ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons

Inhibition of glycogen synthase kinase-3beta (GSK3beta) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3beta in cortical neurons and that both ERK1/2 and PI3K were required for brain-derived neurotrophic factor (BDNF) suppression of GSK3beta activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3beta was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3beta. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3beta was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3beta activity, neither had an effect on GSK3beta phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3beta at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3beta(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3beta-induced apoptosis and that inhibition of GSK3beta may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3beta activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.     

GSK3beta modulates PACAP-induced neuritogenesis in PC12 cells by acting downstream of Rap1 in a caveolae-dependent manner

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic peptide. Here, we show that PACAP recruits Rap1 into caveolin-enriched membrane subdomains in PC12 cells and activates Rap1, nuclear ERK1/2, Elk-1 and CREB in a caveolae-dependent manner. We reveal that GSK3beta is a novel modulator in PACAP signalling. PACAP induces phosphorylation of serine 9 in GSK3beta, which is inhibited by silencing Rap1. Lithium and valproate promote but wortmannin and LY294002 attenuate PACAP-induced phosphorylation of both GSK3beta and ERK1/2, whereas MEK inhibitor PD98059 inhibits nerve growth factor- but not PACAP-induced phosphorylation of GSK3beta, suggesting that GSK3beta operates downstream of Rapt 1 but upstream of ERK1/2 in PACAP signalling. Inhibition or stimulation of GSK3beta results in a 2-fold increase and 6-fold decrease in PACAP-induced neurite outgrowth, respectively. These results reveal an important role of caveolae in the signal transduction of PACAP and that GSK3beta is a critical regulator in PACAP-induced neuronal differentiation.     

The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils

Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.     

Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.     

P2Y receptors activate MAPK/ERK through a pathway involving PI3K/PDK1/PKC-zeta in human vein endothelial cells.

AIMS: In this study we investigated the effects of P2 receptors in the regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in human umbilical vein endothelial cells (HUVEC). METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2/AM, and MAPK/ ERK phosphorylation using Western blot analysis. RESULTS: ATP, 2-meSATP, UTP and UDP cause a rapid and transitory increase in the phosphorylation of MAPK/ERK. In contrast, negligible response was seen for a,Beta-meATP, a general P2X receptors agonist. ATP-dependent activation of MAPK/ERK was prevented by pretreatment of HUVEC with pertussis toxin or MEK inhibitor PD98059. In addition, activation of the MAPK/ ERK cascade by ATP was blocked in cells pretreated with wortmannin and LY294002, but not by U73122, BAPTA or a Ca(2+)-free medium. Furthermore, an inhibition of ATP-dependent MAPK/ERK phosphorylation was observed in HUVEC pretreated with high doses of GF109203X or myristoylated PKC- zeta pseudosubstrate. Similar results were observed when cells were pretreated with the Src tyrosine kinase inhibitor PP2. However, ATP-stimulated MAPK/ERK activation was unaffected in cells pretreated with AG1478 or perillic acid. We also found that ATP stimulates both the phosphorylation of 3- phosphoinositide-dependent protein kinase-1 (PDK1) and its translocation to plasma membrane in a time-dependent manner. CONCLUSION: These observations suggest that the effects mediated by ATP in HUVEC occur via PTX-sensitive G-protein-coupled P2Y receptors through PI3K-dependent mechanisms, in which PDK1 and PKC-zeta are two key molecules within signal cascade leading to MAPK/ERK activation.     

NMDA neuroprotection against a phosphatidylinositol-3 kinase inhibitor, LY294002 by NR2B-mediated suppression of glycogen synthase kinase-3beta-induced apoptosis

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 microm protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 microm suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3beta (GSK3beta). GSK3beta activation by LY294002 was associated with decreased levels of inhibitory GSK3beta phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3beta, suggesting that NMDA suppressed GSK3beta in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3betawt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3beta. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3beta are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA.     

