C5a differentially stimulates the ERK1/2 and p38 MAPK phosphorylation through independent signaling pathways to induced chemotactic migration in RAW264.7 macrophages

We elucidate the roles of various protein kinases involved in complement 5a (C5a)-induced cell migration. Results showed that extracellular signal-regulated kinase1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinase (P13K) were necessary for C5a-induced migration, whereas protein kinase C and c-Jun N-terminal kinase (JNK) were nonessential. C5a-induced migration was also suppresses by phospholipase C (PLC) inhibitor U73122 and pertussis toxin (PTX). We found that C5a-induced, time-dependent (1) ERK1/2 phosphorylation was markedly diminished by PTX, U73122, P13K inhibitors wortmannin and LY294002 and ERK1/2 inhibitor PD98059; (2) Akt phosphorylation was also attenuated by the above inhibitors except PD98059; (3) p38 MAPK phosphorylation was only affected by PTX. Furthermore, C5a also stimulated PLCbeta(2) membrane translocation in a time-dependent manner that occurred early prior to Akt phosphorylation and could be abolished only by PTX and U73122. These results suggest that C5a, through the activation of PTX-sensitive G protein, to differentially stimulate ERK1/2 and p38 MAPK phosphorylation and evoke cell migration. That is, ERK1/2 but not p38 MAPK phosphorylation is down stream of P13K/Akt and modulated by PLC. Additionally, beta(2) isoform may be one of the participates in C5a signal and acts more upstream of P13K/Akt.     

Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y1 receptor in a Ca2+ independent manner in differentiated human skeletal muscle cells

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca2+ transients in these cells via P2Y receptors which were not attributable to Ca2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC50 of 21.3 microM. The Ca2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca2+ pool was not involved in ATP triggered Ca2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Substantially, ATP activated IP formation via a P2Y1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP3 receptor mediated Ca2+ transients and Ca2+ insensitive phosphorylation of ERK1/2.     

Fundamental role of nitric oxide in neuritogenesis of PC12h cells

1 We investigated the neuritogenic action of nitric oxide (NO)-generating agents and their mechanisms of action in a subclone of rat pheochromocytoma, PC12h cells. 2 NO donors such as sodium nitroprusside (SNP, 0.05-1 microM), NOR1 (5-100 microM), NOR2 (5-20 microM), NOR3 (5-20 microM), NOR4 (5-100 microM), or S-nitroso-N-acetyl-DL-penicillamine (SNAP, 10-100 microM) significantly induced neurite outgrowth. 3 NOR4-induced neurite outgrowth was accompanied by expression of neurofilament 200 kDa subunit (NF200) protein, an axonal marker, and was significantly inhibited by an NO scavenger, a soluble GC inhibitor, and a PKG inhibitor: 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (carboxy-PTIO, 20-100 microM), 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, 100 microM) and KT5823 (0.2-1 microM), respectively. 4 The intracellular cGMP concentration of cells was markedly increased by treatment with NOR4 (100 microM). 5 A mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059 (10-50 microM), abolished the NOR4-induced neurite outgrowth. In agreement with this observation, NOR4 did phosphorylate extracellular signal-regulated kinase (ERK) 1 and 2, substrates of MAPK kinase. 6 A membrane-permeable cGMP analog, 8-Br-cGMP (1 mM) also induced significant neurite outgrowth. The 8-Br-cGMP-induced neurite outgrowth was almost completely inhibited by both KT5823 (0.5 microM) and PD98059 (50 microM). Moreover, sustained ERK phosphorylation was observed in the 8-Br-cGMP-treated PC12h cells. 7 These results suggest that NO itself has the ability to induce neurite outgrowth and that NO-induced ERK activation involves the NO-cGMP-PKG signaling pathway in PC12h cells.     

Activation of phosphoinositide 3-kinase, protein kinase C, and extracellular signal-regulated kinase is required for oridonin-enhanced phagocytosis of apoptotic bodies in human macrophage-like U937 cells

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity time- dependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB/Akt) and the increased expression of PLCgamma2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNFalpha and anti-IL-1beta antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of ERK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNFalpha or IL-1beta only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and ERK-dependent pathways.     

Activation of osteoblastic functions by a mediator of pain, bradykinin

We investigated the effects of bradykinin (BK) on the production of interleukin (IL)-6 and prostaglandin PGE(2), whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as the signal transduction systems involved, in human osteoblasts (SaM-1 cells). BK receptors B1 (B1R) and B2 (B2R) were expressed in SaM-1 and osteosarcoma (SaOS-2, HOS, and MG-63) cells. Treatment of SaM-1 cells with BK increased the synthesis of both IL-6 and PGE(2) and the increase in both was blocked by HOE140 (B2R antagonist), but not by Des-Arg(9)-[Leu(8)]-BK (B1R antagonist). U-73122, a phospholipase C (PLC) inhibitor, suppressed BK-induced IL-6 and PGE(2) synthesis in SaM-1 cells. In addition, BK caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i), which was inhibited by pretreatment with HOE140 or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) blocker. Furthermore, both SB203580 (an inhibitor of p38 mitogen-activated protein kinase [MAPK]) and PD98059 (an inhibitor of MEK, upstream of ERK) attenuated the BK-induced IL-6 and PGE(2) synthesis. BK treatment resulted in the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK)1/2, and 2-APB could suppress BK-induced phosphorylation of ERK1/2. These findings suggest that BK increased both IL-6 and PGE(2) synthesis in osteoblastic cells via B2R and that PLC, IP(3)-induced [Ca(2+)]i, MEK, and MAPKs were involved in the signal transduction in these cells.     

Cross-talk between angiotensin II and glucagon receptor signaling mediates phosphorylation of mitogen-activated protein kinases ERK 1/2 in rat glomerular mesangial cells

We have recently shown that the pancreatic hormone glucagon-induced phosphorylation of mitogen-activated protein (MAP) kinase ERK 1/2 as well as growth and proliferation of rat glomerular mesangial cells (MCs) via activation of cAMP-dependent protein kinase A (PKA)- and phospholipase C (PLC)/Ca2+-mediated signaling pathways. Since circulating glucagon and tissue angiotensin II (Ang II) levels are inappropriately elevated in type 2 diabetes, we tested the hypothesis that glucagon induces phosphorylation of ERK 1/2 in MCs by interacting with Ang II receptor signaling. Stimulation of MCs by glucagon (10 nM) induced a marked increase in intracellular [Ca2+]i that was abolished by [Des-His1, Glu9]-glucagon (1 microM), a selective glucagon receptor antagonist. Both glucagon and Ang II-induced ERK 1/2 phosphorylation (glucagon: 214+/-14%; Ang II: 174+/-16%; p<0.001 versus control), and these responses were inhibited by the AT1 receptor blocker losartan (glucagon + losartan: 77+/-14%; Ang II + losartan: 84+/-18%; p<0.01 versus glucagon or Ang II) and the AT2 receptor blocker PD 123319 (glucagon + PD: 78+/-7%; Ang II + PD: 87+/-7%; p<0.01 versus glucagon or Ang II). Inhibition of cAMP-dependent PKA with H89 (1 microM) or PLC with U73122 (1 microM) also markedly attenuated the phosphorylation of ERK 1/2 induced by glucagon (glucagon + U73122: 109+/-15%; glucagon + H89: 113+/-16%; p<0.01 versus glucagon) or Ang II (Ang II + U73122: 111+/-13%; Ang II + H89: 86+/-10%; p<0.01 versus Ang II). Wortmannin (1 microM), a selective PI 3-kinase inhibitor, also blocked glucagon- or Ang II-induced ERK 1/2 phosphorylation. These results suggest that AT1 receptor-activated cAMP-dependent PKA, PLC and PI 3-kinase signaling is involved in glucagon-induced MAP kinase ERK 1/2 phosphorylation in MCs. The inhibitory effect of PD 123319 on glucagon-induced ERK 1/2 phosphorylation further suggests that AT2 receptors also play a similar role in this response.     

