GDNF prevents TGF-β-induced damage of the plasma membrane in cerebellar granule neurons by suppressing activation of p38-MAPK via the phosphatidylinositol 3-kinase pathway

Transforming growth factor-β (TGF-β) and glial-cell-line-derived neurotrophic factor (GDNF) have been shown to synergize in several paradigms of neuronal survival. We have previously shown that cerebellar granule neurons (CGN) degenerate in low potassium via ERK1/2 (extra-cellular-regulated kinase)-dependent plasma membrane (PM) damage and caspase-3-dependent DNA fragmentation. Here, we have investigated the putative synergistic function of GDNF and TGF-β in CGN degeneration. GDNF alone prevents low-potassium-induced caspase-3 activation and DNA fragmentation but does not affect either low-potassium-induced ERK activation or PM damage. TGF-β alone does not affect low-potassium-induced DNA fragmentation but potentiates low-potassium-induced PM damage. This effect of TGF-β is independent of ERK1/2 activation but dependent on p38-MAPK (mitogen-activated protein kinase) activation. When co-applied with TGF-β, GDNF paradoxically antagonizes TGF-β-induced potentiation of PM damage by inhibiting TGF-β-induced p38-MAPK activation. In addition, PI3K (phosphatidylinositol 3-kinase) inhibitors abolish the GDNF effect. This study thus demonstrates a differential mechanism of action of GDNF and TGF-β on CGN degeneration. GDNF inhibits caspase-3-dependent DNA fragmentation but does not affect ERK-dependent PM damage. However, GDNF can attenuate TGF-β-induced p38-MAPK-dependent PM damage via the PI3K pathway. This work was supported by the Deutsche Forschungsgemeinschaft (STR 616/1–2) and by a fellowship (Young Investigator Award) from the Medical Faculty, University of Heidelberg, Germany to S. Subramaniam.     

Activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphostin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.     

MEK�CERK-dependent multiple caspase activation by mitochondrial proapoptotic Bcl-2 family proteins is essential for heavy ion irradiation-induced glioma cell death

Recently developed heavy ion irradiation therapy using a carbon beam (CB) against systemic malignancy has numerous advantages. However, the clinical results of CB therapy against glioblastoma still have room for improvement. Therefore, we tried to clarify the molecular mechanism of CB-induced glioma cell death. T98G and U251 human glioblastoma cell lines were irradiated by CB, and caspase-dependent apoptosis was induced in both cell lines in a dose-dependent manner. Knockdown of Bax (BCL-2-associated X protein) and Bak (BCL-2-associated killer) and overexpression of Bcl-2 or Bcl-xl (B-cell lymphoma-extra large) showed the involvement of Bcl-2 family proteins upstream of caspase activation, including caspase-8, in CB-induced glioma cell death. We also detected the activation of extracellular signal-regulated kinase (ERK) and the knockdown of ERK regulator mitogen-activated protein kinase kinase (MEK)1/2 or overexpression of a dominant-negative (DN) ERK inhibited CB-induced glioma cell death upstream of the mitochondria. In addition, application of MEK-specific inhibitors for defined periods showed that the recovery of activation of ERK between 2 and 36 h after irradiation is essential for CB-induced glioma cell death. Furthermore, MEK inhibitors or overexpression of a DN ERK failed to significantly inhibit X-ray-induced T98G and U251 cell death. These results suggested that the MEK–ERK cascade has a crucial role in CB-induced glioma cell death, which is known to have a limited contribution to X-ray-induced glioma cell death.     

Role for apoptosis-inducing factor in the physiological death of cerebellar neurons

Apoptosis-inducing factor (AIF) is implicated in caspase-independent apoptotic-like death. AIF released from mitochondria translocates to the nucleus, where it mediates some apoptotic events such as chromatin condensation and DNA degradation. Here, the role of AIF in the neuronal death was studied under physiological conditions. When we analyzed the cellular localization of AIF during cerebellar development, we found a significant increase in the number of neurons with nuclear AIF localization in an age-dependent manner. On the other hand, cerebellar granule neurons (CGN) chronically cultured in low concentration of potassium (5 mM; K5) die with apoptotic-like characteristics after five days. In the present study we found that K5 induces a caspase-dependent apoptotic-like death of CGN as well as a late nuclear translocation of AIF. When CGN death induced by K5 was carried out in the presence of a general inhibitor of caspases, there was a slight decrement of cell death, but neurons eventually died by showing apoptotic-like features such as phosphatidylserine translocation and nuclear condensation. Besides, there was a significant increment of nuclear AIF translocation. These findings support the idea that AIF could be involved in apoptotic-like death of CGN and that it could be an alternative mechanism of neuronal death during cerebellar development.     

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X₇ nucleotide receptor (P2X₇R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X₇R agonist BzATP were inhibited by the P2X₇R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X₇R antisense oligonucleotide indicating a direct involvement of the P2X₇R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X₇R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X₇Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.     

Kaempferol induces cell death through ERK and Akt-dependent down-regulation of XIAP and survivin in human glioma cells

The present study was undertaken to determine the molecular mechanism by which kaempferol induces cell death in human glioma cells. Kaempferol resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which were largely attributed to cell death. Kaempferol caused an increase in reactive oxygen species (ROS) generation and the kaempferol-induced cell death was prevented by antioxidants, suggesting that ROS generation is involved in kaempferol-induced cell death. Kaempferol caused depolarization of mitochondrial membrane potential. Western blot analysis showed that kaempferol treatment caused a rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. The ERK inhibitor U0126 and the Akt inhibitor LY984002 increased the kaempferol-induced cell death and overexpression of MEK, the upstream kinase of ERK, and Akt prevented the cell death. The expression of anti-apoptotic proteins XIAP and survivin was down-regulated by kaempferol and its effect was prevented by overexpression of MEK and Akt. Kaempferol induced activation of caspase-3 and kaempferol-induced cell death was prevented by caspase inhibitors. Taken together, these findings suggest that kaempferol results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of XIAP and survivin regulating by ERK and Akt.     

Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species-mediated activation of ERK

Sphingosylphosphorylcholine (SPC) produces reactive oxygen species (ROS) in MS1 pancreatic islet endothelial cells. In the present study, we explored the physiological significance of the SPC-induced ROS generation in endothelial cells. SPC induced cell death of MS1 cells at higher than 10 microM concentration through a caspase-3-dependent pathway. SPC treatment induced sustained activation of an extracellular signal-regulated kinase (ERK), in contrast to transient activation of ERK in response to platelet-derived growth factor (PDGF)-BB, which stimulated proliferation of MS1 cells. Both the SPC-induced cell death and ERK activation were abolished by pretreatment of the cells with the MEK inhibitor U0126 or by overexpression of a dominant negative mutant of MEK1 (DN-MEK1). Pretreatment of the cells with N-acetylcysteine, an antioxidant, completely prevented the SPC-induced ROS generation, apoptosis, and ERK activation, whereas the ROS generation was not abrogated by treatment with U0126. Consistent with these results, SPC induced cell death of human umbilical vein endothelial cells (HUVECs) through ROS-mediated activation of ERK. These results suggest that the SPC-induced generation of ROS plays a crucial role in the cell death of endothelial cells through ERK-dependent pathway.     

Bcl-xL prevents staurosporine-induced hepatocyte apoptosis by restoring protein kinase B/mitogen-activated protein kinase activity and mitochondria integrity

Our study reports that staurosporine induces apoptosis in cultured rat hepatocytes in a dose- and time-dependent fashion. Staurosporine induced apparent cleavage of caspase-8, caspase-9, and caspase-3. The release of cytochrome c from mitochondria, and Bid activation were also detected in staurosporine-treated primary hepatocytes. These results suggest that mitochondria-mediated cell death signaling may be involved in staurosporine-induced hepatocyte apoptosis. Bcl-x(L) overexpression protected from "loss of" mitochondrial transmembrane potential and prevented staurosporine-induced caspase-3 and caspase-8 cleavage. Overexpression of constitutively active ERK and PKB inhibited staurosporine-induced caspase-3 activation and hepatocyte death. PI3K inhibitor (LY294002) and ERK inhibitor (PD98059) significantly reversed the protective effects of Bcl-x(L) on staurosporine-induced hepatocyte death. Our data suggest that Bcl-x(L) prevents staurosporine-induced hepatocyte apoptosis by modulating protein kinase B and p44/42 mitogen-activated protein kinase activity and disrupts mitochondria death signaling.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells

Protein kinase C (PKC) isoforms play distinct roles in cellular functions. We have previously shown that ionizing radiation activates PKC isoforms (alpha, delta, epsilon, and zeta), however, isoform-specific sensitivities to radiation and its exact mechanisms in radiation mediated signal transduction are not fully understood. In this study, we showed that overexpression of PKC isoforms (alpha, delta, epsilon, and zeta) increased radiation-induced cell death in NIH3T3 cells and PKC epsilon overexpression was predominantly responsible. In addition, PKC epsilon overexpression increased ERK1/2 activation without altering other MAP-kinases such as p38 MAPK or JNK. Co-transfection of dominant negative PKC epsilon (PKC epsilon -KR) blocked both PKC epsilon -mediated ERK1/2 activation and radiation-induced cell death, while catalytically active PKC epsilon construction augmented these phenomena. When the PKC epsilon overexpressed cells were pretreated with PD98059, MEK inhibitor, radiation-induced cell death was inhibited. Co-transfection of the cells with a mutant of ERK1 or -2 (ERK1-KR or ERK2-KR) also blocked these phenomena, and co-transfection with dominant negative Ras or Raf cDNA revealed that PKC epsilon -mediated ERK1/2 activation was Ras-Raf-dependent. In conclusion, PKC epsilon -mediated ERK1/2 activation was responsible for the radiation-induced cell death.     

Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells

Endoplasmic reticulum (ER) stress has increasingly come into focus as a factor contributing to neuronal injury. Although caspase-dependent mechanisms have been implicated in ER stress, the signaling pathways involved remain unclear. In this study, we examined the role of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase pathway that is highly conserved in many systems for balancing cell survival and death. Prolonged treatment of the human neuroblastoma cell line SH-SY5Y with thapsigargin, an inducer of ER stress, increased cell death over 24-48 h, as measured by LDH release. Caspases were involved; increased levels of active caspase-3 and cleaved caspase substrate PARP were detected, and treatment with Z-VAD-FMK reduced thapsigargin-induced cytotoxicity. In contrast, inhibition of calpain was not protective, although calpain was activated following thapsigargin treatment. An early and transient phosphorylation of ERK1/2 occurred after thapsigargin-induced ER stress, and targeting this pathway with the MEK inhibitors U0126 or PD98059 significantly reduced cell death. Similar cytoprotection was obtained against brefeldin A, another ER stress agent. However, protection against ER stress via ERK inhibition was not accompanied by amelioration of caspase-3 activation, PARP cleavage, or DNA laddering. These data indicate that ERK may contribute to non-caspase-dependent pathways of injury after ER stress.     

Nuclear factor-kappaB p65 inhibits mitogen-activated protein kinase signaling pathway in radioresistant breast cancer cells

The molecular mechanism by which tumor cells increase their resistance to therapeutic radiation remains to be elucidated. We have previously reported that activation of nuclear factor-kappaB (NF-kappaB) is causally associated with the enhanced cell survival of MCF+FIR cells derived from breast cancer MCF-7 cells after chronic exposure to fractionated ionizing radiation. The aim of the present study was to reveal the context of NF-kappaB pathways in the adaptive radioresistance. Using cell lines isolated from MCF+FIR populations, we found that the elevated NF-kappaB activity was correlated with enhanced clonogenic survival, and increased NF-kappaB subunit p65 levels were associated with a decrease in phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK in all radioresistant MCF+FIR cell lines. Further irradiation with 30 fractions of radiation also inhibited MEK/ERK phosphorylation in paired cell lines of MCF+FIR and parental MCF-7 cells. Activation of ataxia-telangiectasia mutated (ATM) protein, a sensor to radiation-induced DNA damage, was elevated with increased interaction with NF-kappaB subunits p65 and p50. The interaction between p65 and MEK was also enhanced in the presence of activated ATM. In contrast, both interaction and nuclear translocation of p65/ERK were reduced. Inhibition of NF-kappaB by overexpression of mutant IkappaB increased ERK phosphorylation. In addition, MEK/ERK inhibitor (PD98059) reduced the interaction between p65 and ERK. Taken together, these results suggest that NF-kappaB inhibits ERK activation to enhance cell survival during the development of tumor adaptive radioresistance.     

Amylin-induced cytotoxicity is associated with activation of caspase-3 and MAP kinases

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.     

ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.     

Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation,ROS generation and mitochondrial dysfunction

RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis.     

Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).