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.     

Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles

Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.     

Physical and functional interaction between protein kinase C delta and Fyn tyrosine kinase in human platelets

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCdelta and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCdelta on tyrosine and is specific in that other isoforms of PKC, PKCepsilon and lambda, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCdelta does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCdelta and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCdelta are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCdelta activity, did not block translocation of either PKCdelta or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.     

c-Jun NH(2)-terminal kinase pathway in growth-promoting effect of the G protein-coupled receptor cholecystokinin B receptor: a protein kinase C/Src-dependent-mechanism.

The proliferative effects of gastrin on normal and malignant gastrointestinal tissues have been shown to be mediated by a G protein-coupled receptor (GPCR), the cholecystokinin B receptor. The c-Jun NH(2)-terminal kinase (JNK) pathway has been implicated in the regulation of mitogenesis by growth factors or cytokines. However, the contribution of this signaling cascade to the proliferative effects of GPCR remains largely unknown. Here, we show that cholecystokinin B receptor occupancy by gastrin leads to the activation of the JNK pathway. The mechanism involves certain protein kinase C isoforms and Src family kinases other than p60Src. The complex p130Cas/CrkII, known to be involved in JNK activation, is also activated in response to gastrin by a protein kinase C- and Src-dependent mechanism. However, gastrin-induced CrkII and JNK pathways are independent. Using a dominant negative mutant of c-Jun, we blocked the ability of gastrin to induce DNA synthesis, demonstrating a major role of the JNK pathway in the growth-promoting effect of a GPCR agonist.     

Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration.

Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-gamma, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation.     

Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions

Oleic acid (OA) affects assembly of gap junctions in neonatal cardiomyocytes. Adherens junction (AJ) regulates the stability of gap junction integrity; however, the effect of OA on AJ remains largely unexplored. The distribution of N-cadherin and catenins at cell–cell junction was decreased by OA. OA induced activation of protein kinase C(PKC)-α and -? and Src family kinase, and all three kinases were involved in the oleic acid-induced disassembly of the adherens junction, since it was blocked by pretreatment with Gö6976 (a PKCα inhibitor), ?V1–2 (a PKC? inhibitor), or PP2 (a Src family kinase inhibitor). Src family kinase appeared to be the downstream of PKC-α and -?, as blockade of either PKC-α or -? activity prevented the OA-induced activation of Src family kinase. Immunoprecipitation analyses showed that OA activated Fyn and Fer. OA promoted the association of p120 catenin/β-catenin with Fyn and Fer and caused increased tyrosine phosphorylation of p120 catenin and β-catenin, resulting in decreased binding of the former to N-cadherin and of the latter to α-catenin. Pretreatment with PP2 abrogated this OA-induced tyrosine phosphorylation of p120 catenin and β-catenin and restored the association of N-cadherin with p120 catenin and that of β-catenin with α-catenin. In conclusion, these results show that OA activates the PKC-Fyn signaling pathway, leading to the disassembly of the AJ. Therefore, inhibitors of PKC-α/-? and Src family kinase are potential candidates as cardioprotection agents against OA-induced heart injury during ischemia-reperfusion.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.     

Hck is a key regulator of gene expression in alternatively activated human monocytes

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.     

Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases

The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck-/-fgr-/-lyn-/- mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Peroxynitrite modulates the activation of p38 and extracellular regulated kinases in PC12 cells

Although peroxynitrite appears to contribute to neuronal dysfunction in several neurodegenerative disorders, little is known about how peroxynitrite affects cellular signaling processes. This study investigated if peroxynitrite affects the mitogen-activated protein kinases, extracellular-regulated kinases 1 and 2 (ERK1/2) and p38. Exposure of PC12 cells to 500 microM peroxynitrite activated ERK1/2 and p38 within 5 min and this was followed by gradual decreases in activation over the next 25 min. Activation of ERK1/2 by peroxynitrite was mediated by activation of the epidermal growth factor (EGF) receptor in a calcium/calmodulin-dependent kinase II- and src family tyrosine kinase-dependent manner, as it was blocked by the selective EGF receptor inhibitor AG1478, by KN62, an inhibitor of calcium/calmodulin-dependent kinase II, and by PP1, a src family tyrosine kinase inhibitor. Activation of p38 by peroxynitrite was independent of the EGF receptor, required activation of calcium/calmodulin-dependent kinase II and src family tyrosine kinases, and was modulated by nerve growth factor (NGF) in a time-dependent manner. Pretreatment with NGF (2 h) attenuated, whereas cotreatment with NGF potentiated, peroxynitrite-induced activation of p38. Thus, peroxynitrite activates ERK1/2 and p38, activation of EGF receptors, calcium/calmodulin-dependent kinase II, and src family tyrosine kinases participate in these signaling responses to peroxynitrite, and peroxynitrite- and NGF-induced signaling activities converge on p38.     

Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells

The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G(i)-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCbeta activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCbeta. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.     

Interaction of Bruton's tyrosine kinase and protein kinase Ctheta in platelets. Cross-talk between tyrosine and serine/threonine kinases.

The nonreceptor Bruton's tyrosine kinase (Btk) has been previously shown to associate physically and functionally with members of the protein kinase C (PKC) family of serine/threonine kinases in a variety of cell types. Here we show evidence for a novel interaction between Btk and PKCtheta; in platelets activated through the adhesion receptors GP Ib-V-IX and GP VI. Alboaggregin A, a snake venom component capable of activating both receptors in combination, leads to tyrosine phosphorylation of Btk downstream of Src family kinases. Inhibition of Btk by the selective antagonist LFM-A13 causes a reduction in calcium entry, although secretion of 5-hydroxytryptamine is potentiated. Btk is also phosphorylated on threonine residues in a PKC-dependent manner and associates with PKCtheta; upon platelet activation by either alboaggregin A or activation of GP Ib-V-IX alone by von Willebrand factor/ristocetin. PKCtheta; in turn becomes tyrosine-phosphorylated in a manner dependent upon Src family and Btk kinase activity. Inhibition of Btk activity by LFM-A13 leads to enhancement of PKCtheta; activity, whereas nonselective inhibition of PKC activity by bisindolylmaleimide I leads to reduction in Btk activity. We propose a reciprocal feedback interaction between Btk and PKCtheta; in platelets, in which PKCtheta; positively modulates activity of Btk, which in turn feeds back negatively upon PKCtheta;.     

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.