Oxidant stress incites spreading of macrophages via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase

Cultured macrophages exhibit spreading in response to external stimuli. It is relevant to in vivo morphologic changes of macrophages during extravasation, migration, and differentiation. The present study was performed to elucidate molecular mechanisms that regulate spreading of macrophages. Redox is a crucial factor that modulates a wide range of cell function. We found that macrophages undergo spreading in response to oxidant stress caused by hydrogen peroxide or an oxidant generating agent menadione. To identify signaling pathways involved, a role of mitogen-activated protein (MAP) kinases was investigated. Western blot analysis showed that treatment of macrophages with menadione rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK1, ERK2) and p38 MAP kinase, but not c-Jun N-terminal kinase (JNK). Pharmacologic inhibition of either ERK or p38 activation blunted the macrophage spreading. Similarly, transfection with dominant-negative mutants of ERKs or a mutant p38 significantly suppressed the oxidant-triggered spreading. ERKs and p38 are known to activate serum response element (SRE) via phosphorylation of the ternary complex factor Elk-1. To further identify downstream events, we focused on a role of SRE. Stimulation of macrophages with menadione induced activation of SRE. Intervention in the SRE activation by a dominant-negative mutant of Elk-1 inhibited the menadione-induced spreading. These results suggest that oxygen radical metabolites, the well-known mediators for tissue injury, incite spreading of macrophages via the MAP kinase-SRE signaling pathways.     

Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms

The p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that are induced in response to lipopolysaccharide, hyperosmolarity, and interleukin 1. p38 MAP kinase appears to play a role in regulating inflammatory responses, including cytokine secretion and apoptosis. Here we show that diverse classes of DNA-damaging agents such as cisplatinum, 1-beta-D-arabinofuranosylcytosine, UV light, ionizing radiation, and methyl methanesulfonate activate p38 MAP kinase. We also demonstrate that cells deficient in c-Abl fail to activate p38 MAP kinase after treatment with cisplatinum and 1-beta-D-arabinofuranosylcytosine but not after exposure to UV and methyl methanesulfonate. Reconstitution of c-Abl in the Abl-/- cells restores that response. Similar results were obtained for induction of the Jun-NH2-kinase/stress-activated protein kinase. These findings indicate that p38 MAP and Jun-NH2-kinase/stress-activated protein kinases are differentially regulated in response to different classes of DNA-damaging agents.     

Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.     

Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases

Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.     

Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells

We have reported that treatment with CdCl2 at 40-100 microM induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 microM) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38(MAPK)), while that in cells treated with a low concentration (60 microM) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 microM cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38(MAPK). By contrast, in 60 microM cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38(MAPK) and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38(MAPK) and ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.     

Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1

Cells respond to environmental stress and proinflammatory cytokines by stimulating the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase cascades. Infection of eukaryotic cells with herpes simplex virus type 1 (HSV-1) resulted in stimulation of both JNK/SAPK and p38 mitogen-activated protein kinase after 3 h of infection, and activation reached a maximum of 4-fold by 9 h post-infection. By using a series of mutant viruses, we showed that the virion transactivator protein VP16 stimulates p38/JNK, whereas no immediate-early, early, or late viral expressed gene is involved. We identified the stress-activated protein kinase kinase 1 as an upstream activator of p38/JNK, and we demonstrated that activation of AP-1 binding proceeded p38/JNK stimulation. During infection, the activated AP-1 consisted mainly of JunB and JunD with a simultaneous decrease in the cellular levels of Jun protein. We suggest that activation of the stress pathways by HSV-1 infection either represents a cascade triggered by the virus to facilitate the lytic cycle or a defense mechanism of the host cell against virus invasion.     

Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete. Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts. Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae. The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.     

Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses

"Stress-regulated" mitogen-activated protein kinases (SR-MAPKs) comprise the stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the agent(s) directly responsible is unclear, reactive oxygen species are generated during ischemia/reperfusion. We have assessed the ability of oxidative stress (as exemplified by H2O2) to activate SR-MAPKs in the perfused heart and compared it with the effect of ischemia/reperfusion. H2O2 activated both SAPKs/JNKs and p38-MAPK. Maximal activation by H2O2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by H2O2 was comparable to that of ischemia and ischemia/reperfusion, activation of the SAPKs/JNKs was less than that of ischemia/reperfusion. As with ischemia/reperfusion, there was minimal activation of the ERK MAPK subfamily by H2O2. MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by H2O2 to a similar extent as with ischemia or ischemia/reperfusion. In all instances, activation of MAPKAPK2 in perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs. Perfusion of hearts at high aortic pressure (20 kilopascals) also activated the SR-MAPKs and MAPKAPK2. Free radical trapping agents (dimethyl sulfoxide and N-t-butyl-alpha-phenyl nitrone) inhibited the activation of SR-MAPKs and MAPKAPK2 by ischemia/reperfusion. These data are consistent with a role for reactive oxygen species in the activation of SR-MAPKs during ischemia/reperfusion.     

Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase

The changes in airway osmolarity have been described to contribute to the production of exercise- induced bronchoconstriction (EIB) and the development of the late-phase response (LPR). The mechanism has been investigated; however, the responsiveness of bronchial epithelial cells (BEC) to hyperosmolarity and the intracellular signals leading to cell activation have not been determined. In this study, we examined the effect of hyperosmolar medium on interleukin-8 (IL-8) expression and the role of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2 terminal kinase ( JNK) in human BEC in this response in order to clarify the intracellular signals regulating IL-8 expression in hyperosmolarity-stimulated BEC. The results showed that hyperosmolarity induced IL-8 expression in a concentration dependent manner, p38 MAP kinase phosphorylation and activation, and JNK activation whether NaCl or mannitol was used as the solute. SB 203580 as the specific p38 MAP kinase inhibitor inhibited hyperosmolarity-induced p38 MAP kinase activation and partially inhibited hyperosmolarity-induced IL-8 expression. These results indicate that p38 MAP kinase, at least in part, regulates hyperosmolarity-induced IL-8 expression in BEC. However, other signals such as JNK are possibly also involved. These results provide new evidence on the mechanism responsible for the development of the LPR induced by EIB, and a strategy for treatment with the specific p38 MAP kinase inhibitor.     

Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis

Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.     

Phosphatidylinositol 3-kinase-gamma activates Bruton's tyrosine kinase in concert with Src family kinases

Bruton's tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-gamma. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-gamma and Src family kinases.     

In vitro enhancement of p38 mitogen-activated protein kinase activity by phosphorylated glia maturation factor

We previously demonstrated that glia maturation factor (GMF), a 17-kDa brain protein, can be phosphorylated in test tube by several protein kinases, and that endogenous GMF is rapidly phosphorylated upon stimulation of astrocytes by phorbol 12-myristate 13-acetate. We further observed that protein kinase A (PKA)-phosphorylated GMF is a potent inhibitor (IC50 = 3 nM) of the ERK1/ERK2 (p44/p42) subfamily of mitogen-activated protein (MAP) kinase. We now report that, by contrast, PKA-phosphorylated GMF strongly enhances the activity of a related but distinct subfamily of MAP kinase, the p38 MAP kinase, showing an increase of 60-fold over baseline and an EC50 of 7 nM. Non-phosphorylated GMF or GMF phosphorylated by other kinases exhibits only minimal effect. The intracellular interaction of PKA, GMF, and p38 is supported by the phosphorylation of GMF upon cellular stimulation by forskolin (blocked by PKA inhibitor) and by the co-immunoprecipitation of p38 with GMF from cell lysates. Withdrawal of nerve growth factor from PC12 leads to increased GMF phosphorylation with a time course similar to that reported for p38 activation. The results correlate well with a previous report that ERK and p38 carry out opposing functions and implicate GMF as a regulator of major cellular events.     

Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase

p38 is a member of the mitogen-activated protein (MAP) kinase superfamily activated by stress signals and implicated in cellular processes involving inflammation and apoptosis. Unlike the extracellular signal-regulated kinases (p42 and p44 MAP kinases), which are stimulated by insulin in many cell types, p38 activity is inhibited by insulin in postmitotic fetal neurons for which insulin is a potent survival factor (Heidenreich, K. A., and Kummer, J. L. (1996) J. Biol. Chem. 271, 9891-9894). These data suggested that insulin's effects on neuronal survival are mediated by inhibition of a p38-mediated apoptotic pathway. To better understand the relationship between p38 activity and cell survival, we induced apoptosis in two cell lines and examined the ability of insulin or a specific p38 inhibitor (a pyridinyl imidazole compound PD169316) to block p38 activity and cell death. In Rat-1 fibroblasts grown in the presence of serum, p38 activity was undetectable by immune complex assays, and the number of apoptotic cells was very low (<0.5%). After the removal of serum for 16 h, p38 activity was markedly elevated, and apoptosis increased by 14-15-fold. Insulin (50 ng/ml) inhibited p38 activity by approximately 70% and blocked apoptosis by at least 80%. PD169316 also blocked p38 enzyme activity and apoptosis by approximately 80%. Similar results were obtained in differentiated PC12 cells that were deprived of nerve growth factor (NGF) for 16 h. In the presence of NGF, p38 activity and the number of apoptotic cells was very low (approximately 1.0%). After NGF withdrawal, p38 activity was selectively elevated and apoptosis increased to 15%. Both insulin and PD169316 markedly blocked the increase in p38 activity and apoptosis. The MAP kinase kinase inhibitor, PD98059, had no effect on apoptosis in Rat-1 fibroblasts and only partially blocked apoptosis in PC12 cells. PD98059 did not influence insulin's ability to block apoptosis, indicating that the extracellular signal-regulated kinase pathway does not mediate insulin's survival effects. These data further support the role of p38 in cellular apoptosis and support the hypothesis that insulin promotes cell survival, at least in part, by inhibiting the p38 pathway.     

Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.