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MAPK, cell survival, and apoptosis
The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K that is necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. Granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity; when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. In contrast, Interleukin 3 (IL-3) requires PI3K for survival, and blocking PI3K partially inhibits Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it does not stimulate Bad phosphorylation. Finally, although these data suggest that the MAPK pathway is not required for inhibition of apoptosis, evidence is provided that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. These data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect (Scheid, 1998).
Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal leads to sustained activation of the JNK (Drosophila homolog: JNK/Basket) and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrate that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis (Xia, 1995).
The cellular and signaling mechanisms of angiotensin II (Ang II) type 2 (AT2) receptor-induced apoptosis were examined in PC12W (rat pheochromocytoma cell line) cells that express abundant AT2 receptor but not Ang II type 1 receptor. In these cells, nerve growth factor (NGF) inhibits the internucleosomal DNA fragmentation induced by serum depletion, whereas Ang II antagonizes this NGF cell survival action and induces apoptosis. AT2 receptor activation affects intracellular Bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue) protein levels. Bcl-2 phosphorylation is stimulated by NGF, whereas AT2 receptor activation blocks this NGF effect. Pretreatment with antisense oligonucleotide of mitogen-activated protein (MAP) kinase phosphatase-1 enhances the effects of NGF on MAP kinase activation and Bcl-2 phosphorylation, but attenuates the inhibitory effects of AT2 receptor on MAP kinase, Bcl-2 phosphorylation, and apoptosis. Taken together, these results suggest that MAP kinase plays a critical role in inhibiting apoptosis by phosphorylating Bcl-2. The AT2 receptor inhibits MAP kinase activation, resulting in the inactivation of Bcl-2 and the induction of apoptosis (Horiuchi, 1997).
The regional selectivity and mechanisms underlying the toxicity of the serine/threonine protein phosphatase inhibitor okadaic acid (OA) were investigated in hippocampal slice cultures. Image analysis of propidium iodide-labeled cultures reveals that okadaic acid caused a dose- and time-dependent injury to hippocampal neurons. Pyramidal cells in the CA3 region and granule cells in the dentate gyrus are much more sensitive to okadaic acid than the pyramidal cells in the CA1 region. Electron microscopy reveals ultrastructural changes in the pyramidal cells that are not consistent with an apoptotic process. Treatment with okadaic acid leads to a rapid and sustained tyrosine phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2 [p44/42(mapk)]. The phosphorylation is markedly reduced after treatment of the cultures with the microbial alkaloid K-252a (a nonselective protein kinase inhibitor) or the MAP kinase kinase (MEK1/2) inhibitor PD98059. K-252a and PD98059 also ameliorates the okadaic acid-induced cell death. Inhibitors of protein kinase C, Ca2+/calmodulin-dependent protein kinase II, or tyrosine kinase are ineffective. These results indicate that sustained activation of the MAP kinase pathway, as seen after ischemia, for example, may selectively harm specific subsets of neurons. The susceptibility to MAP kinase activation of the CA3 pyramidal cells and dentate granule cells may provide insight into the observed relationship between cerebral ischemia and dementia in Alzheimer's disease (Runden, 1998).
Oxidative stress is implicated in the nerve cell death that occurs in a variety of neurological disorders, and the loss of protein kinase C (PKC) activity has been coupled to the severity of the damage. The functional relationship between stress, PKC, and cell death is, however, unknown. Using an immortalized hippocampal cell line that is particularly sensitive to oxidative stress, it has been shown that activation of PKC by the phorbol ester tetradecanoylphorbol acetate (TPA) inhibits cell death via the stimulation of a complex protein phosphorylation pathway. TPA treatment leads to the rapid activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), the inactivation of p38 mitogen-activated protein kinase (MAPK), and the downregulation of PKCdelta. Inhibition of either ERK or JNK activation blocks TPA-mediated protection, whereas p38 MAPK and PKCdelta inhibitors block stress-induced nerve cell death. Both p38 MAPK inactivation and JNK activation appear to be downstream of ERK because an agent that blocks ERK activation also blocks the modulation of these other MAP kinase family members by TPA treatment. Thus, the protection from oxidative stress afforded nerve cells by PKC activity requires the combined modulation of multiple enzyme pathways and suggests why the loss of PKC activity contributes to nerve cell death (Maher, 2001).
Olfactory sensory neurons (OSNs) can be sensitized to odorants by repeated exposure, suggesting that an animal's responsiveness to olfactory cues can be enhanced at the initial stage of detection. However, because OSNs undergo a regular cycle of apoptosis and replacement by ostensibly naive, precursor-derived neurons, the advantage of sensitization would be lost in the absence of a mechanism for odorant-enhanced survival of OSNs. Using recombinant adenoviruses in conjunction with surgical and electrophysiological techniques, OSN survival and function were monitored in vivo; odorant exposure selectively rescues populations of OSNs from apoptosis. Odorant stimuli rescue OSNs in a cAMP-dependent manner by activating the MAPK/CREB-dependent transcriptional pathway, possibly as a result of expression of Bcl-2 (Watt, 2004).
Odorant activation of the MAPK pathway and CRE-mediated transcription as well as CREB phosphorylation in OSNs suggest that these neurons may possess some form of neuroplasticity akin to that observed in the central nervous system. Given the role of this pathway in the survival of certain neuronal tissues, the possibility that it could contribute to odorant-stimulated survival of peripheral OSNs was tested. Cotransduction of the olfactory turbinates with AdLacZ and Ad-HA dominant-negative MEK1 K97M (AdDNMEK) has no discernible effect on ß-gal expression, which was robust and uniform. However, inhibition of MAPK signaling by coexpression of this construct attenuates the ability of odorants to stimulate OSN survival in the Obx (olfactory bulbectomy) ipsilateral turbinates, demonstrating a requirement for MAPK activity in odorant-stimulated rescue. Cotransduction of the turbinates with AdLacZ and adenovirus carrying a constitutive-active FLAG-MEK1 R4F (AdCAMEK), without odorant exposure, produced the greatest survival of ß-gal-expressing OSNs, which was paralleled by persisting neuron-specific tubulin (NST) expression. The robust effect of the odorant citralva is likely due to its activation of numerous odorant receptor subtypes in these experiments (Watt, 2004).
In addition, electro-olfactogram analysis experiments have demonstrated that transduction with AdDNMEK blocks the ability of citralva to rescue odorant responsiveness after Obx, and AdCAMEK rescues responsiveness to all odorants tested without rescue odorant exposure. Thus, while in C. elegans the Ras/MAPK pathway appears to play a role in acute odorant detection, the current observations reveal effects that may be more likely to occur via downstream signaling events, perhaps resulting in gene transcription (Watt, 2004).
Activated MAPK has numerous targets in neurons including ion channels, cytoskeletal elements, and multiple transcription factors. To test the possibility that odorant-stimulated MAPK rescues OSNs from apoptosis by activation of its downstream target, the transcription factor CREB, the olfactory turbinates were cotransduced with AdLacZ and either the dominant-negative CREB-M1 (AdDNCREB) or constitutive-active FLAG-VP16-CREB (AdCACREB). Expression of DNCREB blocks the ability of odorants to rescue OSNs from apoptosis, while CACREB rescues a large number of OSNs in the absence of odorant stimuli, as well as robust NST expression. Furthermore, use of these viruses in EOG experiments has revealed that blockade of normal CREB function abolishes the ability of citralva to rescue OE responsiveness to odorants after Obx, while constitutive activation of CREB activity is sufficient to preserve responsiveness in the absence of exogenous rescue odorant (Watt, 2004).
One established mechanism for the survival of CNS neurons following apoptotic stimuli is the MAPK/CREB-regulated transcription of the protooncogene bcl-2. Importantly, OSNs in transgenic mice ectopically overexpressing Bcl-2 are refractory to Obx-induced apoptosis. Repeatedly exposing mice to odorants stimulates expression of Bcl-2 in OSNs; this stimulation was blocked by transduction with either AdDNMEK or AdDNCREB. Both AdCAMEK and AdCACREB induce expression of Bcl-2 in the absence of odorant stimuli. These results provide a mechanistic basis for the ability of odorant stimuli to rescue OSNs and indicate that activity-dependent regulation of Bcl-2 expression in neurons may have a more ubiquitous role in neuronal survival than previously thought (Watt, 2004).
MAPK and integrin signaling
A characteristic feature of certain integrins is their ability to modulate their affinity for extracellular ligands in response to intracellular signals, a process termed "activation" or inside-out signaling". Using a screen for suppressors of integrin activation, the small GTP-binding protein H-Ras, and its effector kinase, Raf-1 were identified as negative regulators of integrin activation. HRas inhibits the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression is not associated with integrin phosphorylation and is independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlates with activation of the ERK MAP kinase pathway. It is possible that the integrin suppression pathway forms a local negative feedback loop for the regulation of integrin function. Ras activation through integrins might occur via the formation of a complex of FAK (Drosophila homolog Focal adhesion kinase-like), GRB-2 and SOS. Cells derived from FAK-deficient mice show enhanced focal adhesion formation, suggesting that these cells may have lost a negative regulator of integrin function. In addition, dominant negative Ras can enhance focal adhesion formation. Further evidence for the existence of a negative feedback loop comes from observations that integrin occupancy or the expression of isolated beta subunit cytoplasmic domains can suppress the function of other integrins. It is likely that a cytoplasmic substrate of a MAP kinase is involved in suppression (Hughes, 1997).
Focal adhesion kinase (FAK) overexpression enhances ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. Focal adhesion kinase associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family protein tyrosine kinases (PTKs) and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in Fibronectin-stimulated signaling events to ERK2, wild type and mutant forms of FAK were expressed in human 293 epithelial cells by transient transfection. FAK overexpression enhances FN-stimulated activation of ERK2 approximately 4-fold. This is blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 is Ras-dependent. FN-stimulated c-Src PTK activity is enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 is blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhances activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK does not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhances p130(Cas) association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of a FAK.c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation (Schlaepfer, 1997).
Using immunoprecipitation and phosphotyrosine detection by Western blotting, intracellular signaling intermediates were analyzed in human primary dermal fibroblasts, either seeded as monolayers on collagen I coats (2D) or seeded within three-dimensional collagen I lattices (3D). Integrin activation in these systems results in a cascade of protein tyrosine phosphorylation, including focal adhesion kinase. Further downstream signaling events have now been shown to include coordinate activation of ERK1 and ERK2 at 2 h after cell-collagen contact, irrespective of 2D or 3D culture conditions. Application of U-73122, an inhibitor of PLC, inhibits collagen lattice contraction in a dose-dependent fashion. Immunoprecipitation identified the isoform PLCgamma-1 as a signaling intermediate in fibroblast-collagen interactions. PLCgamma-1 becomes phosphorylated within 10 min after culture initiation and declines after 2 h. So far, no qualitative differences in signaling intermediates between 2D and 3D cultures have been identified (Langholz 1997).
There are contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation. Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. The mechanisms by which changes in integrin alpha subunit ratios regulate this decision are addressed. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway, whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (1) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (2) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (3) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase (Sastry, 1999).
The role of integrins in leukocyte apoptosis is unclear: some studies suggest enhancement, others inhibition. ß2-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alphaMß2 delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alphaMß2 results in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins does not show this proapoptotic effect and continues to inhibit apoptosis. This discrepancy can be attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alphaMß2 is capable of stimulating the kinases ERK and Akt. Activated alphaMß2 stimulates Akt, but not ERK. When proapoptotic stimuli are combined with either alphaMß2 clustering or activation, Akt activity is blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alphaMß2 continues to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, ß2-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes (Whitlock, 2000).
In epithelial cell movements, which occur during wound healing or embryonic morphogenesis, sheets of cells move together as a unit. Molecular mechanisms that regulate this sheet movement have been largely unknown, although cell locomotion or movement mechanisms for individual cells, such as for fibroblastic cells, have been extensively studied. During wound healing, sheets of MDCK epithelial cells migrate coordinately as a unit, and wound-induced activation of ERK MAP kinase (ERK1/2) propagates in cell sheets in accordance with the cell sheet movement. Inhibition of ERK1/2 activation by specific MEK inhibitors or by expressing dominant-negative ERK2 results in marked inhibition of the sheet movement during wound healing, and inhibition of the cell sheet movement by disrupting actin cytoskeleton suppresses propagation of ERK1/2 activation. These results indicate that cell movement and ERK1/2 activation form a positive feedback loop, which facilitates cell sheet migration. Moreover, Src family kinase inhibitors suppress both cell migration and propagation of ERK1/2 activation, suggesting that Src family kinase may participate in this feedback loop. Interestingly, neither cell sheet migration as a unit nor migration-dependent propagation of ERK1/2 activation occurs during wound healing in fibroblastic 3Y1 cells. Thus, these results identify specific requirements of ERK1/2 MAP kinase for epithelial cell sheet movement (Matsubayashi, 2004).
ERK1/2 was found to be activated in a remarkable 'two wave' fashion after wounding: a rapid and transient activation (the 'first wave') and a slow and sustained activation that propagates from the wound margin to the submarginal cells (the 'second wave'). Both the cell sheet migration and the propagation of the second wave started immediately after wounding and continued at almost constant rates. During the propagation of the second wave, there was a remarkable correlation between ERK1/2 activation and cell motility: those cells that had higher concentrations of phosphorylated ERK1/2 showed a higher motility, which reflected in an elongated cell shape. Moreover, once the opposing edges of the wound confronted one another, ERK1/2 was inactivated around the closing edges of the wound, while ERK1/2 was still activated around the open wound. Taken together, these results suggest that ERK1/2 is activated in those cells in a migratory phase (Matsubayashi, 2004).
Two types of inhibitors were found to specifically inhibit the second wave of ERK1/2 activation: one is Src family kinase inhibitors (PP1 and PP2) and the other actin inhibitors (cytochalasin D and latrunculin B). Src family kinase inhibitors PP1 and PP2 inhibited almost completely the second wave of ERK activation but not the first wave of activation, and they blocked the cell sheet movement during wound healing almost completely. This result also supports the idea that the second wave of ERK1/2 activation is important for cell sheet movement. A recent report has shown that during HGF-induced spreading of split cells, Src phosphorylation of paxillin induces activation of ERK1/2 at focal complexes. Thus, Src family kinase may lie upstream of ERK1/2 activation in the MDCK cell system. Two actin inhibitors, cytochalasin D and latrunculin B, which completely blocked cell migration and cell sheet movement, also inhibited specifically the second wave of ERK1/2 activation. In contrast, microtubule inhibitors nocodazole or taxol, which did not block cell sheet movement, did not inhibit ERK1/2 activation. These results suggest the existence of a positive feedback loop between the cell sheet movement and the second wave of ERK1/2 activation. Recent studies have demonstrated that mechanical stretch is able to induce ERK1/2 activation. It is speculated that mechanical tension, which is induced by neighboring cell movement, leads to activation of ERK1/2, which in turn facilitates cell movement. The propagation of the second wave of ERK1/2 activation thus may occur through this positive feedback loop. Src family kinase may participate in this loop. In summary, the results identify a novel feedback loop, consisting of the cell movement and the propagation of ERK1/2 activation, that regulates cell sheet migration (Matsubayashi, 2004).
Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocks NGF- and TPA-induced tyrosine phosphorylation 42 and 44 kd MAPK proteins. MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, is also regulated by Ras. Ras is not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-gamma 1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs (Thomas, 1992).
In cultured rat PC12 cells, ERK1 and ERK2 are activated by nerve growth factor (NGF), which also induces rapid association between ERK1 and the high affinity gp140prototrk tyrosine kinase NGF receptor. ERKs are associated with the low affinity NGF receptor, p75. ERK1 and, to a lesser extent, ERK2 are found to be constitutively associated with p75. NGF does not modulate the total amount of ERK proteins coimmunoprecipitated with p75 but does markedly stimulate the level of p75-associated ERK catalytic activity. NGF treatment also enhances the tyrosine phosphorylation of a p75-associated species that co-migrates with ERK1 in Western blots. Finally, K-252a, a compound that specifically inhibits activation by NGF of gp140prototrk, abolishes the latter effect. These findings indicate that NGF, via activation of gp140prototrk, leads to association of enzymatically active ERKs with p75 and raise the possibility that this interaction may play a role in the NGF mechanism of action (Volente, 1993).
Plasmalopsychosine, a characteristic fatty aldehyde conjugate of beta-galactosylsphingosine (psychosine) found in brain white matter, enhances p140trk (Trk A) phosphorylation and mitogen-activated protein kinase (MAPK) activity and as a consequence induces neurite outgrowth in PC12 cells. The effect of plasmalopsychosine on neurite outgrowth and its prolonged activation of MAPK is similar to that of nerve growth factor (NGF), and the effect is specific to neuronal cells. Plasmalopsychosine is not capable of competing with high affinity binding of NGF to Trk A, indicating that plasmalopsychosine and NGF differ in terms of Trk A-activating mechanism. Tyrosine kinase inhibitors K-252a and staurosporine, known to inhibit the neurotrophic effect of NGF, also inhibit these effects of plasmalopsychosine, suggesting that plasmalopsychosine and NGF share a common signaling cascade. Plasmalopsychosine prevents apoptosis of PC12 cells caused by serum deprivation, indicating that it has "neurotrophic factor-like" activity. Taken together, these findings indicate that plasmalopsychosine plays an important role in development and maintenance of the vertebrate nervous system (Sakakura, 1996).
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