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Sphingomyelinase (SMase) and its product ceramide have recently attracted a great deal of attention because of their possible role in the signal transduction pathway. However, the role of sphingomyelinase in UV-induced c-Jun N-terminal kinase (JNK) activation remains unclear. A genetic SMase-deficient (2 ~3% residual acid SMase activity) lymphoblast cell line, MS1418, was used to investigate this problem. While UV irradiation markedly induces JNK activation in a normal human lymphoblast cell line (JY), it induces only weak JNK activation in MS1418 cells. This difference of JNK response to UV irradiation between these two cell lines is further observed in time course and dose-response studies. In contrast, phorbol ester-induced JNK activation can be observed in both JY and MS1418 cells. Significant JNK activation can be observed in MS1418 cells by exposure of the cells to SMase or C2-ceramide. In contrast, phospholipase A2 or phospholipase C does not show significant induction of JNK activity, and C2-dihydroceramide and sphingosine induce much weaker JNK activation in MS1418 cells than that induced by C2-ceramide. These data demonstrate that SMase plays an essential role in UV-induced JNK activation (Huang, 1997).
The induction of programmed cell death, or apoptosis, involves activation of a signaling system, many elements of which remain unknown. The sphingomyelin pathway, initiated by hydrolysis of the phospholipid sphingomyelin in the cell membrane to generate the second messenger ceramide, is thought to mediate apoptosis in response to tumour-necrosis factor (TNF)-alpha, to Fas ligand and to X-rays. It is not known whether it plays a role in the stimulation of other forms of stress-induced apoptosis. Given that environmental stresses also stimulate a stress-activated protein kinase (SAPK/JNK), the sphingomyelin and SAPK/JNK signaling systems may be coordinated in induction of apoptosis. Ceramide initiates apoptosis through the SAPK cascade (Verheij, 1996).
Anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway that includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induces expression of CD95-L, cleavage of caspases, and apoptosis. Antisense CD95-L, as well as dominant-negative FADD, inhibit ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, fail to up-regulate CD95-L expression and thus fail to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity is still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts are restorable by exogenously added ceramide. NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis (Herr 1997).
Sphingolipid products such as ceramide (cer), sphingosine (sph), and sphingosine-1-phosphate (SPP) are implicated in the regulation of cell growth and apoptosis. Cer, sph, and SPP differentially modulate ionic events in cultured oligodendrocytes (OLGs). Cer but not sph or SPP inhibits the inward rectifier (IKir) in OLGs. To further investigate the role of sphingolipid products in OLGs, the effects of cer, sph, and SPP on OLG survival and on the regulation of mitogen-activated protein kinases (MAPKs) were studied. Cer, sph, and SPP differentially modulate OLG survival and activation of MAPK members. Cer causes OLG apoptosis, sph causes OLG lysis, and SPP does not affect OLG survival. Cer induces a preferential activation of p38alpha, whereas sph and SPP induce a preferential activation of extracellular signal-regulated kinase 2 (ERK2) in OLGs. In addition, the effect of cer on p38alpha activity is mimicked by the inhibition of IKir with Ba2+. In contrast, exposure to cer results in increased activity of ERK2 but not of p38alpha in astrocytes. Cer-induced OLG apoptosis is attenuated by a p38 inhibitor, SB203580, and by expression of a p38alpha dominant negative mutant. It is concluded that p38alpha is the mediator in cer-induced OLG apoptosis and that cer-induced IKir inhibition may contribute to the sustained activation of p38alpha in OLGs (Hida, 1999).