Carboxymethylated chitosan stimulates proliferation of Schwann cells in vitro via the activation of the ERK and Akt signaling pathways

Proliferation of Schwann cell in the injured peripheral nerve supports axonal regeneration and also is critical for the regeneration of injured nerves. In this publication, carboxymethylated chitosan (CMCS) was studied to determine its capacity (i) to induce proliferation and synthesis of proliferating cell nuclear antigen (PCNA) and (ii) to activate mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) and phosphatidylinositil-3 kinase (PI3K)/Akt signaling pathways in rat Schwann cells. CMCS was found to induce proliferation and PCNA synthesis in Schwann cells in a dose and time dependent manner. CMCS was shown to phosphorylate ERK1/2 and Akt in Schwann cell proliferation. The phosphorylation of ERK1/2 and Akt in Schwann cells was blocked by the MEK inhibitor PD98059 and the PI3K inhibitor wortmannin. In addition, inhibition of the MEK/ERK or the PI3K/Akt signaling pathways significantly decreased the proliferative effects of CMCS in Schwann cells. Overall, the above results indicate that CMCS stimulates proliferation of Schwann cells by activating the intracellular signaling cascades of ERK1/2 and PI3K/Akt.     

Phosphatidylinositol 3-kinase in angiotensin II-induced hypertrophy of vascular smooth muscle cells

Activation of 4E-binding protein 1 (4E-BP1) by growth factors regulates protein synthesis in vascular smooth muscle cells. The interaction between G protein-coupled receptors and activated 4E-BP1 is unclear. We examined phosphadityl inositol (PI) 3-kinase in angiotensin II-induced 4E-BP1 phosphorylation in cultured rat vascular smooth muscle cells. Angiotensin II time and dose dependently stimulated phosphorylation of 4E-BP1 through the angiotensin AT(1) receptor. Pretreatment with wortmannin or 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI 3-kinase inhibitor, suppressed angiotensin II-induced phosphorylation, but a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) kinase-1 (MEK-1) inhibitor, 2'-Amino-3'-methoxyflavone (PD98059), and a p38 MAPK inhibitor, 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), had no effect. With regard to the involvement of mammalian target of rapamycin (mTOR) and p70 S6 kinase, angiotensin II-induced phosphorylation was abolished by pretreatment with rapamycin, but not by tosylphenylalanine chloromethyl ketone or tosyllysine chloromethyl ketone. Ca(2+) was involved, since intracellular Ca(2+) chelation inhibited angiotensin II-induced phosphorylation while a Ca(2+) ionophore, A23187, stimulated phosphorylation. Thus, angiotensin II induces the phosphorylation of 4E-BP1 via the PI 3-kinase/mTOR pathway, but not via ERK or p70 S6 kinase.     

Cryptotanshinone inhibits chemotactic migration in macrophages through negative regulation of the PI3K signaling pathway

BACKGROUND AND PURPOSE: Cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, exhibits anti-inflammatory activity. However, there is no report on the effect of cryptotanshinone on recruitment of leukocytes to inflammatory sites. We therefore assessed the effects of cryptotanshinone on macrophage chemotaxis. EXPERIMENTAL APPROACH: Macrophage migration induced by complement 5a (C5a) or macrophage inflammatory protein-1alpha (MIP-1alpha) was measured in vitro. Intracellular kinase translocation and phosphorylation was assessed by Western blotting. KEY RESULTS: RAW264.7 cell migration towards C5a (1 microg ml(-1)) was significantly inhibited by cryptotanshinone (1, 3, 10 and 30 microM) in a concentration-dependent manner. Primary human macrophages stimulated by C5a were similarly inhibited. C5a-evoked migration in RAW264.7 cells was significantly suppressed by wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor), PD98059 (MEK1/2 inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor), but not by SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), suggesting that activation of PI3K, ERK1/2 and p38 MAPK signal pathways was involved in responses to C5a. Western blotting revealed that cryptotanshinone significantly inhibited PI3K-p110gamma membrane translocation and phosphorylation of Akt (PI3K downstream effector protein) and ERK1/2 induced by C5a. However, neither p38 MAPK nor JNK phosphorylation was affected by cryptotanshinone. Wortmannin significantly attenuated C5a-induced PI3K-p110gamma translocation, Akt and ERK1/2 phosphorylation. PD98059 suppressed ERK1/2 phosphorylation but failed to modify PI3K-p110gamma translocation by C5a stimulation. Furthermore, MIP-1alpha-induced cell migration and PI3K-p110gamma translocation were also inhibited by cryptotanshinone in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.     