NT3 inhibits FGF2-induced neural progenitor cell proliferation via the PI3K/GSK3 pathway

Neurotrophin 3 (NT3), a member of the neurotrophin family, antagonizes the proliferative effect of fibroblast growth factor 2 (FGF2) on cortical precursors. However, the mechanism by which NT3 inhibits FGF2-induced neural progenitor (NP) cell proliferation is unclear. Here, using an FGF2-dependent rat neurosphere culture system, we found that NT3 inhibits both FGF2-induced neurosphere growth and bromodeoxyuridine (BrdU) incorporation in a dose-dependent manner. U0126, a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, both inhibited FGF2-induced BrdU incorporation, suggesting that the extracellular signal-regulated kinase1/2 (ERK1/2) and PI3K pathways are required for FGF2-induced NP cell proliferation. NT3 significantly inhibited FGF2-induced phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta), a downstream kinase of Akt, whereas phosphorylation of ERK1/2 was unaffected. The inhibitory effect of NT3 on FGF2-induced NP cell proliferation was abolished by LY294002, and treatment with SB216763, a specific GSK3 inhibitor, antagonized the NT3 effect, rescuing both neurosphere growth and BrdU incorporation. Moreover, experiments with anti-NT3 antibody revealed that endogenous NT3 also plays a role in inhibiting FGF2-induced NP cell proliferation, and that anti-NT3 antibody enhanced phospho-Akt and phospho-GSK3beta levels in the presence of FGF2. These findings indicate that FGF2-induced NP cell proliferation is inhibited by NT3 via the PI3K/GSK3 pathway.     

Activation of MAP kinase by muscarinic cholinergic receptors induces cell proliferation and protein synthesis in human breast cancer cells

Carbachol (Cch), a muscarinic acetylcholine receptor (mAChR) agonist, increases intracellular-free Ca(2+) mobilization and induces mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in MCF-7 human breast cancer cells. Pretreatment of cells with the selective phospholipase C (PLC) inhibitor U73122, or incubation of cells in a Ca(2+)-free medium did not alter Cch-stimulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by phorbol 12-myristate acetate (PMA), an activator of protein kinase C (PKC), but Cch-evoked MAPK/ERK activation was unaffected by down-regulation of PKC or by pretreatment of cells with GF109203X, a PKC inhibitor. However, Cch-stimulated MAPK/ERK phosphorylation was completely blocked by myristoylated PKC-zeta pseudosubstrate, a specific inhibitor of PKC-zeta, and high doses of staurosporine. Pretreatment of human breast cancer cells with wortmannin or LY294002, selective inhibitors of phosphoinositide 3-kinase (PI3K), diminished Cch-mediated MAPK/ERK phosphorylation. Similar results were observed when MCF-7 cells were pretreated with genistein, a non-selective inhibitor of tyrosine kinases, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF-7 human breast cancer cells mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen activated kinase kinase. In conclusion, analyses of mAChR downstream effectors reveal that PKC-zeta, PI3K, and Src family of tyrosine kinases, but not intracellular-free Ca(2+) mobilization or conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK activation. In addition, MAPK/ERK are involved in the regulation of growth and proliferation of MCF-7 human breast cancer cells.     

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.     

NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

Neuroprotective properties of ciliary neurotrophic factor for cultured adult rat dorsal root ganglion neurons

We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 mum) and large neurons (soma diameter >/=30 mum), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.     

Ghrelin stimulates angiogenesis via GHSR1a-dependent MEK/ERK and PI3K/Akt signal pathways in rat cardiac microvascular endothelial cells

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), is thought to exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function such as cell proliferation, migration, survival and angiogenesis. However, the effect of ghrelin on angiogenesis and the corresponding mechanisms have not yet been extensively studied in cardiac microvascular endothelial cells (CMECs) isolated from left ventricular myocardium of adult Sprague-Dawley (SD) rats. In our study, we found that ghrelin and GHSR are constitutively expressed in CMECs. Ghrelin significantly increases CMECs proliferation, migration, and in vitro angiogenesis. The ghrelin-induced angiogenic process was accompanied by phosphorylation of ERK and Akt. MEK inhibitor PD98059 abolished ghrelin-induced phosphorylation of ERK, but had no effect on Akt phosphorylation. PI3K inhibitor LY294002 abolished ghrelin-induced phosphorylation of Akt, but had no effect on ERK phosphorylation. Ghrelin-induced angiogenesis was partially blocked by treatment with PD98059 or LY294002. In addition, this angiogenic effect was almost completely inhibited by PD98059+LY294002. Pretreatment with GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced phosphorylation of ERK, Akt and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates CMECs angiogenesis through GHSR1a-mediated MEK/ERK and PI3K/Akt signal pathways, indicating that two pathways are required for full angiogenic activity of ghrelin. This study suggests that ghrelin may play an important role in myocardial angiogenesis.     

Enteric neuroblasts require the phosphatidylinositol 3‐kinase pathway for GDNF‐stimulated proliferation

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial‐derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest‐derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation. We found that GDNF stimulates the phosphorylation of both the PI 3‐kinase downstream substrate Akt and the MAP kinase substrate ERK in cultures of immunoaffinity‐purified embryonic avian enteric crest‐derived cells. The selective PI 3‐kinase inhibitor LY‐294002 blocked GDNF‐stimulated Akt phosphorylation in purified crest cells, and reduced proliferation in cultures of dissociated quail gut. The ERK kinase (MEK) inhibitors PD 98059 and UO126 did not reduce GDNF‐stimulated proliferation, although PD 98059 blocked GDNF‐stimulated phosphorylation of ERK. We conclude that the PI 3‐kinase pathway is necessary for the GDNF‐stimulated proliferation of enteric neuroblasts. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 306–317, 2001     

Insulin-like growth factor-1 effects on bovine retinal endothelial cell glucose transport: role of MAP kinase

In order to maintain normal metabolism, the neuroretina is completely dependent on the constant delivery of glucose across the retinal microvascular endothelial cells comprising the inner blood-retinal barrier. Glucose uptake into these cells is influenced by various stimuli, including hypoxia and growth factors. Recently, insulin-like growth factor-1 (IGF-1) was shown to enhance retinal endothelial glucose transport in a process that is dependent on protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3 kinase). In the current study, the role of mitogen-activated protein kinase (MAP kinase) in regulating IGF-1 effects on retinal endothelial cell glucose transport was investigated in a bovine retinal endothelial cell (BREC) culture model. IGF-1 (25 ng/mL) caused a rapid increase in MAP-kinase activity and ERK phosphorylation. Inhibition of MAP kinase with PD98059 (100 microm) blocked IGF-1 enhancement of 2-deoxyglucose uptake. In order to clarify the relationship between PKC, PI3 kinase and MAP kinase in IGF-1 signaling in retinal endothelial cells, the effects of selective inhibitors of MAP kinase (PD98059), PKC (GF109203X), and PI3 kinase (wortmannin, LY294002) on signal transduction by IGF-1 were studied. Inhibition of MAP kinase abolished IGF-1 stimulation of PKC but had no effect on PI3 kinase activity, whereas inhibition of either PKC and PI3 kinase had no effect on MAP kinase phosphorylation or activity in IGF-1-treated cells. Taken together, these data demonstrate that IGF-1 stimulation of BREC glucose transport requires activation of MAP kinase and that MAP kinase is upstream from PKC but is independent of PI3 kinase in mediating the actions of IGF-1 on retinal endothelial cells.     

ERK1/2 mediates insulin stimulation of Na(+),K(+)-ATPase by phosphorylation of the alpha-subunit in human skeletal muscle cells

Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na(+),K(+)-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive (86)Rb(+) uptake and [(3)H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na(+),K(+)-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 microm PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nm wortmannin) and protein kinase C (10 microm GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na(+),K(+)-ATPase in vitro. In conclusion, insulin stimulates Na(+),K(+)-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.     

Embryonic testis cord formation and mesonephric cell migration requires the phosphotidylinositol 3-kinase signaling pathway

Mesonephric cell migration and seminiferous cord formation are critical processes in embryonic testis development at the time of male sex determination. Extracellular growth factors shown to influence seminiferous cord formation such as neurotropin-3 utilize in part the phosphotidylinositol 3-kinase (PI3K) signal transduction pathway. The current study investigates the hypothesis that the PI3K pathway is critical in seminiferous cord formation and testis development. The role of the PI3K signaling pathway in testicular cord formation was examined using an Embryonic Day 13 organ culture system and a PI3K-specific inhibitor LY294002. The actions of a mitogen-activated protein (MAP) kinase-specific inhibitor PD98059 was also examined. The PI3K inhibitor blocked cord formation or reduced the number of cords in a concentration-dependent manner. The actions of LY294002 were found to have a developmental stage specificity in that cord inhibition was observed in organs from embryos with 16-17 tail somites, while organs from embryos with 19 or more tail somites had no block in cord formation and only a small reduction in cord number. In contrast, the MAP kinase inhibitor PD98059 did not block cord formation and only caused a slight reduction in cord number. Neither PI3K or MAP kinase inhibitor altered apoptotic cell number, suggesting apoptosis was not the reason for the inhibition of cord formation. Embryonic testis cell migration assays showed that the PI3K inhibitor LY294002 blocked mesonephros cell migration into the testis, while the MAP kinase inhibitor had no effect. Observations suggest the interference of cell migration is the cause for the inhibition of cord formation. Western blot analysis confirmed that LY294002 and PD98509 inhibited phosphorylation of Akt and ERK1/ERK2, respectively. Combined observations demonstrate that the PI3K signaling pathway is involved in embryonic testis cord formation and mesonephros cell migration.