This study demonstrates that cer, sph, and SPP differentially modulate OLG survival and the activation of MAPK members, although these sphingolipid products are interconvertible. Cer is deacylated to form sph, which is then phosphorylated to form SPP. The forward reactions are catalyzed by ceramidase and sph kinase, whereas the reverse reactions are catalyzed by phosphatidate phosphohydrolase and cer synthase, respectively. The mechanisms involved in the regulation of cell growth and survival by sphingolipid products are not completely understood. Cer and SPP cause OLG depolarization, whereas sph elicits OLG hyperpolarization. Sph consistently induces Cai increases in OLGs, whereas Cai responses are observed infrequently with cer or SPP. In addition, inhibition of OLG IKir underlies cer-induced depolarization but not SPP-induced depolarization. Both cer and SPP induce OLG depolarization, yet OLG apoptosis is enhanced only by cer. This study asked whether downstream effectors such as MAPK members play a role in determining whether conditions are permissive for apoptotic stimuli. JNK and ERK2 are differentially regulated by sphingolipid products in airway smooth muscle cells and rat mesangial cells, supporting the concept that the dynamic balance between ERK2 and JNK/p38 cascades is important in determining cell survival. Similar although not identical results were observed in OLGs. p38alpha is activated by cer only, whereas ERK2 is activated by sph and SPP. There is no difference in the JNK1 activity in cer-, sph-, and SPP-treated OLGs. One interpretation would be that cer-induced activation of p38alpha, but not of ERK2, is permissive to OLG apoptosis; conversely, SPP-induced ERK2 activation, but not p38alpha activation, is not permissive. In addition, the effect of cer on p38alpha activity is mimicked by Ba2+, a known IKir blocker, but not by high K+, suggesting that IKir inhibition rather than depolarization per se is a contributory signal to the differential activation of MAPK members. Failure of SPP to inhibit IKir, despite its depolarizing action, correlates with the absence of p38alpha induction and absence of apoptosis in SPP-treated cells. Based on MAPK cascades activated by sph, sph should not induce cell death in OLGs. However, sph also causes sustained Cai increases in OLGs, which can lead to cell death. Hence, the mechanisms underlying sph-induced OLG lysis and necrosis differ from those of cer-induced OLG apoptosis (Hida, 1999 and references).
In general, JNK and p38 kinase pathways are considered key mediators of the inflammatory response and are activated by both Fas and TNF receptor oligomerization or other stressful stimuli; however, their respective roles in apoptosis remains controversial. The p38 subfamily consists of at least four isoforms: p38alpha-delta. p38alpha (also known as p38) and p38beta, but not p38gamma and p38delta, are inhibited by pyridinyl imidazole compounds, such as SB203580. Activation of p38alpha induces apoptosis in Jurkat T cells and cardiac myocytes, whereas activation of p38beta inhibits apoptosis or induces a hypertrophic response. Cer is shown to cause sustained activation of p38alpha in OLGs; cer-induced apoptosis is inhibited by SB203580 and by p38alpha dominant negative mutant, indicating that activated p38alpha mediates cer-induced OLG apoptosis. In view of the uniform, modest activation of JNK1 by cer, sph, and SPP, the role of JNK1 in cer-induced OLG apoptosis in this study appears to be less significant than p38alpha. Other stimuli that activate JNK in OLGs include NGF, TNF-alpha, IL, UV light, and heat shock. Studies from Jurkat T cells and other cell lines suggest that JNK activation is associated with apoptosis. But other investigators have stressed that activation of JNK alone is not sufficient to induce apoptosis. Transfection with c-Jun dominant negative mutant or with SEK1 dominant negative mutant protects neurons against apoptosis induced by withdrawal of trophic factors and protects U937 cells against cer-induced apoptosis but does not protect Jurkat T cells or human breast carcinoma cells against Fas- or TNF-induced apoptosis. It is plausible that the role of JNK1 versus p38alpha in apoptosis depends on the cell type and the apoptotic trigger. In murine fibroblast cell line L9290cyt16, neither JNK nor p38alpha appears to be required for Fas- or TNF-induced apoptosis (Hida, 1999 and references).
In contrast to p38alpha and JNK1, activation of ERK1/2 is generally associated with cell proliferation or differentiation, depending on whether activation is sustained or transient. ERK1 and ERK2 are activated by mitogenic factors (PDGF and basic FGF) and phorbol esters in OLGs and progenitors. OLGs treated with PD098059 have a limited number of processes, suggesting a role for ERKs in process extension. ERK2 activity is transiently enhanced by cer in astrocytes but not in OLGs, whereas p38alpha is enhanced by cer in OLGs but not in astrocytes. These results are in agreement with the concept that cell survival is regulated by opposing actions of ERK and p38/JNK pathways. However, simultaneous activation of both ERK1/2 and p38 cascades appears to be required for maximal endotoxin-induced astroglial cell activation and for NGF-induced neuronal differentiation of PC12 cells. One interpretation of the apparent discrepancy would be that the pattern of activation of MAPK members is a crucial, but not the sole determinant of cell survival, activation, and differentiation. Other important factors that influence cell survival include Bcl2, BAD, and other related mitochondrial proteins, intracellular glutathione content, and ionic fluxes (Hida, 1999 and references).
