p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells

It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) modulate vascular smooth muscle cell functions. In the present study, we investigated the effect of simvastatin on vascular endothelial growth factor (VEGF) release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. Administration of simvastatin increased the VEGF level in rat plasma in vivo. In cultured cells, simvastatin significantly stimulated VEGF release in a dose-dependent manner. Simvastatin induced the phosphorylation of p44/p42 MAP kinase but not p38 MAP kinase or SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). PD98059 and U-0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly reduced the simvastatin-induced VEGF release in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by simvastatin was reduced by PD98059 or U-0126. Moreover, a bolus injection of PD98059 truly suppressed the simvastatin-increased VEGF level in rat plasma in vivo. These results strongly suggest that p44/p42 MAP kinase plays a role at least partly in the simvastatin-stimulated VEGF release in vascular smooth muscle cells.     

Substance P-induced activation of p42/44 mitogen-activated protein kinase associated with cell proliferation in human tracheal smooth muscle cells

Substance P (SP) released from sensory nerve endings in the airways induces several responses including cell proliferation. However, the mechanisms were not completely understood in tracheal smooth muscle cells (TSMCs). We therefore investigated the effect of SP on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. SP stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Both DNA synthesis and phosphorylation of MAPK in response to SP were attenuated by pretreatment with pertussis toxin, genistein, D609, U73122, staurosporine, removal of Ca(2+) by BAPTA/AM plus EGTA, PD98059, and SB202190. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by SP and PDGF-BB. These results conclude that the mitogenic effect of SP was mediated through the activation of Ras/Raf/MEK/MAPK pathway, which was modulated by PC-PLC, PI-PLC, Ca(2+), and PKC in cultured human TSMCs.     

Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.     

Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin-1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin-1 but contain normal amounts of caveolin-2. NIH 3T3 cells harboring antisense caveolin-1 exhibit anchorage-independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin-1 downregulation is reversed when caveolin-1 protein levels are restored to normal by loss of the caveolin-1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin-1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin-1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.     

p38 MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells

We have previously reported that interleukin (IL)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.     

Human endotoxemia activates p38 MAP kinase and p42/44 MAP kinase, but not c-Jun N-terminal kinase.

BACKGROUND: All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to which these kinases are actually activated during inflammation in humans in vivo has not been investigated. We employed experimental human endotoxemia, a model of systemic inflammation, to address this question. MATERIALS AND METHODS: Male volunteers were intravenously infused with 4 ng/kg bw lipopolysaccharide (LPS). Directly before LPS infusion and up to 24 h thereafter, activation of p38 MAPK, p42/p44 MAPK and JNK was assessed in peripheral blood, using Western blot and in vitro kinase assays. RESULTS: We observed that LPS induced a strong but transient phosphorylation and activation of p38 MAPK and p42/p44 MAPK, maximal activity being reached after 1 hr of LPS infusion. Strikingly, no JNK phosphorylation or activation was detected under these circumstances. CONCLUSIONS: These results suggest that both inhibitors of p38 MAPK and p42/p44 MAPK but not JNK are potentially useful for anti-inflammatory therapy.     

Role of Ras in ethanol modulation of angiotensin II activated p42/p44 MAP kinase in rat hepatocytes

Angiotensin II plays a role in both liver cell proliferation and liver injury but the effects of ethanol on angiotensin II signaling in liver are not clearly understood. We have investigated the role of Ras in ethanol modulation of p42/p44 mitogen-activated protein kinase (MAPK) stimulated by angiotensin II (Ang II) in primary cultures of rat hepatocytes. Hepatocytes were incubated with ethanol (100 mM) for 24 h, then stimulated with Ang II (100 nM). The level of p42/p44 MAPK phosphorylation was measured by Western blot analysis and Ras activation was assessed by specific binding of Ras-GTP (activated form) to a GST-RBD fusion protein containing Ras-binding domain (RBD) of Raf-1. Ethanol potentiated p42/p44 MAPK activation by Ang II, whereas ethanol alone did not significantly affect phosphorylation of p42/p44 MAPK. Ang II increased Ras activity by about 2 fold. Ethanol exposure increased Ang II stimulated Ras activity by an additional about 2 fold. Ethanol alone elicited a small increase in basal Ras activity. Pretreatment with manumycin A (10 microM), a Ras farnesylation inhibitor, partially blocked both Ang II-activated and ethanol-potentiated MAPK activities. These data provided the first evidence that ethanol potentiation of Ang II stimulated p42/p44 MAPK is mediated, in part, by Ras in hepatocytes.     

Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, G?-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca(2+) chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca(2+)](i), supporting the involvement of Ca(2+) mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca(2+), MEK, and PI 3-kinase.     

Up-regulation by zinc of FGF-2-induced VEGF release through enhancing p44/p42 MAP kinase activation in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether zinc affects the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ZnSO(4) but not Na(2)SO(4). The enhancing effect of ZnSO(4) was dose-dependent between 1 and 100 muM. ZnSO(4) markedly enhanced the FGF-2-induced phosphorylation of p44/p42 MAP kinase while having little effect on the SAPK/JNK phosphorylation. PD98059 significantly reduced the amplification by ZnSO(4) of the FGF-2-stimulated VEGF release. Taken together, our findings strongly suggest that zinc enhances FGF-2-stimulated VEGF release resulting from up-regulating activation of p44/p42 MAP kinase in osteoblasts.     

Heme oxygenase-1 contributes to the cytoprotection of alpha-lipoic acid via activation of p44/42 mitogen-activated protein kinase in vascular smooth muscle cells

Alpha-lipoic acid (ALA) is a natural antioxidant that scavenges reactive oxygen species (ROS) and regenerates or recycles endogenous antioxidants. ALA has recently been reported to protect against oxidative injury in various disease processes. The aim of this study was to investigate whether the antioxidant effect of ALA is mediated by the induction of heme oxygenase (HO)-1 in rat aortic smooth muscle cells (A10 cells). ALA significantly induced HO-1 expression accompanied by an increase in HO activity in A10 cells. Pretreatment with ALA increased the resistance of A10 cells to hydrogen-peroxide-induced oxidant stress. This protection of ALA was abrogated in the presence of the HO inhibitor zinc protoporphyrin IX. ALA significantly increased ROS, and this effect was blocked by N-acetyl-cysteine, which also inhibited ALA-induced activation of p44/42 mitogen-activated protein kinase (MAPK) and AP-1, HO-1 expression, and HO activity. These results suggest that ALA induces HO-1 expression through the production of ROS and subsequent activation of the p44/42 MAPK pathway and AP-1 in vascular smooth muscle cells. This study demonstrated that ALA increases the expression of HO-1, a critical cytoprotective molecule, and identified a novel pleiotropic effect of ALA on cardiovascular protection.     

(-)-Epigallocatechin gallate suppresses endothelin-1-induced interleukin-6 synthesis in osteoblasts: inhibition of p44/p42 MAP kinase activation

We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade.     

细胞外信号调节激酶活化在慢性支气管哮喘大鼠气道平滑肌细胞增殖中的作用

本文旨在探讨细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）在慢性支气管哮喘大鼠气道平滑肌细胞（airway smooth muscle cells,ASMCs）增殖中的作用。建立慢性哮喘大鼠模型,用ERK激动剂表皮生长因子（epidermal growth factor,EGF）和抑制剂PD98059干预慢性哮喘大鼠ASMCs的培养。采用流式细胞仪、四甲基偶氮唑盐（MTT）法、^3H-thymidine（TdR）掺入法和增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）免疫组织化学法检测ASMCs增殖情况,观察ERK信号通路对ASMCs增殖的影响。RT-PCR和Western blot检测ERK mRNA和ERK1／2、磷酸化ERK1／2（p-ERK1／2）蛋白的表达。与正常对照组ASMCs比较,慢性哮喘组ASMCs的G0/G1期细胞所占比例明显减少,S＋G2／M期细胞所占比例增高;吸光度（A490）值、细胞DNA合成量和PCNA阳性表达量均明显增加,ERK mRNA、ERK1／2蛋白、P-ERK1／2蛋白的表达量以及ERK活化率显著增高。经PD98059干预之后,慢性哮喘组ASMCs的S＋G2／M期细胞所占比例、A490值、细胞DNA合成量和PCNA阳性表达量明显降低,ERK mRNA、ERK1／2蛋白、p-ERK1／2蛋白的表达量以及ERK活化率显著降低。经EGF干预后,慢性哮喘组ASMCs的S＋G2／M期细胞所占比例、A490值、细胞DNA合成量和PCNA阳性表达量进一步增高,而这一作用可以被PD98059抑制。以上结果提示,慢性哮喘大鼠ASMCs内源性增殖活性增加,ERK1／2参与其增殖活性的调控,ERK信号通路在哮喘气道重建的ASMCs增殖调控中具有重要作用。     

