The mechanism of heat shock activation of ERK mitogen-activated protein kinases in the interleukin 3-dependent ProB cell line BaF3

We have investigated heat shock stimulation of MAPK cascades in an interleukin 3-dependent cell line, BaF3. Following exposure to 42 degrees C, the stress-activated JNK MAPKs were phosphorylated and activated, but p38 MAPKs remained unaffected. Surprisingly, heat shock also activated ERK MAPKs in a potent (>60-fold), delayed (>30 min), and sustained (>/=120 min) manner. These characteristics suggested a novel mechanism of ERK MAPK activation and became the focus of this study. A MEK-specific inhibitor, PD98059, inhibited heat shock ERK MAPK activation by >75%. Surprisingly, a role for Ras in the heat shock response was eliminated by the failure of a dominant-negative Ras(Asn-17) mutant to inhibit ERK MAPK activation and the failure to observe increases in Ras.GTP. Heat shock also failed to stimulate activation of A-, B-, and c-Raf. Instead, a serine/threonine phosphatase inhibitor, okadaic acid, activated ERK MAPK in a similar manner to heat shock. Furthermore, pretreatment with suramin, generally recognized as a broad range inhibitor of growth factor receptors, inhibited both okadaic acid-stimulated and heat shock-stimulated ERK MAPK activity by >40%. Inhibiting ERK MAPK activation during heat shock with PD98059 enhanced losses in cell viability. These results demonstrate Ras- and Raf-independent ERK MAPK activation maintains cell viability following heat shock.     

Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG‐63 cells

Insulin has been proposed to be an anabolic agent in bone, but the mechanisms underlying insulin effects on osteoblast differentiation are still not clear. To explore the mechanisms of action of insulin on osteoblast growth and differentiation, human osteoblastic cell line‐MG‐63 was used and stimulated by insulin in the presence or absence of ERK inhibitor PD98059, PI3‐K inhibitor LY294002, or inhibitor PD98059 + LY294002. The results showed that insulin positively regulated the expression of its receptor. Insulin stimulated the proliferation of MG‐63 cells in a time‐ and dose‐dependent manner and blockade of both MAPK and PI3K pathways could inhibit the cell proliferation. In addition, ALP activity, the secretion of type I collagen, OC gene expression, and mineralized nodule formation were increased in the insulin treated group, whereas these indicators were decreased after treatment with blocking agents. However, treatment with PI3‐K inhibitor LY294002 significantly reversed the down‐regulation of Runx2 expression and treatment with ERK inhibitor PD98059 remarkably decreased up‐regulation of Osx and IGF‐1 expression after insulin treatment. Therefore, the data obtained from this study suggested that insulin promoted osteoblast proliferation and differentiation through MAPK and PI3K pathway in MG‐63 cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Intracellular signalling pathways of okadaic acid leading to mitogenesis in Rat1 fibroblast overexpressing insulin receptors: okadaic acid regulates Shc phosphorylation by mechanisms independent of insulin

Okadaic acid is a powerful inhibitor of serine/threonine protein phosphatases 1 and 2A. Although it is known as a potent tumour promoter, the intracellular mechanism by which okadaic acid mediates its mitogenic effect remains to be clarified. We investigated the effect of okadaic acid on the activation of mitogenesis in Rat1 fibroblasts overexpressing insulin receptors. As previously reported, insulin induced Shc phosphorylation, Shc-Grb2 association, MAP kinase activation, and BrdU incorporation. Okadaic acid also stimulated tyrosine phosphorylation of Shc and its subsequent association with Grb2 in a time- and dose-dependent manner without affecting tyrosine phosphorylation of insulin receptor beta-subunit and IRS. However, to a lesser extent, okadaic acid stimulated MAP kinase activity and BrdU incorporation. Interestingly, preincubation of okadaic acid potentiated insulin stimulation of tyrosine phosphorylation of Shc (213% of control), Shc-Grb2 association (150%), MAP kinase activity (152%), and BrdU incorporation (148%). These results further confirmed the important role of Shc, but not IRS, in cell cycle progression in Rat1 fibroblasts. Furthermore, serine/ threonine phosphorylation appears to be involved in the regulation of Shc tyrosine phosphorylation leading to mitogenesis by mechanisms independent of insulin signalling.     

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.     

Insulin and oxidative stress modulate proliferation of rat ovarian theca-interstitial cells through diverse signal transduction pathways

Insulin and moderate oxidative stress stimulate proliferation of ovarian theca-interstitial cells. The effects of these agents on selected signal transduction pathways were examined. PD98059 (inhibitor of MAP2K1, also known as MEK-1, upstream of extracellular signal-regulated protein kinases MAPK3/1, also known as ERK1/2), wortmannin (inhibitor of PIK3C2A, also known as PI3K), and rapamycin (inhibitor of FRAP1, also known as mTOR, upstream of RPS6KB1) each significantly decreased insulin and oxidative stress-induced proliferation of theca-interstitial cells. The greatest inhibition was observed in the presence of rapamycin; this effect occurred without a significant change in cell viability. Phosphorylation of AKT was stimulated by insulin only, while phosphorylation of MAPK3/1 and RPS6KB1 was increased by insulin and oxidative stress. Insulin-induced and oxidative stress-induced phosphorylation of RPS6KB1 was partly inhibited by wortmannin and partly by PD98059; the greatest inhibition was observed in the presence of a combination of wortmannin plus PD98059. Effects of insulin and oxidative stress on phosphorylation of RPS6KB1 were confirmed by kinase activity assays. These findings indicate that actions of insulin and oxidative stress converge on MAPK3/1 and RPS6KB1. Furthermore, we speculate that activation of RPS6KB1 may be in part induced via the MAPK3/1 pathway.     

Insulin-induced beta-arrestin1 Ser-412 phosphorylation is a mechanism for desensitization of ERK activation by Galphai-coupled receptors

Beta-arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of beta-arrestin1 at position Ser-412 is a regulator of beta-arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in beta-arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated beta-arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with beta-arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, beta-arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased beta-arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by beta-arrestin1-dependent receptors with no effect on beta-adrenergic receptor Gs-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by Galphai-coupled receptors.     

Effects of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, on glucose transport and metabolism in skeletal muscle.

The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.     

Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.     

Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.     

Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases jun N-terminal kinase and p38

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase 1,2 (ERK 1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK 1,2, SAPKβ, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.     

Interaction of the insulin receptor kinase with serine/threonine kinases in vitro

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.     

Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis

Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose-dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl-inositol-3-kinase (PI-3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin-stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI-3K- and MEK-dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI-3K rather than MEK supports insulin-mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473-phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI-3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome-c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin-dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.     

Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.     

Mechanisms of activation of NHE by cell shrinkage and by calyculin A in Ehrlich ascites tumor cells

The Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 were all found to be expressed in Ehrlich ascites tumor cells, as evaluated by Western blotting and confocal microscopy. Under unstimulated conditions, NHE1 was found predominantly in the plasma membrane, NHE3 intracellularly, and NHE2 in both compartments. Osmotic cell shrinkage elicited a rapid intracellular alkalinization, the sensitivity of which to EIPA (IC50 0.19 microM) and HOE 642 (IC50 0.85 microM) indicated that it predominantly reflected activation of NHE1. NHE activation by osmotic shrinkage was inhibited by the protein kinase C inhibitors chelerythrine (IC50 12.5 microM), G? 6850 (5 microM), and G? 6976 (1 microM), and by the p38 MAPK inhibitor SB 203580 (10 microM). Furthermore, hypertonic cell shrinkage elicited a biphasic increase in p38 MAPK phosphorylation, with the first significant increase detectable 2 minutes after the hypertonic challenge. Neither myosin light chain kinase-specific concentrations of ML-7 (IC50 40 microM) nor ERK1/2 inhibition by PD 98059 (50 microM) had any effect on NHE activation. Under isotonic conditions, the serine/threonine protein phosphatase inhibitor calyculin A elicited an EIPA- and HOE 642-inhibitable intracellular alkalinization, indicating NHE1 activation. Similarly, shrinkage-induced NHE activation was potentiated by calyculin A. The calyculin A-induced alkalinization was not associated with an increase in the free, intracellular calcium concentration, but was abolished by chelerythrine. It is concluded that shrinkage-induced NHE activation is dependent on PKC and p38 MAPK, but not on MLCK or ERK1/2. NHE activity under both iso- and hypertonic conditions is increased by inhibition of serine/threonine phosphatases, and this effect appears to be PKC-dependent.     

Adaptive increase of amino acid transport system A requires ERK1/2 activation.

Amino acid starvation markedly stimulates the activity of system A, a widely distributed transport route for neutral amino acids. The involvement of MAPK (mitogen-activated protein kinase) pathways in this adaptive increase of transport activity was studied in cultured human fibroblasts. In these cells, a 3-fold stimulation of system A transport activity required a 6-h amino acid-free incubation. However, a rapid tyrosine phosphorylation of ERK (extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium with amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced after a 15-min amino acid-free incubation and maintained at stimulated values thereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivation. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaffected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the presence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimulation of ERK1/2 was observed. To identify the stimulus that maintains MAPK activation, cell volume was monitored during amino acid-free incubation. It was found that amino acid deprivation caused a progressive cell shrinkage (30% after a 6-h starvation). If proline was added to amino acid-starved, shrunken cells, normal values of cell volume were rapidly restored. However, proline-dependent volume rescue was hampered if cells were pretreated with PD98059. It is concluded that (a) the triggering of adaptive increase of system A activity requires a prolonged activation of ERK1 and 2 and that (b) cell volume changes, caused by the depletion of intracellular amino acid pool, may underlie the activation of MAPKs.     

TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes

Tumor necrosis factor-alpha (TNF-alpha) increases adipocyte lipolysis after 6-12 h of incubation. TNF-alpha has been demonstrated to activate mitogen-activated protein (MAP) kinases including extracellular signal-related kinase (ERK) and N-terminal-c-Jun-kinase (JNK) in different cell types. To determine if the MAP kinases have a role in TNF-alpha-induced lipolysis, 3T3-L1 adipocytes were treated with the cytokine (10 ng/ml), in the presence or absence of PD98059 or U0126 (100 micromoles), specific inhibitors of ERK activity. We demonstrated that U0126 or PD98059 blocked TNF-alpha-induced ERK activity and decreased TNF-alpha-induced lipolysis by 65 or 76% respectively. The peroxisome-proliferator-activated receptor gamma (PPARgamma) agonists, rosiglitazone (ros), and 15-deoxy-Delta-(12,14)- prostaglandin J(2) (PGJ2) have been demonstrated to block TNF-alpha-induced lipolysis. Pretreatment of adipocytes with these agents almost totally blocked TNF-alpha-induced ERK activation and reduced lipolysis by greater than 90%. TNF-alpha also stimulated JNK activity, which was not affected by PD98059 or PPARgamma agonist treatment. The expression of perilipin, previously proposed to contribute to the mechanism of lipolysis, is diminished in response to TNF-alpha treatment. Pretreatment of adipocytes with PD98059 or ros significantly blocked the TNF-alpha-induced reduction of perilipin A protein level as determined by Western analysis. These data suggest that activation of the ERK pathway is an early event in the mechanism of TNF-alpha-induced lipolysis.