Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Preconditioning-induced activation of ERK5 is dependent on moderate Ca2+ influx via NMDA receptors and contributes to ischemic tolerance in the hippocampal CA1 region of rats

Accumulating evidence implicates activation (phosphorylation) of mitogen-activated protein kinases (MAPK) during nonlethal ischemic preconditioning in the protection of hippocampal CA1 neuron against subsequent ischemic events. In this paper, we undertook to identify the role of extracellular signal regulated kinase (ERK) 5 in cerebral ischemic preconditioning (CIP). Three minutes of ischemia was induced as preconditioning stimulus. Three days later, 6 min of ischemia was induced. The levels of ERK5 protein expression and its activation were detected with or without the CIP in hippocampal CA1 and the dentate gyrus (DG) regions. Our results showed that ERK5 was activated selectively in hippocampal CA1 region with, but not without, the ischemic preconditioning. Notably, during the later phase of reperfusion, the rise in ERK5 activation was strong and persistent with a peak occurring at the third day. The activation peak was effectively prevented and ERK5 protein expression was significantly decreased by intracerebroventricular infusion of ERK5 antisense oligonucleotide (every 24 h for 3 days before the preconditioning), but not by sense oligonucleotide or vehicle. Subsequently, the CA1 neuronal loss was largely elevated. Moreover, both MK801 (10 microM), an antagonist of NMDA receptor, and EGTA (100 mM, but neither 50 nor 150 mM), an extracellular Ca2+ chelator, not only effectively inhibited the ERK5 activation but also markedly abolished CIP-induced survival of the CA1 neurons. These results suggested that activation of the ERK5 pathway by CIP was at least partly dependent on moderate Ca2+ influx via NMDA receptor, which might contribute to ischemic tolerance in hippocampal CA1 region of rats.     

The role of mitochondrial reactive oxygen species in cartilage matrix destruction

According to a compelling body of evidence anesthetic preconditioning (APC) attenuates the deleterious consequences of ischemia–reperfusion and protects the heart through a mechanism similar to ischemic preconditioning. The present study was purported to investigate the intracellular signaling pathways activated in human myocardium in response to a preconditioning protocol with two different volatile anesthetics, namely isoflurane and sevoflurane. To this aim, phosphorylation of PKCα and -δ, ERK1/2, Akt, and GSK3β was determined at the end of the APC protocol, in human atrial samples harvested from patients undergoing open-heart surgery. The results demonstrate that preconditioning with volatile anesthetics triggers the activation of PKCδ and -α isoforms and of prosurvival kinases, ERK1/2, and Akt, while inhibiting their downstream target GSK3β during the memory phase.     

Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation and protein synthesis in human breast cancer cells

Carbachol (Cch), a muscarinic acetylcholine receptor (mAChR) agonist, increases intracellular-free Ca(2+) mobilization and induces mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in MCF-7 human breast cancer cells. Pretreatment of cells with the selective phospholipase C (PLC) inhibitor U73122, or incubation of cells in a Ca(2+)-free medium did not alter Cch-stimulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by phorbol 12-myristate acetate (PMA), an activator of protein kinase C (PKC), but Cch-evoked MAPK/ERK activation was unaffected by down-regulation of PKC or by pretreatment of cells with GF109203X, a PKC inhibitor. However, Cch-stimulated MAPK/ERK phosphorylation was completely blocked by myristoylated PKC-zeta pseudosubstrate, a specific inhibitor of PKC-zeta, and high doses of staurosporine. Pretreatment of human breast cancer cells with wortmannin or LY294002, selective inhibitors of phosphoinositide 3-kinase (PI3K), diminished Cch-mediated MAPK/ERK phosphorylation. Similar results were observed when MCF-7 cells were pretreated with genistein, a non-selective inhibitor of tyrosine kinases, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF-7 human breast cancer cells mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen activated kinase kinase. In conclusion, analyses of mAChR downstream effectors reveal that PKC-zeta, PI3K, and Src family of tyrosine kinases, but not intracellular-free Ca(2+) mobilization or conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK activation. In addition, MAPK/ERK are involved in the regulation of growth and proliferation of MCF-7 human breast cancer cells.     

Role of G proteins and modulation of p38 MAPK activation in the protection by nitric oxide against ischemia-reoxygenation injury

