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.     

Catalytic activation of extracellular signal-regulated kinases induces cyclin D1 expression in primary tracheal myocytes

We have demonstrated that extracellular signal-regulated kinases (ERKs) and cyclin D1 are required for bovine tracheal myocyte DNA synthesis. We hypothesized that catalytic activation by ERKs may regulate cyclin D1 expression in these cells. To test this hypothesis, we examined the effects of two inhibitors of ERKs and two reagents that increase the level of activated ERKs on cyclin D1 protein abundance and promoter activity. ERK activity was inhibited either by PD98059, a synthetic inhibitor of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), the upstream signaling intermediate required and sufficient for ERK activation, or by transient transfection with a dominant-negative mutant of MEK1 (MEK-2A). The level of activated ERKs was increased by transient transfection with either a constitutively active form of MEK1 (MEK-2E) or wild-type ERK2 (MAPKwt). Cyclin D1 expression was assessed either by immunoblot or cotransfection with the full-length cyclin D1 promoter subcloned into a luciferase reporter. We found that pretreatment of bovine tracheal myocytes with PD98059 significantly attenuated platelet- derived growth factor (PDGF)-induced cyclin D1 protein abundance. Furthermore, transfection with MEK-2A reduced PDGF-induced cyclin D1 promoter activity. Finally, transfection with either MEK-2E or MAPKwt induced cyclin D1 promoter activity in the absence of growth factor treatment. We conclude that catalytic activation of ERKs regulates cyclin D1 expression in airway smooth-muscle cells.     

Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor

The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 microM PD98059, with maximal effects at 10-100 microM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140trk receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3'-kinase. However, PD98059 completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.     

Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current

Astrocytes swell during neuronal activity as they accumulate K+ to buffer the increase in external K+ released from neurons. This swelling activates volume-sensitive Cl- channels, which are thought to be important in regulatory volume decrease and in the response of the CNS to trauma and excitotoxicity. Mitogen-activated protein (MAP) kinases also are activated by cell volume changes, but their roles in volume regulation are unknown. We have investigated the role of tyrosine and MAP kinases in the activation of volume-activated Cl- channels in cultured astrocytes, using whole-cell patch-clamp recording and Western immunoblots. As previously described, hypo-osmotic solution induced an outwardly rectifying Cl- current, which was blocked by NPPB and SITS. This Cl- current did not depend on [Ca2+ ]i because it was still observed when 20 mM BAPTA was included in the pipette, but it did exhibit rundown when ATP was omitted. Inhibition of tyrosine kinases with genistein or tyrphostin A23 (but not the inactive agents daidzein and tyrphostin A1) blocked the Cl- current. The MAP kinase kinase (MEK) inhibitor PD 98059 reversibly inhibited activation of the Cl- current by hypo-osmotic solution. Western immunoblots showed that genistein or PD 98059 blocked activation of Erk-1 and Erk-2 by hypo-osmotic solution in astrocytes. Therefore, activation of tyrosine and MAP kinases by swelling is a critical step in the opening of volume-sensitive Cl- channels.     

Importance of MEK in neutrophil microbicidal responsiveness

Exposure of neutrophils to inflammatory stimuli such as the chemoattractant FMLP leads to activation of responses including cell motility, the oxidative burst, and secretion of proteolytic enzymes. A signaling cascade involving sequential activation of Raf-1, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) is also rapidly activated after agonist exposure. The temporal relationship between these events suggests that the kinases may be involved in triggering the effector functions, but direct evidence of a causal relationship is lacking. To assess the role of the MEK/ERK pathway in the activation of neutrophil responses, we studied the effects of PD098059, a potent and selective inhibitor of MEK. Preincubation of human neutrophils with 50 microM PD098059 almost completely (>90%) inhibited the FMLP-induced activation of MEK-1 and MEK-2, the isoforms expressed by neutrophils. This dose of PD098059 virtually abrogated chemoattractant-induced tyrosine phosphorylation and activation of ERK-1 and ERK-2, implying that MEKs are the predominant upstream activators of these mitogen-activated protein kinases. Pretreatment of neutrophils with the MEK antagonist inhibited the oxidative burst substantially and phagocytosis only moderately. In addition, PD098059 antagonized the delay of apoptosis induced by exposure to granulocyte-macrophage CSF. However, the effects of PD098059 were selective, as it failed to inhibit other responses, including chemoattractant-induced exocytosis of primary and secondary granules, polymerization of F-actin, chemotaxis, or activation of phospholipase A2. We conclude that MEK and ERK contribute to the activation of the oxidative burst and phagocytosis, and participate in cytokine regulation of apoptosis.     