Cer has been shown to inhibits IKir via a ras- and raf-1-dependent pathway in cultured OLGs. Yet, cer activates p38alpha instead of ERK2. The experiments with Ba2+ indicate that IKir inhibition may contribute to cer-induced activation of p38alpha and perhaps prevent an increase in ERK2 activity as well. A working model whereby proinflammatory cytokines, hypoxia, or other apoptotic stimuli lead to OLG apoptosis is presented. Although cer-induced IKir inhibition and increased p38alpha activity may constitute two simultaneous independent signals required for OLG apoptosis, it is proposed that IKir inhibition contributes to the cer-induced sustained activation of p38alpha, perhaps via activation of caspases. Cer-induced IKir inhibition, by reducing K+ influx, leads to diminished [K+]i, a condition linked to caspase activation and apoptosis. Interleukin 1beta-converting enzyme (ICE) family proteases are required for activation of p38 by Fas but not by sorbitol or etoposide. These studies support the concept that sphingomyelin cycle is an important regulator of cell survival and that the ultimate cellular outcome depends on the integration of multiple signals, including activation of MAPK members and modulation of ionic events at the plasma membrane (Hida, 1999 and references).
Compelling evidence indicates that activation of the JNK/SAPK signaling pathway is obligatory for apoptosis induction by multiple cell stresses which activate the sphingomyelin cycle. Ectopic expression of bcl-2 can impair apoptosis signaling by most of the cell stresses that activate the ceramide/JNK pathway (for information about Bcl-2, see Death caspase-1: Evolutionary homologs section). Enforced expression of bcl-2 protects prostate carcinoma cells against the induction of apoptosis by exogenous C2-ceramide. Enforced bcl-2 expression blocks the capacity of C2-ceramide to activate JNK1, indicating bcl-2 functions at the level of JNK1 or upstream of JNK1 in the ceramide/JNK pathway. The contribution of bcl2 to the regulation of the arachidonate pathway for prostate carcinoma cell survival was also investigated using highly selective inhibitors of arachidonate metabolism. These results indicate that bcl-2 can protect cells against the diminished availability of arachidonic acid, 12-HETE, and 15-HETE. Arachidonic acid substantially suppresses the induction of apoptosis by C2-ceramide, providing evidence for the opposing influences of these lipid signaling pathways in the mediation of prostate carcinoma cell survival. These results provide evidence for the opposing influences of the ceramide and arachidonate signaling pathways on the mediation of cell death and cell survival, respectively, in prostate carcinoma cells, and suggest a dual role for bcl-2 in this context (Herrmann, 1997).
Purified Bcl-2 was found to be phosphorylated by the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) p54-SAPKbeta; this is specific insofar as the extracellular signal-regulated kinase 1 (ERK1) and p38/RK/CSBP (p38) catalyzed only weak modification. Bcl-2 undergoes similar phosphorylation in COS-7 when coexpressed together with p54-SAPKbeta and the constitutive Rac1 mutant G12V. This is seen with 32PO4 labeling as well as in the appearance of five discrete Bcl-2 bands with reduced gel mobility. As anticipated, both intracellular p54-SAPKbeta activation and Bcl-2 phosphorylation are blocked by co-transfection with the MAP kinase specific phosphatase MKP3/PYST1. MAP kinase specificity is also seen in COS-7 cells as Bcl-2 undergoes only weak phosphorylation when co-expressed with enzymatically activated ERK1 or p38. Four critical residues undergoing phosphorylation in COS-7 cells were identified by expression of the quadruple Bcl-2 point mutant T56A,S70A,T74A, S87A. Sequencing phosphopeptides derived from tryptic digests of Bcl-2 indicates that purified GST-p54-SAPKbeta phosphorylates identical sites in vitro. This is the first report of Bcl-2 phosphorylation by the JNK/SAPK class of MAP kinases and could indicate a key modification allowing control of Bcl-2 function by cell surface receptors, Rho family GTPases, and/or cellular stresses (Maundrell, 1997).
