Kinetics of ERK1/2 activation determine sensitivity of acute myeloid leukaemia cells to the induction of apoptosis by the novel small molecule ingenol 3-angelate (PEP005)

The novel small molecule ingenol 3-angelate (PEP005) has been shown previously to induce apoptosis in leukaemic cell lines and primary AML cells, an effect that requires the expression of protein kinase C-delta (PKCδ). Here we have investigated signalling events downstream of PKCδ that determine sensitivity of AML cells to PEP005. We show that activation of ERK1/2 MAP kinase occurred in both sensitive and resistant cells and that induction of apoptosis required sustained signalling through the ERK1/2 pathway. Inhibition of ERK1/2 signalling using the MEK inhibitor PD98059 inhibited PEP005-induced apoptosis and activation of ERK1/2 was shown to occur downstream of PKC activation. The data show that PEP005-induced apoptosis is both PKC and ERK1/2 dependent and indicate that chronic activation of ERK1/2 in leukaemic cells delivers a pro-apoptotic rather than a proliferative or survival signal.     

Non-steroidal anti-inflammatory drugs inhibit nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes independent of cyclooxygenase activity

Nitric oxide (NO) causes apoptosis and dedifferentiation of articular chondrocytes by the modulation of extracellular signal-regulated kinase (ERK), p38 kinase, and protein kinase C (PKC) alpha and -zeta. In this study, we investigated the effects and mechanisms of non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, ketoprofen, ibuprofen, sulindac sulfide, and flurbiprofen, in NO-induced apoptosis and dedifferentiation of articular chondrocytes. We found that all of the examined NSAIDs inhibited apoptosis and dedifferentiation. NO production in chondrocytes caused activation of ERK-1/2 and p38 kinase, which oppositely regulate apoptosis and dedifferentiation. NO production also caused inhibition of PKCalpha and -zeta independent of and dependent on, respectively, p38 kinase, which is required for apoptosis and dedifferentiation. Among the signaling molecules modulated by NO, NSAIDs blocked NO-induced activation of p38 kinase, potentiated ERK activation, and blocked inhibition of PKCalpha and -zeta. NSAIDs also inhibited some of the apoptotic signaling that is downstream of p38 kinase and PKC, such as NFkappaB activation, p53 accumulation, and caspase-3 activation. The inhibitory effects of NSAIDs on apoptosis and dedifferentiation were independent of the inhibition of cyclooxygenase (COX)-2 and prostaglandin E(2) (PGE(2)) production, as evidenced by the observation that specific inhibition of COX-2 activity and PGE(2) production or exogenous PGE(2) did not affect NO-induced apoptosis and dedifferentiation. Taken together, our results indicate that NSAIDs block NO-induced apoptosis and dedifferentiation of articular chondrocytes by the modulation of ERK, p38 kinase, and PKCalpha and -zeta in a manner independent of their ability to inhibit COX-2 and PGE(2) production.     

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.     

High-density lipoprotein induces cyclooxygenase-2 expression and prostaglandin I-2 release in endothelial cells through sphingosine kinase-2

High-density lipoprotein (HDL) has a significant cardioprotective effects. HDL induces cyclooxygenase-2 (COX-2) expression and prostacyclin I-2 (PGI-2) release in vascular endothelial cells, which contributes to its anti-atherogenic effects. However, the underlying mechanisms are not fully understood. In the present study, we observed that HDL-stimulated COX-2 expression and PGI-2 production in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. These effects triggered by HDL were inhibited by pertussis toxin (PTX), protein kinase C (PKC) inhibitor GF109203X, and ERK inhibitor PD98059, suggesting that Gαi/Gαo-coupled GPCR, PKC, and ERK pathways are involved in HDL-induced COX-2/PGI-2 activation. More importantly, we found that silencing of sphingosine kinase 2 (SphK-2) also blocked HDL-induced COX-2/PGI-2 activation. In addition, HDL-activated SphK-2 phosphorylation accompanied by increased S1P level in the nucleus. Our ChIP data demonstrated that SphK-2 is associated with CREB at the COX-2 promoter region. Collectively, these results indicate that HDL induces COX-2 expression and PGI-2 release in endothelial cells through activation of PKC, ERK1/2, and SphK-2 pathways. These findings implicate a novel mechanism underlying anti-atherothrombotic effects of HDL.     

