Sphingosine 1-phosphate induces EGFR expression via Akt/NF-kappaB 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-kappaB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P-induced activation of NF-kappaB was blocked by wortmannin, but not by U0126, suggesting that activation of NF-kappaB 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-kappaB 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.     

Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.     

EV71 induces VCAM-1 expression via PDGF receptor, PI3-K/Akt, p38 MAPK, JNK and NF-kappaB in vascular smooth muscle cells

Enterovirus 71 (EV71) is a widespread virus that causes severe and fatal diseases in patients, including circulation failure. The mechanisms underlying EV71-initiated intracellular signaling pathways to influence host cell functions remain unknown. In this study, we identified a requirement for PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB in the regulation of VCAM-1 expression by rat vascular smooth muscle cells (VSMCs) in response to viral infection. EV71 induced VCAM-1 expression in a time- and viral concentration-dependent manner. Infection of VSMCs with EV71 stimulated VCAM-1 expression and phosphorylation of PDGFR, Akt, and p38 MAPK which were attenuated by AG1296, wortmannin, and SB202190, respectively. The phosphorylation of JNK stimulated by EV71 was not detected under present conditions. In contrast, JNK inhibitor SP600125 inhibited EV71-induced VCAM-1 expression. Furthermore, VCAM-1 expression induced by EV71 was significantly attenuated by a selective NF-kappaB inhibitor (helenalin). Consistently, EV71-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha as well as VCAM-1 mRNA expression was blocked by helenalin, AG1296, SB202190, SP600125, wortmannin, and LY294002. Moreover, the involvement of p38 MAPK, PI3-K/Akt, and NF-kappaB in EV71-induced VCAM-1 expression was reveled by that transfection with dominant negative plasmids of p38 MAPK, p85, Akt, NIK, IKK-alpha, and IKK-beta attenuated these responses. These findings suggest that in VSMCs, EV71-induced VCAM-1 expression was mediated through activation of PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB pathways.     

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.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.     

BK-induced COX-2 expression via PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB in astrocytes

Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

Intracellular signalings underlying bradykinin-induced matrix metalloproteinase-9 expression in rat brain astrocyte-1

Bradykinin (BK), an inflammatory mediator, 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. Here we report that the mitogen-activated protein kinase (MAPK) and NF-kappaB pathways participate in the induction of MMP-9 expression induced by BK in RBA cells. Zymographic, Western blotting, and RT-PCR analyses showed that 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-kappaB inhibitor helenalin. In accordance with these findings, BK-induced phosphorylation of p42/p44 MAPK and Akt and activation of NF-kappaB 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 B(2) receptor antagonist Hoe 140, indicating the involvement of B(2) receptors revealed by [(3)H]-BK binding assay. Furthermore, BK-stimulated translocation of NF-kappaB 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 Akt cascades mediated through NF-kappaB pathway are essential for BK-induced MMP-9 gene expression. This study may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation and brain astrocytoma.     

Involvement of MAPKs and NF-kappaB in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells

Lipopolysaccharide (LPS) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for LPS-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in HTSMCs. LPS-induced expression of VCAM-1 protein and mRNA in a time-dependent manner, was significantly inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and c-Jun-N-terminal kinase (JNK; SP600125). The involvement of p42/p44 MAPK and p38 in these responses was further confirmed by that transfection with small interference RNAs (siRNA) direct against MEK, p42, and p38 significantly attenuated LPS-induced VCAM-1 expression. Consistently, LPS-stimulated phosphorylation of p42/p44 MAPK and p38 was attenuated by pretreatment with U0126 or SB202190, and transfection with these siRNAs, respectively. In addition, LPS-induced VCAM-1 expression was significantly blocked by a specific NF-kappaB inhibitor helenalin. LPS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, U0126, SB202190, or SP600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to monolayer of HTSMCs which was blocked by pretreatment with helenalin, U0126, or SP600125 prior to LPS exposure. Taken together, these results suggest that in HTSMCs, activation of p42/p44 MAPK, p38, and JNK pathways, at least in part, mediated through NF-kappaB, is essential for LPS-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of LPS action that bacterial toxins may promote inflammatory responses in the airway disease.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.     

BK-induced cytosolic phospholipase A2 expression via sequential PKC-delta, p42/p44 MAPK, and NF-kappaB activation in rat brain astrocytes

Bradykinin (BK), an inflammatory mediator, has been shown to induce cytosolic phospholipase A2 (cPLA2) expression implicating in inflammatory responses in various cell types. However, the detailed mechanisms underlying BK-induced cPLA2 expression in astrocytes remain unclear. RT-PCR and Western blotting analysis showed that BK induced the expression of cPLA2 mRNA and protein, which was inhibited by Hoe140, suggesting the involvement of B2 BK receptors, confirmed by immunofluorescence staining using anti-B2 BK receptor antibody. BK-induced cPLA2 expression and phosphorylation of p42/p44 MAPK was attenuated by PD98059, indicating the involvement of MEK1/2-p42/p44 MAPK in these responses. BK-induced cPLA2 expression might be due to the translocation of NF-kappaB into nucleus which was inhibited by Hoe140, helenalin, and PD98059, implying the involvement of NF-kappaB. Moreover, BK-induced cPLA2 expression was attenuated by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was supported by the transfection with a dominant negative plasmid of PKC-delta significantly attenuated BK-induced response. In addition, BK-stimulated translocation of PKC-delta from cytosol to membrane fraction was inhibited by rottlerin but not by PD98059, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Accordingly, BK-induced phosphorylation of p42/p44 MAPK was attenuated by rottlerin but not by helenalin. These results suggest that in RBA-1 cells, BK-induced cPLA2 expression was sequentially mediated through activation of PKC-delta, p42/p44 MAPK, and NF-kappaB. Understanding the regulation of cPLA2 expression induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.     

PKC-delta/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.     

Induction of cyclooxygenase-2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB pathways

Lipopolysaccharide (LPS) was found to induce inflammatory responses in the airways and exerted as a potent stimulus for PG synthesis. This study was to determine the mechanisms of LPS-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). LPS markedly increased the expression of COX-2 and release of PGE(2) in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Both the expression of COX-2 and the generation of PGE(2) in response to LPS 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. Furthermore, LPS-induced NF-kappaB activation correlated with the degradation of IkappaB-alpha, COX-2 expression, and PGE(2) synthesis, was inhibited by transfection with dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. LPS-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK inhibitor), but these two inhibitors had no effect on LPS-induced NF-kappaB activation, indicating that NF-kappaB is activated by LPS independently of activation of p42/p44 MAPK and p38 MAPK pathways in TSMCs. Taken together, these findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from LPS-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways. LPS-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

Negative regulation of the protein kinase C activator-induced ICAM-1 expression in the human bronchial epithelial cell line NCI-H292 by p44/42 mitogen-activated protein kinase

The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one.