Li-Der Hsiao

Mitogenic effect of oxidized low-density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway

It has been demonstrated that oxidized low‐density lipoprotein (OX‐LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms of OX‐LDL‐induced cell proliferation are not completely understood. Therefore, we investigated the effect of OX‐LDL on cell proliferation associated with mitogen‐activated protein kinase (MAPK) activation in rat cultured VSMCs. Both native‐LDL (N‐LDL) and OX‐LDL induced a time‐ and concentration‐dependent incorporation of [3H]‐thymidine in VSMCs. OX‐LDL induced time‐ and concentration‐dependent phosphorylation of p42/p44 MAPK. Pretreatment of these cells with pertussis toxin or U73122 attenuated the OX‐LDL‐induced responses. Pretreatment with PMA for 24 h, preincubation with a PKC inhibitor staurosporine or the tyrosine kinase inhibitors, genistein and herbimycin A for 1 h, substantially reduced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation induced by OX‐LDL. Removal of Ca2+ by BAPTA/AM or depletion of the internal Ca2+ pool by thapsigargin significantly inhibited OX‐LDL‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation. OX‐LDL‐induced [3H]‐thymidine incorporation and p42/p44 MAPK phosphorylation was inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK) in a concentration‐dependent manner. Overexpression of dominant negative mutants of Ras (H‐Ras‐15A) and Raf (Raf‐N4) significantly suppressed MEK1/2 and p42/p44 MAPK activation induced by OX‐LDL and PDGF‐BB, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the mitogenic effect of OX‐LDL is mediated through a PTX‐sensitive G protein‐coupled receptor that involves the activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF‐BB in rat cultured VSMCs.

Interleukin-1β-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS, and NF-κB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.

Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes

BackgroundJapanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear.MethodsIn this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs.ResultsHere, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs.ConclusionFrom these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases.

Cigarette smoke extract induces COX-2 expression via a PKCα/c-Src/EGFR, PDGFR/PI3K/Akt/NF-κB pathway and p300 in tracheal smooth muscle cells

Exposure to cigarette smoke extract (CSE) leads to airway or lung inflammation, which may be mediated through cyclooxygenase-2 (COX-2) expression and its product prostaglandin E2 (PGE2) synthesis. The aim of this study was to investigate the molecular mechanisms underlying CSE-induced COX-2 expression in human tracheal smooth muscle cells (HTSMCs). Here, we describe that COX-2 induction is dependent on PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB signaling in HTSMCs. CSE stimulated the phosphorylation of c-Src, EGFR, PDGFR, and Akt, which were inhibited by pretreatment with the inhibitor of PKCalpha (Gö6976 or Gö6983), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), or PI3K (LY294002). Moreover, CSE induced a significant increase in COX-2 expression, which was reduced by pretreatment with these inhibitors or transfection with siRNA of PKCalpha, Src, or Akt. Furthermore, CSE-stimulated NF-kappaB p65 phosphorylation and translocation were also attenuated by pretreatment with Gö6976, PP1, AG1478, AG1296, or LY294002. CSE-induced COX-2 expression was also mediated through the recruitment of p300 associated with NF-kappaB in HTSMCs, revealed by coimmunoprecipitation and Western blot analysis. In addition, pretreatment with the inhibitors of NF-kappaB (helenalin) and p300 (garcinol) or transfection with p65 siRNA and p300 siRNA markedly inhibited CSE-regulated COX-2 expression. However, CSE-induced PGE2 generation was reduced by pretreatment with the inhibitor of COX-2 (NS-398). These results demonstrated that in HTSMCs, CSE-induced COX-2-dependent PGE2 generation was mediated through PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt leading to the recruitment of p300 with NF-kappaB complex.