The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal

Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor- and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin-Darby canine kidney cells stably expressing an estrogen-inducible activated-Raf-1 construct (DeltaRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the DeltaRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.     

Reactive oxidant and p42/44 MAP kinase signaling is necessary for mechanical strain-induced proliferation in pulmonary epithelial cells.

Mechanical strain is necessary for normal lung growth and development. Individuals with respiratory failure are supported with mechanical ventilation, leading to altered lung growth and injury. Understanding signaling pathways initiated by mechanical strain in lung epithelial cells will help guide development of strategies aimed at optimizing strain-induced lung growth while mitigating ventilator-induced lung injury. To study strain-induced proliferative signaling, focusing on the role of reactive oxidant species (ROS) and p42/44 mitogen-activated protein (MAP) kinase, human pulmonary epithelial H441 and MLE15 cells were exposed to equibiaxial cyclic mechanical strain. ROS were increased within 15 min of strain. N-acetylcysteine inactivated strain-induced ROS and inhibited p42/44 MAP kinase phosphorylation and strain-induced proliferation. PD98059 and UO126, p42/44 MAP kinase inhibitors, blocked strain-induced proliferation. To verify the specificity of p42/44 MAP kinase inhibition, cells were transfected with dominant-negative mitogen-activated protein kinase kinase-1 plasmid DNA. Transfected cells did not proliferate in response to mechanical strain. To determine whether strain-induced tyrosine kinase activity is necessary for strain-induced ROS-p42/44 MAP kinase signaling, genistein, a tyrosine kinase inhibitor, was used. Genistein did not block strain-induced ROS production or p42/44 MAP kinase phosphorylation. Gadolinium, a mechanosensitive calcium channel blocker, blocked strain-induced ROS production and p42/44 MAP kinase phosphorylation but not strain-induced tyrosine phosphorylation. These data support ROS production and p42/44 MAP kinase phosphorylation being involved in a common strain-induced signaling pathway, necessary for strain-induced proliferation in pulmonary epithelial cells, with a parallel strain-induced tyrosine kinase pathway.     

Bradykinin-stimulated p42/p44 MAPK activation associated with cell proliferation in corneal keratocytes

Bradykinin (BK) is released into the tear-film in ocular allergic patients. BK has been shown to exert mitogenic effects on several cell types. However, the mechanisms underlying its action on corneal keratocytes (CKs) were largely unknown. This study was to investigate the mitogenic effect of BK on rabbit CKs linked to activation of p42/p44 mitogen-activated protein kinase (MAPK), assessed by [3H]thymidine incorporation and Western blotting analysis, respectively. BK stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner. Pretreatment with pertussis toxin attenuated the BK-induced responses. BK-stimulated responses were attenuated by inhibitors of selective B2 receptor (Hoe 140), phosphatidylinositol (PI)-PLC (U73122), an intracellular Ca2+chelator (BAPTA/AM), PKC (GF109203X), tyrosine kinase (genistein), and MEK1/2 (PD98059). BK also stimulated translocation of p42/p44 MAPK into nucleus and led to expression of c-fos and c-jun in CKs. These results demonstrate that in CKs, BK-stimulated phosphorylation of p42/p44 MAPK is mediated through the activation of BK B2 receptors and leads to cell proliferation.