cell functional changes by cell migration assay.ResultsThe data show that c-Jun/AP-1 mediates LTA-induced MMP-9 expression in RBA-1 cells. Next, we demonstrated that LTA induces MMP-9 expression via a calcium/CaM/CaMKII-dependent transactivation of PDGFR pathway. Transactivation of PDGFR led to activation of PI3K/Akt and JNK1/2 and then activated c-Jun/AP-1 signaling. Activated-c-Jun bound to the AP-1-binding site of the MMP-9 promoter, and thereby turned on transcription of MMP-9. Eventually, up-regulation of MMP-9 by LTA enhanced cell migration of astrocytes.ConclusionsThese results demonstrate that in RBA-1 cells, activation of c-Jun/AP-1 by a CaMKII-dependent PI3K/Akt-JNK activation mediated through transactivation of PDGFR is essential for up-regulation of MMP-9 and cell migration induced by LTA. Understanding the regulatory mechanisms underlying LTA-induced MMP-9 expression and functional changes in astrocytes may provide a new therapeutic strategy for Gram-positive bacterial infections in brain disorders.

Oxidized Low-Density Lipoprotein-Induced Matrix Metalloproteinase-9 Expression via PKC-δ/p42/p44 MAPK/Elk-1 Cascade in Brain Astrocytes

After ischemic injury to brain, disruption of the blood–brain barrier (BBB) raises the possibility of exposing the central nervous system (CNS) to oxidized low-density lipoprotein (oxLDL), a risk factor implicated in neurodegenerative diseases. Matrix metalloproteinases (MMPs), especially MMP-9, contribute to extracellular matrix (ECM) remodeling during the CNS diseases. However, the molecular mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes remained unclear. Here, we reported that oxLDL induced MMP-9 expression via a PKC-δ/p42/p44 MAPK-dependent Elk-1 activation in rat brain astrocyte (RBA)-1 cells, revealed by gelatin zymography, RT-PCR, and Western blotting analyses. These responses were attenuated by pretreatment with pharmacological inhibitors and transfection with dominant negative mutants. Moreover, Elk-1-mediated MMP-9 gene transcription was confirmed by transfection with an Elk-1 binding site-mutated MMP-9 promoter construct (mt-Ets-MMP9), which blocked oxLDL-stimulated MMP-9 luciferase activity. Understanding the regulatory mechanisms by which oxLDL induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain diseases.

Endothelin-1 enhances cell migration via matrix metalloproteinase-9 up-regulation in brain astrocytes.

J. Neurochem. (2010) 113, 1133–1149.