Interleukin-1β induces ICAM-1 expression enhancing leukocyte adhesion in human rheumatoid arthritis synovial fibroblasts: Involvement of ERK, JNK, AP-1, and NF-κB

Interleukin‐1β (IL‐1β) has been shown to induce the expression of adhesion molecules on various cell types and contributes to inflammatory responses. However, the molecular mechanisms by which IL‐1β induced intercellular adhesion molecule (ICAM)‐1 expression remain unclear in human rheumatoid arthritis synovial fibroblasts (RASFs). Here, we demonstrated that IL‐1β induces ICAM‐1 gene expression via the de novo protein synthesis through transcription and translation, which is attenuated by pretreatment with actinomycin D and cycloheximide, respectively. IL‐1β‐induced ICAM‐1 expression, extracellular signal‐regulated kinase (ERK) and c‐Jun‐N‐terminal kinase (JNK) phosphorylation, AP‐1 activation, and nuclear factor‐κB (NF‐κB) p65 translocation were attenuated by the inhibitors of MEK1/2 (U0126), JNK (SP600125), AP‐1 (tanshinone IIA), and NF‐κB (helenalin) or transfection with respective short hairpin RNA plasmids. Moreover, IL‐1β‐stimulated NF‐κB p65 translocation was blocked by helenalin, but not by U0126 or SP600125, revealing that MAPKs and NF‐κB pathways were independent on these responses. IL‐1β‐stimulated AP‐1 activation was blocked by U0126 or SP600125, revealing that ERK and JNK linked to AP‐1 on these responses. IL‐1β‐stimulated ICAM‐1 gene expression was attenuated by pretreatment with U0126, SP600125, tanshinone IIA, or helenalin, revealed by ICAM‐1 promoter assay and real‐time RT‐PCR analysis. Finally, up‐regulation of ICAM‐1 enhanced the adhesion of leukocytes to RASFs exposed to IL‐1β. These results suggest that in human RASFs, activation of ERK, JNK, AP‐1, and NF‐κB are essential for IL‐1β‐induced ICAM‐1 expression and leukocyte adhesion. J. Cell. Physiol. 224: 516–526, 2010.

Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cells

In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non‐receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3‐kinase (PI3‐K) have been implicated in linking a variety of G‐protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK‐induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK‐ and EGF‐dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK‐ and EGF‐dependent effects. Inhibition of PI3‐K by LY294002 attenuated BK‐induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3‐K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK‐induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK‐induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK‐stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3‐K/Akt. Copyright

Tumour necrosis factor‐α‐ and interleukin‐1β‐stimulated cell proliferation through activation of mitogen‐activated protein kinase in canine tracheal smooth muscle cells

The elevated levels of inflammatory cytokines such as tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF‐α and IL‐1β on cell proliferation and activation of p42/p44 mitogen‐activated protein kinase (MAPK) in these cells. TNF‐α and IL‐1β induced [3H]‐thymidine incorporation in a time‐ and concentration‐dependent manner. The maximal stimulation of [3H]‐thymidine incorporation induced by TNF‐α and IL‐1β was seen 12 h after incubation with these cytokines. In response to TNF‐α and IL‐1β, p42/p44 MAPK was activated with a concentration‐dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF‐α and IL‐1β. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)‐phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)‐PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca2+ by addition of BAPTA/AM plus EGTA. TNF‐α‐ and IL‐1β‐induced [3H]‐thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF‐α and IL‐1β were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF‐α‐ and IL‐1β‐mediated responses were modulated by PLC, Ca2+, PKC, and tyrosine kinase associated with cell proliferation in TSMCs.

Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway.

Elevated levels of several cytokines including interleukin-1beta (IL-1beta) have been detected in airway fluid of asthmatic patients. Inhalation of IL-1beta induced a bronchial hyper-reactivity to contractile agonists. However, the implication of IL-1beta in the pathogenesis of bronchial hyper-reactivity is not completely understood. Therefore, we investigated the effect of IL-1beta on bradykinin (BK)-induced inositol phosphate [Ins(X)P] accumulation and Ca2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth-muscle cells (TSMCs). Treatment of TSMCs with IL-1beta potentiated BK-induced Ins(X)P accumulation and Ca2+ mobilization. However, there was no effect on the Ins(X)P response induced by endothelin-1, 5-hydroxytryptamine or carbachol. Treatment with platelet-derived growth factor B-chain homodimer (PDGF-BB) also enhanced the BK-induced Ins(X)P response. These enhancements by IL-1beta and PDGF-BB might be due to an up-regulation of BK B(2) receptor density (B(max)), since [(3)H]BK binding to TSMCs was inhibited by the B(2)-selective agonist and antagonist, BK and Hoe 140, but not by B(1)-selective reagents. The enhancing effects of IL-1beta and PDGF-BB on Ins(X)P accumulation, Ca2+ mobilization and B(max) were attenuated by PD98059 [an inhibitor of activation of mitogen-activated protein kinase (MAPK) kinase, MEK] and cycloheximide (an inhibitor of protein synthesis), suggesting that IL-1beta may share a common signalling pathway with PDGF-BB via protein synthesis. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed the up-regulation of BK receptors induced by IL-1beta, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by IL-1beta might be, at least in part, mediated through activation of the Ras/Raf/MEK/MAPK pathway in TSMCs.

Involvement of MAPKs and NF-κB in tumor necrosis factor α–induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts

OBJECTIVE To investigate the roles of MAPKs and NF-kappaB in tumor necrosis factor alpha (TNFalpha)-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). METHODS Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-kappaB in TNFalpha-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase-polymerase chain reaction, and gene promoter assay. NF-kappaB translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. RESULTS TNFalpha-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-kappaB were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-kappaB (helenalin) or by transfection with their respective shRNA. TNFalpha-stimulated translocation of NF-kappaB into the nucleus and NF-kappaB promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNFalpha in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with helenalin, U0126, SP600125, or SB202190 prior to exposure to TNFalpha or by anti-VCAM-1 antibody before the addition of lymphocytes. CONCLUSION In RASFs, TNFalpha-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches.

NADPH oxidase-mediated redox signal contributes to lipoteichoic acid-induced MMP-9 upregulation in brain astrocytes.

BackgroundLipoteichoic acid (LTA) is a component of gram-positive bacterial cell walls and may be elevated in the cerebrospinal fluid of patients suffering from meningitis. Among matrix metalloproteinases (MMPs), MMP-9 has been observed in patients with brain inflammatory diseases and may contribute to the pathology of brain diseases. Moreover, several studies have suggested that increased oxidative stress is implicated in the pathogenesis of brain inflammation and injury. However, the molecular mechanisms underlying LTA-induced redox signal and MMP-9 expression in brain astrocytes remain unclear.ObjectiveHerein we explored whether LTA-induced MMP-9 expression was mediated through redox signals in rat brain astrocytes (RBA-1 cells).MethodsUpregulation of MMP-9 by LTA was evaluated by zymographic and RT-PCR analyses. Next, the MMP-9 regulatory pathways were investigated by pretreatment with pharmacological inhibitors or transfection with small interfering RNAs (siRNAs), Western blotting, and chromatin immunoprecipitation (ChIP)-PCR and promoter activity reporter assays. Moreover, we determined the cell functional changes by migration assay.ResultsThese results showed that LTA induced MMP-9 expression via a PKC(α)-dependent pathway. We further demonstrated that PKCα stimulated p47phox/NADPH oxidase 2 (Nox2)-dependent reactive oxygen species (ROS) generation and then activated the ATF2/AP-1 signals. The activated-ATF2 bound to the AP-1-binding site of MMP-9 promoter, and thereby turned on MMP-9 gene transcription. Additionally, the co-activator p300 also contributed to these responses. Functionally, LTA-induced MMP-9 expression enhanced astrocytic migration.ConclusionThese results demonstrated that in RBA-1 cells, activation of ATF2/AP-1 by the PKC(α)-mediated Nox(2)/ROS signals is essential for upregulation of MMP-9 and cell migration enhanced by LTA.