Effect of bradykinin on bradykinin-B2 receptor in rat aortic vascular smooth muscle cells and the involved signal transduction pathways

The aim of this paper is to study the effect of bradykinin (BK) on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells (VSMCs). Rat aortic VSMCs were cultured. Cells after 4–6 passages were used in the experiment. VSMCs were incubated with BK, BK + B2 receptor inhibitor (HOE-140), BK + MEK inhibitor (PD98059), BK + mitogen-activated protein kinase (MAPK) inhibitor (apigenin), BK + phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), and BK + Akt inhibitor to evaluate the expression of B2 receptor and phosphorylation of signaling molecules MAPK, Akt, and PI3K by Western blot. (1) BK markedly up-regulated the expression of B2 receptor in VSMC. (2) Apigenin, PD98059, Akt inhibitor, and LY294002 inhibited upregulation of B2 receptor induced by BK. (3) Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK mediation. Results suggest that bradykinin can up-regulate the expression of B2 receptor in VSMCs.     

Desnitro-imidacloprid activates the extracellular signal-regulated kinase cascade via the nicotinic receptor and intracellular calcium mobilization in N1E-115 cells

Imidacloprid (IMI) is the principal neonicotinoid (the only major new class of synthetic insecticides of the past three decades). The excellent safety profile of IMI is not shared with a metabolite, desnitro-IMI (DNIMI), which displays high toxicity to mammals associated with agonist action at the alpha4beta2 nicotinic acetylcholine receptor (nAChR) in brain. This study examines the hypothesis that IMI, DNIMI, and (-)-nicotine activate the extracellular signal-regulated kinase (ERK) cascade via primary interaction with the alpha4beta2 nAChR in mouse neuroblastoma N1E-115 cells. These three nicotinic agonists induce phosphorylation of ERK (p44/p42) in a concentration-dependent manner with an optimal incubation period of 30 min. DNIMI (1 microM)-induced ERK activation is blocked by nicotinic antagonist mecamylamine but not by alpha-bungarotoxin and muscarinic antagonist atropine. This activation is prevented by intracellular Ca(2+) chelator BAPTA-AM but not by removal of external Ca(2+) using EGTA and Ca(2+)-free medium. 2-Aminoethoxy-diphenylborate, a blocker for inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from intracellular stores, inhibits DNIMI-induced ERK activation but a high level of ryanodine (to block ryanodine receptor-mediated Ca(2+) release) does not. The inhibitor U-73122 for phospholipase C (to suppress IP(3) production) prevents ERK activation evoked by DNIMI. Inhibitors for protein kinase C (PKC) (GF109203X) and ERK kinase (PD98059) block this activation whereas an inhibitor (H-89) for cyclic AMP-dependent protein kinase does not. Thus, neonicotinoids activate the ERK cascade triggered by primary action at the alpha4beta2 nAChR with an involvement of intracellular Ca(2+) mobilization possibly mediated by IP(3). It is further suggested that intracellular Ca(2+) activates a sequential pathway from PKC to ERK.     

Activation of extracellular-signal regulated kinase by platelet-derived growth factor is potentiated by phenylephrine in primary cultures of adult rat hepatocytes

We investigated the effects of the α(1)-adrenergic agonist phenylephrine on platelet-derived growth factor (PDGF)-stimulated extracellular signal-regulated kinase (ERK) in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with PDGF (10 ng/ml) and/or α-adrenergic agonist. Phosphorylated ERK isoforms (ERK1 and ERK2) were detected by Western blotting analysis using anti-phospho mitogen-activated protein kinase (MAPK) antibody. PDGF stimulated phosphorylation of ERK2 (42 kDa MAPK) by 2.0-fold within 3-5 min. The PDGF-induced ERK activation was abolished by AG1296 (10(-7) M) or LY294002 (10(-7) M) treatment. MAPK kinase inhibitor, PD98059 (10(-6) M), completely inhibited the PDGF-induced increase in ERK activity. In addition, PDGF-induced mammalian target of rapamycin activity was completely inhibited by AG1296, LY294002, PD98059, or rapamycin treatment. Phenylephrine alone showed no effects on ERKs, but significantly increased phosphorylation of ERK2 induced by PDGF. Moreover, a synthetic analog of diacylglycerol (DG), phorbol 12-myristate 13 acetate (TPA; 10(-7) M), potentiated PDGF-induced ERK2 phosphorylation, while ionomycin had no effect (10(-6) M). The effects of phenylephrine and TPA were antagonized by the phospholipase C (PLC) inhibitor U73122 (10(-7) M), and the protein kinase C (PKC) inhibitor GF109203X (10(-7) M), respectively. Accordingly, PDGF-induced DNA synthesis and proliferation in the presence or absence of phenylephrine or TPA were completely inhibited by AG1296, LY294002, PD98059, or rapamycin treatment. These results suggest that activation of PLC/PKC by phenylephrine represent an indirect positive regulatory mechanism for stimulating ERK induced by 10 ng/ml PDGF.     

Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells

To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.     

p38 mitogen-activated protein kinase contributes to angiotensin II-stimulated migration of rat aortic smooth muscle cells

In this study, we clarified the intracellular mechanism of angiotensin II (Ang II) in promoting migration in rat aortic smooth muscle cells (RASMCs). RASMC migration was measured with the Boyden chamber assay, and the result was confirmed with an aortic sprout assay. The activities of kinases were investigated by western blot analysis. Ang II enhanced RASMC migration, which was chemotaxis directed, and induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and heat shock protein 27 (Hsp27). Ang II-enhanced cell migration was inhibited by SB203580 (a p38 MAPK inhibitor) and piceatannol (a spleen tyrosine kinase inhibitor), but only partially by PD98059 (an ERK inhibitor) and PP2 (a Src inhibitor). The Ang II-stimulated phosphorylation of p38 MAPK and Hsp27 in RASMCs was inhibited by piceatannol and SB203580. The phosphorylation of ERK1/2 stimulated by Ang II was suppressed by PD98059, piceatannol, and PP2. Ang II increased the sprout outgrowth from aortic rings and this response was attenuated by pretreatment with SB203580, PD98059, PP2, or piceatannol. These results suggest that p38 MAPK contributes to the regulation of the Ang II-induced chemotactic migration of vascular smooth muscle cells, which is mediated by Hsp27 phosphorylation.     

Calcineurin contributes to the enhancing effect of adenosine on nerve growth factor-induced neurite outgrowth via the decreased duration of p38 mitogen-activated protein kinase phosphorylation

Adenosine enhances nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. We found that adenosine increases NGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), but decreases the duration of phosphorylation of p38 mitogen-activated protein (MAP) kinase. Therefore, we further examined the involvement of protein phosphatase in these effects of adenosine. FK506, a specific calcineurin inhibitor, inhibited the enhancing effect of adenosine on the NGF-induced neurite outgrowth and increased the duration of p38 MAP kinase phosphorylation without affecting ERK phosphorylation. These results suggest that adenosine decreases the duration of p38 MAP kinase via calcineurin activation, which contributes to the enhancement of NGF-induced neurite outgrowth.     

Sansoninto as evidence-based remedial medicine for depression-like behavior

In vitro screening methods using cultured Neuro2a cells to examine the activation (phosphorylation) of extracellular signal-regulated kinase (ERK) 1/2 and promotion of neurite outgrowth revealed that the extracts of 5 Kampo (Japanese traditional) formulations have potential as medicines for the treatment of behavioral abnormalities. Since sansoninto (SAT) extract exerted stronger effects than the other candidates tested, we investigated whether its oral administration ameliorates the pathologies of some mouse models of behavioral impairments. The results obtained suggested that SAT extract exerted anti-depression-like effects in the forced swim test, which may be mediated by the up-regulated expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. They may also be mediated by the enhanced phosphorylation of the cAMP response element-binding protein (CREB) via the mitogen-activated protein kinase (MAPK) cascade and Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) cascade, a downstream signaling cascade of the N-methyl-d-aspartate (NMDA) receptor. These results indicate that the extract of SAT has potential as a new remedial medicine in the treatment of depression-like behavior.     

Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes

UTP causes interleukin (IL)-6 production via mRNA expression through P2Y(2)/P2Y(4) receptors in human HaCaT keratinocytes. In the present study, we analyzed the mechanism of UTP-induced IL-6 production in these cells. UTP, an agonist of P2Y(2)/P2Y(4) receptors, induced phosphorylation of extracellular signal-regulated kinase (ERK) in a concentration- and time-dependent manner. PD98059, a MEK (mitogen-activated protein kinase kinase) inhibitor, and BAPTA-AM [O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester], an intracellular Ca(2+) chelator, reduced UTP-induced ERK phosphorylation and IL-6 mRNA expression. 2-APB [(2-aminoethoxy)diphenylborane], an inositol 1,4,5-trisphosphate (IP(3))-receptor antagonist, inhibited UTP-induced IL-6 mRNA expression; and the action of A23187, a Ca(2+) ionophore, resembled the action of UTP. In contrast, protein kinase C (PKC) downregulation and pertussis toxin did not affect UTP-induced IL-6 mRNA expression, suggesting that PKC and G(i) are not involved in the UTP-induced IL-6 production. However, AG1478, an epidermal growth factor (EGF)-receptor inhibitor, partially decreased UTP-induced ERK phosphorylation and IL-6 expression. These results suggest that UTP-induced IL-6 production is in part mediated via phosphorylation of ERK through G(q/11)/IP(3)/[Ca(2+)](i) and transactivation of the EGF receptor.     

Fluorofenidone inhibits transforming growth factor-beta1-induced cardiac myofibroblast differentiation

Cardiac myofibroblast differentiation, characterized by expression of alpha-smooth muscle actin (alpha-SMA) and fibrillar collagens, plays a key role in the adverse myocardial remodeling. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD) is a novel pyridone antifibrotic agent, which exerts a strong antifibrotic effect. This study investigated the potential role of AKF-PD in suppressing cardiac myofibroblast conversion induced by transforming growth factor-beta1 (TGF-beta1) and the related mitogen-activated protein kinase (MAPK) signaling pathways in neonatal rat cardiac fibroblasts. The MAPK inhibitors used for pathway determination are c-Jun NH(2)-terminal kinase (JNK) inhibitor II (JNK inhibitor), PD98059 (extracellular signal-regulated kinase inhibitor (ERK) inhibitor) and SB203580 (p38 MAPK inhibitor). Cell proliferation was evaluated by multiply-table tournament (MTT) assay. The expressions of fibronectin (FN), alpha-SMA, phosphorylated ERK1/2 (pERK1/2) and ERK1/2 were investigated using Western blot analysis. AKF-PD remarkablely reduced the proliferative response of cardiac fibroblasts by 27.57% compared with TGF-beta1 stimulated group. AKF-PD, PD98059, and JNK inhibitor II completely prevented TGF-beta1-induced FN protein production. In addition, AKF-PD, PD98059 and SB203580 greatly attenuated alpha-SMA expression induced by TGF-beta1. Furthermore, AKF-PD significantly blocked TGF-beta1-induced phosphorylation of ERK. These results indicate that (1) AKF-PD inhibits TGF-beta1-induced myofibroblast differentiation; (2) the anti-fibrotic effects of AKF-PD are partially mediated by ERK phosphorylation.     

Angiotensin-induced EGF receptor transactivation inhibits insulin signaling in C9 hepatic cells

To investigate the potential interactions between the angiotensin II (Ang II) and insulin signaling systems, regulation of IRS-1 phosphorylation and insulin-induced Akt activation by Ang II were examined in clone 9 (C9) hepatocytes. In these cells, Ang II specifically inhibited activation of insulin-induced Akt Thr³⁰⁸ and its immediate downstream substrate GSK-3α/β in a time-dependent fashion, with ∼70% reduction at 15 min. These inhibitory actions were associated with increased IRS-1 phosphorylation of Ser⁶³⁶/Ser⁶³⁹ that was prevented by selective blockade of EGFR tyrosine kinase activity with AG1478. Previous studies have shown that insulin-induced phosphorylation of IRS-1 on Ser⁶³⁶/Ser⁶³⁹ is mediated mainly by the PI3K/mTOR/S6K-1 sequence. Studies with specific inhibitors of PI3K (wortmannin) and mTOR (rapamycin) revealed that Ang II stimulates IRS-1 phosphorylation of Ser⁶³⁶/Ser⁶³⁹ via the PI3K/mTOR/S6K-1 pathway. Both inhibitors blocked the effect of Ang II on insulin-induced activation of Akt. Studies using the specific MEK inhibitor, PD98059, revealed that ERK1/2 activation also mediates Ang II-induced S6K-1 and IRS-1 phosphorylation, and the impairment of Akt Thr³⁰⁸ and GSK-3α/β phosphorylation. Further studies with selective inhibitors showed that PI3K activation was upstream of ERK, suggesting a new mechanism for Ang II-induced impairment of insulin signaling. These findings indicate that Ang II has a significant role in the development of insulin resistance by a mechanism that involves EGFR transactivation and the PI3K/ERK1/2/mTOR-S6K-1 pathway.