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.     

Activation of ERK during DNA damage-induced apoptosis involves protein kinase Cdelta

We have previously shown that protein kinase C (PKC) acts upstream of caspases to regulate cisplatin-induced apoptosis. Since extracellular signal-regulated kinases (ERKs) have also been implicated in DNA damage-induced apoptosis, we have examined if ERK signaling pathway acts downstream of PKC in the regulation of cisplatin-induced apoptosis. PKC activator PDBu induced ERK1/2 phosphorylation which was inhibited by general PKC inhibitor bisindolylmaleimide and G? 6983 as well as the MEK inhibitor U0126 but not by the PKCdelta inhibitor rottlerin. Cisplatin caused a concentration-dependent activation of ERK1/2 in HeLa cells. The level of ERK2 was decreased in HeLa cells that acquired resistance to cisplatin (HeLa/CP). The MEK inhibitor U0126 inhibited cisplatin-induced ERK activation and attenuated cisplatin-induced cell death. Inhibition of PKCdelta by rottlerin or depletion of PKCdelta by siRNA inhibited cisplatin-induced ERK activation. These results suggest that cisplatin-induced DNA damage results in activation of ERK1/2 via PKCdelta.     

JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes

Interleukin (IL)-6 decreases cardiac contractility via a nitric oxide (NO)-dependent pathway. However, mechanisms underlying IL-6-induced NO production remain unclear. JAK2/STAT3 and ERK1/2 are two well known signaling pathways activated by IL-6 in non-cardiac cells. However, these IL-6-activated pathways have not been identified in adult cardiac myocytes. In this study, we identified activation of these two pathways during IL-6 stimulation and examined their roles in IL-6-induced NO production and decrease in contractility of adult ventricular myocytes. IL-6 increased phosphorylation of STAT3 (at Tyr(705)) and ERK1/2 (at Tyr(204)) within 5 min that peaked at 15-30 min and returned to basal levels at 2 h. Phosphorylation of STAT3 was blocked by genistein, a protein tyrosine kinase inhibitor, and AG490, a JAK2 inhibitor, but not PD98059, an ERK1/2 kinase inhibitor. The phosphorylation of ERK1/2 was blocked by PD98059 and genistein but not AG490. Furthermore, IL-6 enhanced de novo synthesis of iNOS protein, increased NO production, and decreased cardiac contractility after 2 h of incubation. These effects were blocked by genistein and AG490 but not PD98059. We conclude that IL-6 activated independently the JAK2/STAT3 and ERK1/2 pathways, but only JAK2/STAT3 signaling mediated the NO-associated decrease in contractility.     

Integrated pharmacological preconditioning and memory of cardioprotection: role of protein kinase C and phosphatidylinositol 3-kinase

Although protein kinase C (PKC) and phosphatidylinositol 3 (PI3)-kinase are implicated in cardioprotective signal transduction mediated by ischemic preconditioning, their role in pharmacological preconditioning (PPC) has not been determined. Cultured neonatal rat cardiomyocytes (CMCs) were subjected to simulated ischemia for 2 h followed by 15 min of reoxygenation. PPC of CMCs consisted of administration of 50 microM adenosine, 50 microM diazoxide, and 50 microM S-nitroso-N-acetylpenicillamine (SNAP), each alone or in combination, for 15 min followed by 30 min of washout before simulated ischemia. Although PKC-epsilon and PI3-kinase were significantly activated during treatment with adenosine, activation of these kinases dissipated after washout. In contrast, PPC combined with adenosine, diazoxide, and SNAP elicited sustained activation of PKC-epsilon and PI-3 kinase after washout. The combined-PPC, but not the single-PPC, protocol conferred antiapoptotic and antinecrotic effects after reoxygenation. The PKC inhibitor chelerythrine (5 microM) or the PI3-kinase inhibitor LY-294002 (10 microM) given during the washout period partially blocked the activation of PKC-epsilon and PI3-kinase mediated by the combined-PPC protocol, whereas combined addition of chelerythrine and LY-294002 completely inhibited activation of PKC-epsilon and PI3-kinase. Chelerythrine or LY-294002 partially blocked antiapoptotic and antinecrotic effects mediated by the combined-PPC protocol, whereas combined addition of chelerythrine and LY-294002 completely abrogated antiapoptotic and antinecrotic effects. These results suggest that the combined-PPC protocol confers cardioprotective memory through sustained and interdependent activation of PKC and PI3-kinase.     

Chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells in a PKC-independent manner

BackgroundChelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells.MethodsIn this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line.ResultsWe found that 10 μM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular Ca²⁺ levels that mediates calpain activation. In addition, we found that chelerythrine induces ERK1/2- and calpain-independent caspase-3 activation that can be prevented by the Ca²⁺ chelator BAPTA-AM.ConclusionsThis is the first report showing that chelerythrine promotes Ca²⁺-dependent calpain activation in neuronal cells, which has consequences for the interpretation of studies using this compound.General significanceChelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works.     

Actin cytoskeletal architecture regulates nitric oxide-induced apoptosis,dedifferentiation, and cyclooxygenase-2 expression in articular chondrocytes via mitogen-activated protein kinase and protein kinase C pathways

Nitric oxide (NO) in articular chondrocytes regulates differentiation, survival, and inflammatory responses by modulating ERK-1 and -2, p38 kinase, and protein kinase C (PKC) alpha and zeta. In this study, we investigated the effects of the actin cytoskeletal architecture on NO-induced dedifferentiation, apoptosis, cyclooxygenase (COX)-2 expression, and prostaglandin E2 production in articular chondrocytes, with a focus on ERK-1/-2, p38 kinase, and PKC signaling. Disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, COX-2 expression, and prostaglandin E2 production in chondrocytes cultured on plastic or during cartilage explants culture. CD treatment did not affect ERK-1/-2 activation but blocked the signaling events necessary for NO-induced dedifferentiation, apoptosis, and COX-2 expression such as activation of p38 kinase and inhibition of PKCalpha and -zeta. CD also suppressed activation of downstream signaling of p38 kinase and PKC, such as NF-kappaB activation, p53 accumulation, and caspase-3 activation, which are necessary for NO-induced apoptosis. NO production in articular chondrocytes caused down-regulation of phosphatidylinositol (PI) 3-kinase and Akt activities. The down-regulation of PI 3-kinase and Akt was blocked by CD treatment, and the CD effects on apoptosis, p38 kinase, and PKCalpha and -zeta were abolished by the inhibition of PI 3-kinase with LY294002. Our results collectively indicate that the actin cytoskeleton mediates NO-induced regulatory effects in chondrocytes by modulating down-regulation of PI 3-kinase and Akt, activation of p38 kinase, and inhibition of PKCalpha and -zeta     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

Mitogen-activated protein kinases regulate COX-2 and mucosal recovery in ischemic-injured porcine ileum

Mitogen-activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli. The three primary MAPK-signaling pathways are the extracellular regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Previous research in our laboratory has shown that COX-2-elaborated prostanoids participate in recovery of mucosal barrier function in ischemic-injured porcine ileum. Because COX-2 expression is regulated in part by MAPKs, we postulated that MAPK pathways would play an integral role in recovery of injured mucosa. Porcine mucosa was subjected to 45 min of ischemia, after which tissues were mounted in Ussing chambers, and transepithelial electrical resistance (TER) was monitored as an index of recovery of barrier function. Treatment of tissues with the p38 MAPK inhibitor SB-203580 (0.1 mM) or the ERK1/2 inhibitor PD-98059 (0.1 mM) abolished recovery. Western blot analysis revealed that SB-203580 inhibited upregulation of COX-2 that was observed in untreated ischemic-injured mucosa, whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER recovery by SB-203580 or PD-98059 was overcome by administration of exogenous prostaglandin E(2) (1 microM). The JNK inhibitor SP-600125 (0.1 mM) significantly increased TER and resulted in COX-2 upregulation. COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways, respectively. Alternatively, ERK1/2 appear to be involved in COX-2-independent reparative events that remain to be defined.     