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.     

Interleukin-8 and chemotactic activity of middle ear effusions

Chemotactic factors, i.e., an N-formyl peptide, C5a, interleukin-8, and leukotriene B4, induced neutrophils to activate mitogen-activated protein (MAP) kinases, as defined by the tyrosine phosphorylation and decrease in electrophoretic mobility of immunodetected 44-, 42-, and 40-kDa proteins. PD 98059, an inhibitor of MAP kinase kinase activation, blocked these changes. The drug likewise blocked neutrophil chemotaxis but did not alter superoxide anion production and paradoxically enhanced degranulation responses to the stimuli. The MAP kinase pathway appears to have a highly selective role in mediating motility but not other cellular responses.     

Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway

Hepatocyte growth factor/scatter factor (HGF/SF) treatment of the Madin-Darby canine kidney epithelial cell line causes scattering of cells grown in monolayer culture and the formation of branching tubules by cells grown in collagen gels. HGF/SF causes prolonged activation of both the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 2 (ERK2) and the phosphoinositide 3-OH kinase (PI 3-kinase) target protein kinase B (PKB)/Akt; inhibition of either the MAP kinase pathway by the MAP kinase/ERK kinase inhibitor PD98059 or the PI 3-kinase pathway by LY294002 blocks HGF/SF induction of scattering, although in morphologically distinct ways. Expression of constitutively activated PI 3-kinase, Ras, or R-Ras will cause scattering, but activated Raf will not, indicating that activation of the MAP kinase pathway is not sufficient for this response. Downstream of PI 3-kinase, activated PKB/Akt and Rac are both unable to induce scattering, implicating a novel pathway. Scattering induced by Ras or PI 3-kinase is sensitive to PD98059, as well as to LY294002, suggesting that basal MAP kinase activity is required, but not sufficient, for the scattering response. Induction of MDCK cell tubulogenesis in collagen gels by HGF/SF is inhibited by PD98059; expression of activated Ras and Raf causes disorganized growth in this system, but activated PI 3-kinase or R-Ras causes branching tubule formation similar to that seen with HGF/SF treatment. These data indicate that multiple signaling pathways acting downstream of Met and Ras are needed for these morphological effects; scattering is induced primarily by the PI 3-kinase pathway, which acts through effectors other than PKB/Akt or Rac and requires at least basal MAP kinase function. Elevated PI 3-kinase activity induces tubulogenesis, but total inhibition and excess activation of the MAP kinase pathway both oppose this effect.     

Human metabotropic glutamate receptor 2 couples to the MAP kinase cascade in chinese hamster ovary cells

We have examined the functional coupling of the human metabotropic glutamate receptor type 2 (mGluR2) with the regulation of the mitogen activated protein kinase (MAP kinase) signal transduction cascade. We demonstrated that L-glutamate stimulation of the human mGluR2 receptor transiently expressed in chinese hamster ovary (CHO) cells leads to a rapid increase in the activity of p42/p44 MAP kinase (also known as the extracellular signal regulated kinases, ERK1 and ERK2). Activation of p42/p44 MAP kinase has been demonstrated in a peptide phosphorylation assay and through the demonstration of a shift in electrophoretic mobility of p42 MAP kinase following activation. In both assay systems L-glutamate stimulation of MAP kinase was inhibited by pertussis toxin and by the MEK (MAP/ERK activating kinase) inhibitor PD 98059. We conclude that L-glutamate stimulation of the mGluR2 receptor in CHO cells mediated regulation of p42/p44 MAP kinase following the activation of pertussis toxin-sensitive G alpha(i) G-proteins via a distinct protein kinase signalling pathway that utilizes MEK.     

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.     

Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.     

A 16-element phased-array head coil

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.     

Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.     

p38 Kinase is a negative regulator of angiotensin II signal transduction in vascular smooth muscle cells: effects on Na+/H+ exchange and ERK1/2

Activation of the Na+/H+ exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II-mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na+/H+ exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na+/H+ exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na+/H+ exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na+/H+ exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II-stimulated Na+/H+ exchange. First, angiotensin II activation of ERK1/2 was increased 1. 5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II-stimulated MAP kinases to phosphorylate a glutathione S-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II-mediated regulation of the Na+/H+ exchanger in VSMC.     

Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells

The molecular mechanism(s) by which tumor cells survive after exposure to ionizing radiation are not fully understood. Exposure of A431 cells to low doses of radiation (1 Gy) caused prolonged activations of the mitogen activated protein (MAP) kinase and stress activated protein (SAP) kinase pathways, and induced p21(Cip-1/WAF1) via a MAP kinase dependent mechanism. In contrast, higher doses of radiation (6 Gy) caused a much weaker activation of the MAP kinase cascade, but a similar degree of SAP kinase cascade activation. In the presence of MAP kinase blockade by the specific MEK1 inhibitor (PD98059) the basal activity of the SAP kinase pathway was enhanced twofold, and the ability of a 1 Gy radiation exposure to activate the SAP kinase pathway was increased approximately sixfold 60 min after irradiation. In the presence of MAP kinase blockade by PD98059 the ability of a single 1 Gy exposure to cause double stranded DNA breaks (TUNEL assay) was enhanced at least threefold over the following 24-48 h. The increase in DNA damage within 48 h was also mirrored by a similar decrease in A431 cell growth as judged by MTT assays over the next 4-8 days following radiation exposure. This report demonstrates that the MAP kinase cascade is a key cytoprotective pathway in A431 human squamous carcinoma cells which is activated in response to clinically used doses of ionizing radiation. Inhibition of this pathway potentiates the ability of low dose radiation exposure to induce cell death in vitro.     

Angiotensin II induces superoxide anion production by mesangial cells

BACKGROUND: The recognized role of angiotensin II (Ang II) in the pathogenesis of the progression of renal disease cannot be solely attributed to Ang II's hemodynamic effects. Indeed, growth stimulating signals driven by Ang II promote mesangial cell (MC) hypertrophy and extracellular matrix production, prominent features of progressive glomerular injury. Superoxide anion (O2-) avidly interacts with nitric oxide, an endogenous vasodilator that inhibits growth factor stimulated MC growth and matrix production. In addition, O2- acting as an intracellular signal is linked to growth related responses such as activation of mitogen activated protein (MAP) kinases. The studies reported herein were designed to investigate: (a) whether Ang II induces MC O2-production and (b) if increased O2- production elicits growth responses in MC. METHODS: MC were exposed to Ang II for 24 or 48 hours. In some experiments, in addition to Ang II, MC were exposed to: diphenylenieodonium (DPI), an inhibitor of the flavin containing NADH/NADPH oxidase; losartan (LOS), an Ang II type 1 (AT1) receptor blocker; PD 98059, a MAP kinases inhibitor; the protein kinase C inhibitors Calphostin C or H-7; and the tyrosine kinase inhibitors, herbymycin A or genistein. RESULTS: Ang II (10(-5) M to 10(-8) M) dose dependently increased MC O2- production up to 125% above control (ED 50 5 x 10(-7) M). LOS as well as DPI, and the PKC inhibitors blocked Ang II stimulated MC O2- production. Ang II dose dependently increased MC 3H-leucine incorporation, and MC protein content, two markers of MC hypertrophy, as well as 3H-thymidine incorporation, a marker of MC hyperplasia. PD98059, a specific inhibitor of MAP kinases prevented Ang II induced MC hypertrophy. Moreover, LOS, DPI, and the PKC inhibitors each independently inhibited MC 3H-leucine incorporation, thereby establishing the specificity of Ang II induced O2- in driving MC hypertrophy. CONCLUSIONS: The current studies demonstrate a previously unrecognized link between Ang II and MC O2- production that may participate in the pathophysiology of progressive renal disease by concomitantly affecting the hemodynamics of the glomerular microcirculation as well as growth related responses of MC to injury.     

Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation

Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In the present study, we examined the signaling pathway underlying IGF-I-mediated neurite outgrowth. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induced concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation was observed within 1 min, maximal phosphorylation of ERKs was not reached for 30 min. Both IGF-IR and ERK phosphorylation were maintained for at least 24 h. Also, the concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation paralleled that of IGF-I-mediated neurite outgrowth. We further examined the role of mitogen-activated protein kinase activation in IGF-I-stimulated neuronal differentiation using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059. Whereas PD98059 had no effect on IGF-IR phosphorylation, PD98059 reduced IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produced a parallel reduction of IGF-I-stimulated neurite outgrowth. Finally, consistent with its ability to block neuronal differentiation, PD98059 inhibited IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation.