Chlamydial Antiapoptotic Activity Involves Activation of the Raf/MEK/ERK Survival Pathway

Chlamydiae are obligate intracellular bacteria that cause variety of human diseases. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by many different apoptotic stimuli. The inhibition of apoptosis is thought to be an important immune escape mechanism allowing chlamydiae to productively complete their obligate intracellular growth cycle. Infection with chlamydiae can activate the Raf/MEK/ERK pathway. Because the survival pathway can modulate apoptosis, we used MEK-specific inhibitor U0126 and Raf-specific inhibitor GW5074 to examine the role of Raf/MEK/ERK pathway in chlamydial antiapoptotic activity. Apoptosis was induced by staurosporine (STS) and detected by morphology, DNA fragmentation, caspase-3 activation, and poly (ADP-ribose) polymerase cleavage. Inhibition of the pathway sensitized Chlamydia-infected cells to STS-mediated cell apoptosis. The data indicate that chlamydial antiapoptotic activity involves activation of the Raf/MEK/ERK survival pathway.     

PAK1 is a novel MEK-independent raf target controlling expression of the IAP survivin in M-CSF-mediated osteoclast survival

As activation of the Ras/Raf/MEK/ERK pathway is a critical component of M-CSF-promoted osteoclast survival, determining specific mechanism by which M-CSF activates this signal transduction pathway is paramount towards advancing treatment of pathological conditions resulting in increased bone turnover. The p21 activated kinase PAK1 modulates activation of the Raf/MEK/ERK pathway by either directly activating Raf or priming MEK for activation by Raf. Therefore a role for PAK1 in M-CSF-mediated activation of the MEK/ERK pathway controlling osteoclast survival was assessed. Here we show that PAK1 is activated by M-CSF in a Ras-dependent mechanism that promotes osteoclast survival. Surprisingly, PAK1 did not modulate Raf activation or Raf-mediated MEK activation. M-CSF mediated activation of Raf was required for PAK1 activation and osteoclast survival promoted by PAK1. This survival response was MEK-independent as expression of constitutively active MEK did not rescue osteoclasts from apoptosis induced by blocking PAK1 function. Functionally, PAK1 promoted osteoclast survival by modulating expression of the IAP family member Survivin. M-CSF therefore functions to promote PAK1 activation as a novel MEK-independent Raf target to control Survivin-mediated osteoclast survival.     

Neurotrophins protect against cytosine arabinoside-induced apoptosis of immature rat cerebellar neurons

Neurotrophin-induced neuroprotection against apoptosis was investigated using immature cultured cerebellar granule cells (CGC) from newborn rat pups. Apoptotic cell death induced by treatment with cytosine arabinoside (AraC) was confirmed by DNA fragmentation and quantified by cell survival assays. AraC was most effective in inducing apoptosis when added to CGC on the day of culture preparation, while less or no effect was observed when added at 24 or 48h after plating, respectively. Pretreatment of CGC cultures for 24h with brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), but not neurotrophin-3 (NT-3), robustly protected against AraC neurotoxicity. K252a, an inhibitor of the tropomyosin-related kinase (Trk) tyrosine kinase receptor family which showed no toxicity by itself, blocked BDNF protection of AraC-induced apoptosis in a concentration-dependent manner. Neither protein kinase C activation nor inhibition mimicked or affected BDNF protection against AraC neurotoxicity. BDNF, but not NT-3, treatment of immature CGC caused a marked, but transient activation of Akt through phosphatidylinositol (PI) 3-kinase. The neuroprotective effects of BDNF were suppressed by pretreatment with LY 294002 (a PI 3-kinase inhibitor). BDNF neuroprotection was also preceded by activation of mitogen activated protein kinase (MAPK) and suppressed by two MAPK/ERK (MEK)-selective inhibitors, PD 98059 and U-0126. Moreover, inhibitors of PI 3-kinase and MEK potentiated AraC-induced neurotoxicity. These results show that neurotrophins protect against AraC-induced apoptosis, at least in part, through TrkB-mediated activation of the PI 3-kinase/Akt and MEK signaling pathways.     

Artemisinin attenuated ischemic stroke induced cell apoptosis through activation of ERK1/2/CREB/BCL-2 signaling pathway in vitro and in vivo

Ischemic stroke is characterized by the presence of both brain ischemic and reperfusion-induced injuries in the brain, leading to neuronal dysfunction and death. Artemisinin, an FDA-approved antimalarial drug, has been reported to have neuroprotective properties. However, the effect of artemisinin on ischemic stroke is not known. In the present study, we investigated the effect of artemisinin on ischemic stroke using an oxygen-glucose deprivation/reperfusion (OGD/RP) cellular model and a mouse middle cerebral artery occlusion (MCAO) animal model and examined the underlying mechanisms. The obtained results revealed that a subclinical antimalarial concentration of artemisinin increased cell viability and decreased LDH release and cell apoptosis. Artemisinin also attenuated the production of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (Δψm). Importantly, artemisinin attenuated the infarction volume and the brain water content in the MCAO animal model. Artemisinin also improved neurological and behavioural outcomes and restored grasp strength and the recovery of motor function in MCAO animals. Furthermore, artemisinin treatment significantly inhibited the molecular indices of apoptosis, oxidative stress and neuroinflammation and activated the ERK1/2/CREB/BCL-2 signaling pathway. Further validation of the involved signaling pathway by the ERK1/2 inhibitor PD98059 revealed that inhibiting the ERK1/2 signaling pathway or silencing ERK1/2 reversed the neuroprotective effects of artemisinin. These results indicate that artemisinin provides neuroprotection against ischemic stroke via the ERK1/2/CREB/BCL-2 signaling pathway. Our study suggests that artemisinin may play an important role in the prevention and treatment of stroke.     

Variable apoptotic response of NSCLC cells to inhibition of the MEK/ERK pathway by small molecules or dominant negative mutants

To evaluate the role of the MEK/ERK pathway in NSCLC survival, we analyzed NSCLC cell lines that differed in tumor histology and status of p53, Rb, and K-ras. Constitutive ERK1/2 activity was demonstrated in 17 of 19 cell lines by maintenance of ERK1/2 phosphorylation with serum deprivation. Phosphorylation of ERK1/2 correlated with phosphorylation of MEK1/2 and p90RSK, but was inversely correlated with phosphorylation of c-Raf at S259. With serum deprivation, the MEK inhibitors, PD98059 and U0126, inhibited ERK1/2 activity but did not increase apoptosis. PD98059 and U0126 induced cell cycle arrest in G(0)/G(i) in cells with the highest levels of ERK1/2 activity, which correlated with induction of p27 but not p21. To confirm the cytostatic response to MEK inhibitors, we performed transient transfections with dominant negative forms of MEK or ERK. Surprisingly, dominant negative MEK and ERK mutants increased apoptosis without affecting cell cycle or p27 levels. When combined with paclitaxel, MEK inhibitors had no effect on apoptosis. In contrast, dominant negative ERK2 potentiated paclitaxel-induced apoptosis. Our studies show that constitutive ERK1/2 activity in NSCLC cells promotes cellular survival and chemotherapeutic resistance. Moreover, our data are the first to demonstrate divergent cellular responses to inhibition of the MEK/ERK pathway by small molecule inhibitors or dominant negative mutants.     

Inhibition of ERK1/2 and CREB phosphorylation by caspase-dependent mechanism enhances apoptosis in a fibrosarcoma cell line treated with butyrate

We evaluated the role of MAPKs on apoptosis induced by butyrate in cells derived from a human fibrosarcoma (2C4). Culture of 2C4 cells in 5% of fetal bovine serum (FBS) induced ERK1/2 and CREB phosphorylation and delayed apoptosis induced by butyrate. Butyrate inhibited phosphorylation of ERK1/2 and CREB. Furthermore, the use of specific inhibitors PD98059 (MEK) and H89 (PKA), which block ERK1/2 and CREB phosphorylation, accelerated butyrate induced cell death in 2C4 cells. The butyrate effect was shown to be dependent on caspase activation, once caspase inhibitors restored phosphorylation of ERK1/2 and CREB in 2C4 cells. However, the proteolytic effect of caspases was not directly on ERK1/2 and CREB proteins. In conclusion, butyrate induced apoptosis in 2C4 cells is regulated by the levels of ERK1/2 and CREB phosphorylation in a caspase dependent mechanism.     

The Nitric Oxide-Cyclic GMP Pathway Plays an Essential Role in Both Promoting Cell Survival of Cerebellar Granule Cells in Culture and Protecting the Cells Against Ethanol Neurotoxicity

Abstract: NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N ^G-nitro- l -arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono)bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.     

Calcium-activated RAF/MEK/ERK signaling pathway mediates p53-dependent apoptosis and is abrogated by alpha B-crystallin through inhibition of RAS activation

The ocular lens is the only organ that does not develop spontaneous tumor. The molecular mechanism for this phenomenon remains unknown. Through examination of the signaling pathways mediating stress-induced apoptosis, here we presented evidence to show that different from most other tissues in which the extracellular signal-regulated kinases (ERKs) pathway is generally implicated in mediation of survival signals activated by different factors, the RAF/MEK/ERK signaling pathway alone plays a key role in stress-activated apoptosis of lens epithelial cells. Treatment of N/N1003A cells with calcimycin, a calcium mobilizer, activates the RAF/MEK/ERK pathway through RAS, which is indispensable for the induced apoptosis because inhibition of this pathway by either pharmacological drug or dominant negative mutants greatly attenuates the induced apoptosis. Calcimycin also activates p38 kinase and JNK2, which are not involved in calcium-induced apoptosis. Downstream of ERK activation, p53 is essential. Activation of RAF/MEK/ERK pathway by calcimycin leads to distinct up-regulation of p53. Moreover, overexpression of p53 enhances calcimycin-induced apoptosis, whereas inhibition of p53 expression attenuates calcimycin-induced apoptosis. Up-regulation of p53 directly promotes Bax expression, which changes the integrity of mitochondria, leading to release of cytochrome c, activation of caspase-3 and eventually execution of apoptosis. Overexpression of alphaB-crystallin, a member of the small heat-shock protein family, blocks activation of RAS to inhibit ERK1/2 activation, and greatly attenuates calcimycin-induced apoptosis. Together, our results provide 1) a partial explanation for the lack of spontaneous tumor in the lens, 2) a novel signaling pathway for calcium-induced apoptosis, and 3) a novel antiapoptotic mechanism for alphaB-crystallin.     

Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia

Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.     

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

17beta-estradiol does not protect cerebellar granule cells from excitotoxicity or apoptosis

Mounting evidences have suggested that 17beta-estradiol (E2) could have a neuroprotective action in the CNS. In the present study, we wanted to study whether this estrogen was able to protect cerebellar granule cells (CGCs) from apoptosis or excitotoxicity. Our results suggest that E2 has no anti-apoptotic effect in CGCs cultures. The lack of phosphoinositide 3-kinase/Akt pathway activation in CGCs cultures could be on the basis of the failure of estradiol to protect CGCs from potassium-deprivation and ceramide-mediated apoptosis. Moreover, E2 does not protect CGCs from glutamate-mediated death despite activating the extracellular signal regulated kinase kinase/extracellular signal regulated kinase pathway, which suggests that extracellular signal regulated kinase kinase/extracellular signal regulated kinase pathway activation is not sufficient to sustain an estrogen-mediated neuroprotective effect in CGCs cultures. By contrast, we found that the estrogen had a significant neuroprotective effect against hydrogen peroxide-mediated neuronal death. This effect was due to the antioxidant properties of the chemical structure of estradiol, as the biological inactive isomer 17alpha-estradiol was also able to reduce hydrogen peroxide-mediated neuronal death.     

Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis

Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.     

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

N-methyl-D-aspartate blocks activation of JNK and mitochondrial apoptotic pathway induced by potassium deprivation in cerebellar granule cells

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.