Protein kinase C δ (PKCδ)-extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade regulates glycogen synthase kinase-3 (GSK-3) inhibition-mediated interleukin-10 (IL-10) expression in lipopolysaccharide (LPS)-induced endotoxemia

Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes, including embryonic development, cell differentiation, survival, and apoptosis. Recently, it was reported that a GSK-3 inhibitor attenuates lipopolysaccharide (LPS)-induced septic shock and regulates the mortality of endotoxemic mice. However, the detailed mechanism of reduced mortality via GSK-3 inhibition is not well defined. Herein, we showed that GSK-3 inhibition induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation under LPS-stressed conditions via protein kinase C δ (PKCδ) activation. Furthermore, PKCδ-induced ERK1/2 activation by the inhibition of GSK-3 provoked the production of interleukin (IL)-10, playing a crucial role in regulating endotoxemia. Using a mitogen-activated protein kinase kinase-1 (MEK-1) and PKCδ inhibitor, we confirmed that GSK-3 inhibition induces PKCδ and subsequent ERK1/2 activation, resulting in increased IL-10 expression under LPS-treated conditions. We verified that septic shock caused by LPS is attenuated by GSK-3 inhibition using a GSK-3 inhibitor. This relieved endotoxemia induced by GSK-3 inhibition was restored in an ERK1/2-dependent manner. Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK1/2 activation via PKCδ relieved LPS-mediated endotoxemia. This finding suggests that IL-10 hyperexpression resulting from GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway for endotoxemia.     

Protein kinase C alpha and zeta regulate nitric oxide-induced NF-kappa B activation that mediates cyclooxygenase-2 expression and apoptosis but not dedifferentiation in articular chondrocytes

Nitric oxide (NO) regulates differentiation, survival, and cyclooxygenase (COX)-2 expression in articular chondrocytes. NO-induced apoptosis and dedifferentiation are mediated by p38 kinase activity and p38 kinase-independent and -dependent inhibition of protein kinase C (PKC)alpha and zeta. Because p38 kinase also activates NF-kappa B, we investigated the functional relationship between PKC and NF-kappa B signaling and the role of NF-kappa B in apoptosis, dedifferentiation, and COX-2 expression. We found that NO-stimulated NF-kappa B activation was inhibited by ectopic PKC alpha and zeta expression, whereas NO-stimulated inhibition of PKC alpha and zeta activity was not affected by NF-kappa B inhibition. Inhibition of NO-induced NF-kappa B activity did not affect inhibition of type II collagen expression but did abrogate COX-2 expression and apoptosis. Taken together, our results indicate that NO-induced inhibition of PKC alpha and zeta activity is required for the NF-kappa B activity that regulates apoptosis and COX-2 expression but not dedifferentiation in articular chondrocytes.     

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.     

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.     

Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA2 by m3 and m2 receptors

This study examined the upstream signaling pathways initiated by muscarinic m2 and m3 receptors that mediate sustained ERK1/2- and p38 MAP kinase-dependent phosphorylation and activation of the 85-kDa cytosolic phospholipase (cPL)A(2) in smooth muscle. The pathway initiated by m2 receptors involved sequential activation of Gbetagamma(i3), phosphatidylinositol (PI)3-kinase, Cdc42, and Rac1, p21-activated kinase (PAK1), p38 mitogen-activated protein (MAP) kinase, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (61 +/- 5 to 72 +/- 4%) by the m2 antagonist methoctramine, Gbeta antibody, pertussis toxin, the PI3-kinase inhibitor LY 294002, PAK1 antibody, the p38 MAP kinase inhibitor SB-203580, and a Cdc42/Rac1 GEF (Vav2) antibody and by coexpression of dominant-negative Cdc42 and Rac1 mutants. The pathway initiated by m3 receptors involved sequential activation of Galpha(q), PLC-beta1, PKC, ERK1/2, and cPLA(2), and phosphorylation of cPLA(2) at Ser(505). cPLA(2) activity was inhibited to the same extent (35 +/- 3 to 41 +/- 5%) by the m3 antagonist 4-diphenylacetoxy-N-methylpiperdine (4-DAMP), the phosphoinositide hydrolysis inhibitor U-73122, the PKC inhibitor bisindolylmaleimide, and the ERK1/2 inhibitor PD 98059. cPLA(2) activity was not affected in cells coexpressing dominant-negative RhoA and PLC-delta1 mutants, implying that PKC was not derived from phosphatidylcholine hydrolysis. The effects of ERK1/2 and p38 MAP kinase on cPLA(2) activity were additive and accounted fully for activation and phosphorylation of cPLA(2).