Protein kinase C (PKC)-mediated regulation of the mitogen-activated protein kinases (MAPK) may play a role in the protection afforded by ischemic preconditioning (PC). Nitric oxide (NO) can influence MAPK activation via interaction with PKC or farnesylation of low-molecular-weight (LMWT) G proteins. However, we have recently reported the mechanism of NO-induced cardioprotection to be a PKC-independent process. Therefore, we investigated the role of LMWT G proteins and MAPK signaling in NO-induced cardioprotection against simulated ischemia-reoxygenation (SI-R) injury. Neonatal rat cardiomyocytes treated for 90 min with the NO donor S-nitroso-N-acetyl-l,l-penicillamine (SNAP) 1 mM were protected against 6 h of SI (hypoxic conditions at 37 degrees C with 20 mM lactate, 16 mM KCl at pH 6.2) and 24 h reoxygenation under normal culture conditions. NO-induced protection was blocked by the G protein inhibitor alpha-hydroxyfarnesylphosphonic acid (alphaHFP) 10 microM. We studied the time course of p42/44 and p38 MAPK dual-phosphorylation hourly during SI using phospho-specific antibodies. p38 was phosphorylated during SI and the peak phosphorylation was significantly delayed by SNAP pretreatment. The p38 inhibitor SB203580 1 microM, given during SI, protected against injury. Thus the delay in peak p38 activation may contribute to, rather than be the effect of, NO-induced cardioprotection. We have shown that p38beta does not contribute to the total p38 signal in our extracts. Thus there is no detectable beta isoform. We conclude that the main isoform present in these cells and thought to be responsible for the observed phenomenon, is the alpha isoform.     

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca²⁺]_i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca²⁺]_i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y₂ receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca²⁺-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y₂ receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.     

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C

PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT) at reperfusion protects ischemic hearts, but the mechanism is unknown. We recently proposed that in preconditioned hearts PKC lowers the threshold for adenosine to initiate signaling from low-affinity A2b receptors during early reperfusion thus allowing endogenous adenosine to activate survival kinases phosphatidylinositol 3-kinase (PI3K) and ERK. We tested whether CPT might also sensitize A2b receptors to adenosine. CPT (10 microM) during the first minutes of reperfusion markedly reduced infarction in isolated rabbit hearts undergoing 30-min regional ischemia/2-h reperfusion, and salvage was blocked by MRS 1754, an A2b-selective antagonist. Coadministration of wortmannin (PI3K inhibitor) or PD-98059 (MEK1/2 and therefore ERK1/2 inhibitor) also blocked protection. In nonischemic hearts, 10-min infusion of CPT did not change phosphorylation of Akt or ERK1/2. Neither did a subthreshold dose (2.5 nM) of the nonselective but A2b-potent receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA). However, when 2.5 nM NECA was combined with 10 microM CPT, both phospho-Akt and phospho-ERK1/2 significantly increased, indicating CPT had lowered the threshold for A2b-dependent signaling. The PKC antagonist chelerythrine blocked this phosphorylation induced by CPT + NECA. Chelerythrine also blocked the anti-infarct effect of CPT as did nonselective (glibenclamide) and mitochondrial-selective (5-hydroxydecanoate) K(ATP) channel blockers. A free radical scavenger, N-(2-mercaptopropionyl)glycine, also blocked CPT protection. We propose CPT targets PKG, which activates PKC through mitochondrial K(ATP) channel (mitoKATP)-dependent redox signaling, a sequence mimicking that already documented in preconditioning. Activated PKC then augments sensitivity of normally low-affinity cardiac adenosine A2b receptors so endogenous adenosine can protect by activating Akt and ERK.     

p38 MAPKα/β和ERK1/2在心肌缺氧预处理信号传递中的不同作用

建立培养乳鼠心肌细胞的缺氧／复氧(A／R)损伤模型和缺氧预处理(APC)模型,以细胞存活率、细胞内超氧化物趋化酶(SOD)活性、丙二醛(MDA)含量、培养上清液乳酸脱氢酶(LDH)活性作为反映心肌细胞损伤的指标。采用细胞外信号调节蛋白激酶(ERK1／2)抑制剂PD98059及丝裂素活化蛋白激酶p38α/β(p38α/β)阻滞剂SB203580干预模型,并以胶内原位磷酸化法测定ERK1／2和p38活性,借以探讨ERK1／2和p38α/β在缺氧预处理保护机制中的作用。结果表明：(1)在APC组,于预处理的缺氧时相给予PD98059,可以完全消除APC的延迟保护作用;在A／R组的缺氧时相加入PD98059对细胞损伤无影响;(2)在APC组的预处理缺氧时相给予p38α／β抑制剂SB203580并不能消除APC的保护作用,而在A／R组的持续缺氧时相给予SB203580则可显著减轻缺氧对细胞的损伤;(3)ERK1／2和p38总活性测定表明,缺氧可激活ERK1／2和p38,它们的活性在缺氧后4h时达到高峰,而经过APC处理后,两者活性高峰提前于缺氧后3h时出现,且峰值显著降低。上述结果提示,预处理过程中ERK1／2的激活可能是缺氧预处理延迟保护机制中细胞信号传递的重要环节,预处理阶段p38α/β的活化不参与APC诱导的延迟保护信号传递过程,p38的过度激活可能是缺氧／复氧损伤过程中的一个致损伤参与因素,而预处理抑制随后持续缺氧阶段p38的过度激活可能是其保护机制的一个环节。     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

Sphingosine kinase 1 is involved in dibutyryl cyclic AMP-induced granulocytic differentiation through the upregulation of extracellular signal-regulated kinase, but not p38 MAP kinase, in HL60 cells

The role of sphingosine kinase (SPHK) in the dibutyryl cyclic AMP (dbcAMP)-induced granulocytic differentiation of HL60 cells was investigated. During differentiation, SPHK activity was increased, as were mRNA and protein levels of SPHK1, but not of SPHK2. Pretreatment of HL60 cells with N,N-dimethylsphingosine (DMS), a potent SPHK inhibitor, completely blocked dbcAMP-induced differentiation. The phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK was also increased during dbcAMP-induced differentiation. Pretreatment of HL60 cells with the MEK inhibitor, U0126, but not the p38 MAPK inhibitor, SB203580, completely suppressed dbcAMP-induced ERK1/2 activation and granulocytic differentiation, but did not affect the increase in SPHK activity. DMS inhibited dbcAMP-induced ERK1/2 activation, but had little effect on p38 MAPK activation. DMS had no effect on the dbcAMP-induced membrane translocation of protein kinase C (PKC) isozymes, and PKC inhibitors had no significant effect on ERK activation. The overexpression of wild-type SPHK1, but not dominant negative SPHK1, resulted in high basal levels of ERK1/2 phosphorylation and stimulated granulocytic differentiation in HL60 cells. These data show that SPHK1 participates in the dbcAMP-induced differentiation of HL60 cells by activating the MEK/ERK pathway.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

TNF-induced beta2 integrin activation involves Src kinases and a redox-regulated activation of p38 MAPK

We previously demonstrated that the TNF-alpha-induced inside-out signaling leading to beta(2) integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of beta(2) integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in beta(2) integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to beta(2) integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to beta(2) integrin activation.     

Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H(2)O(2)-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H(2)O(2)-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H(2)O(2)-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H(2)O(2) increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H(2)O(2)-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H(2)O(2)-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.     

Basal cPLA(2) phosphorylation is sufficient for Ca(2+)-induced full activation of cPLA(2) in A549 epithelial cells

The release of [(3)H] arachidonic acid (AA) and its connection with the triggering of the MAP kinase cascade were studied in the human A549 epithelial cell line upon stimulation with thapsigargin. Thapsigargin can increase AA release along with the increase of intracellular calcium concentration, phosphorylation, and activation of extracellular regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)). Both ERK and cPLA(2) phosphorylation in response to thapsigargin were inhibited by PD 98059, a specific inhibitor of MAP kinase kinase of the ERK group (MEK), and EGTA. cPLA(2) phosphorylation was not affected by Ro 31-8220 (an inhibitor of all PKC isoforms) or LY 379196 (a PKCbeta selective inhibitor), while both of them indeed attenuated ERK activation. On the other hand, rottlerin (the selective PKCdelta inhibitor), SB 203580 (the selective p38 MAPK inhibitor), and wortmannin (the PI 3-kinase inhibitor) can affect neither cPLA(2) nor ERK phosphorylation. In A549 cells, PKC activator PMA cannot increase either the basal or thapsigargin-induced (3)H-AA release, while it can induce the phosphorylation of ERK and cPLA(2.) The PMA-induced ERK phosphorylation was inhibited by Ro 31-8220, LY 379196, rottlerin, and PD 98059, but unaffected by SB 203580 and wortmannin. Moreover, the phosphorylation by PMA was non-additive with that of thapsigargin. This implies that intracellular Ca(2+) level is the key factor for induction of cPLA(2) activity and thapsigargin-elicited ERK activation itself is substantially sufficient for cPLA(2) activation upon intracellular Ca(2+) increase.     

Signaling pathways for TNF production induced by human aminoacyl-tRNA synthetase-associating factor,p43

The p43 protein is associated with human macromolecular aminoacyl tRNA synthetase complex and secreted to up-regulate diverse proinflammatory genes including TNF. Here we focused on the p43-induced TNF production and determined its responsible signal pathway. The p43-induced TNF production was mediated by the activation of MAPK family members, ERK and p38 MAPK, and by IkappaB degradation leading to the activation of NFkappaB. We also studied the upstream molecules for ERK and p38 MAPK by using a variety of inhibitors. The inhibitors for protein kinase C (PKC) and phospholipase C (PLC) prevented the p43-induced TNF production. Interestingly, all of the effective drugs inhibited the ERK activity, while the drugs had no effects on p38 MAPK activity and IkappaB degradation. Together, the p43-induced TNF production was controlled by NFkB, p38 MAPK, and ERK that is dependent on the activities of PLC and PKC.     

Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C

The mitogen‐activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal‐regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one‐trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5‐hydroxytryptamine (5‐HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by ‘upstream’ signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5‐HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA‐dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5‐HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA‐AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca²⁺‐dependent PKC activation contributes to ERK phosphorylation, although a PKC‐independent pathway is also involved in 5‐HT‐dependent ERK phosphorylation and activation.     

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A)-AR. α(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited α(1B)-AR- but not α(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.