Bcl-2 is an intracellular membrane-associated protein that prevents cell death, which otherwise would be induced by a variety of apoptotic stimuli. A mechanism by which Bcl-2 exerts an anti-cell death effect is, however, not fully understood. Bcl-2 suppresses cell death of N18TG neuroglioma cells caused by various apoptotic stresses, including etoposide, staurosporine, anisomycin, and ultraviolet irradiation. Concomitantly, Bcl-2 disruptes a signaling cascade to the c-Jun N-terminal kinase activation induced by the apoptotic stresses. Bcl-2 also prevents the etoposide-induced stimulation of MEKK1. Overexpression of c-Jun N-terminal kinase antagonizes the death-protective function of Bcl-2. These data suggest that suppression of the c-Jun N-terminal kinase signaling pathway may be critical for Bcl-2 action (Park, 1997).
Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, has been deleted, do not undergo apoptosis in 5 mM potassium, yet do undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decrease glucose uptake to <20% of control. Protein synthesis also decreases rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increase mRNA levels of c-jun, and caspase 3 (CPP32) and increase phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increase cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells do not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases; they indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, block cleavage of DEVD-AMC and block an increase in DNA degradation. However, these inhibitors have only a marginal effect on the prevention of cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells (Miller, 1997).
The cJun N-terminal kinase (JNK) signal transduction pathway is implicated in the regulation of neuronal function. JNK is encoded by three genes that play partially redundant roles. This study reports the creation of mice with targeted ablation of all three Jnk genes in neurons. Compound JNK-deficient neurons are dependent on autophagy for survival. This autophagic response is caused by FoxO-induced expression of Bnip3 that displaces the autophagic effector Beclin-1 from inactive Bcl-XL complexes. These data identify JNK as a potent negative regulator of FoxO-dependent autophagy in neurons (Xu, 2011).
Studies of nonneuronal cells have implicated JNK in the induction of autophagy. Indeed, this study confirmed the conclusion that JNK can contribute to increased autophagy by examining primary mouse embryonic fibroblasts (MEFs) with compound JNK deficiency. The mechanism of JNK-induced autophagy may be mediated by phosphorylation of Bcl2 by JNK and the subsequent release of the autophagic effector Beclin-1. The sites of JNK phosphorylation on Bcl2 are conserved in the related protein Bcl-XL. This conservation suggests that phosphorylation of Bcl2 and Bcl-XL is functionally important. Phosphorylation of Bcl2 and Bcl-XL by JNK and other protein kinases may represent an important mechanism of autophagy regulation. Indeed, the properties of JNK as a stress-responsive kinase provide an elegant mechanism for coupling stress exposure to the induction of autophagy (Xu, 2011).
Studies of nonneuronal cells demonstrate that JNK is markedly activated from a low basal state when cells are exposed to stress. However, JNK is regulated very differently in neurons. JNK1 remains constitutively activated under basal conditions, while JNK2 and JNK3 exhibit low basal activity and are stress-responsive. The proautophagy role of JNK in nonneuronal cells has been reported to be mediated by JNK1. It is therefore intriguing that JNK1 is constitutively activated in neurons. Based on studies of nonneuronal cells, the constitutive activation of JNK1 in neurons should cause autophagy. A mechanism must therefore exist to prevent autophagy activation by constitutively activated JNK1 in neurons. Although the mechanism is unclear, these considerations indicate that neurons are refractory to the proautophagy JNK1 signaling pathway that has been identified in nonneuronal cells (Xu, 2011).
This analysis of compound JNK-deficient neurons demonstrates that JNK regulates neuronal autophagy. In contrast to the proautophagy role of JNK nonneuronal cells, neuronal JNK acts to suppress autophagy. Loss of neuronal JNK function causes engagement of a transcriptional program that leads to increased expression of autophagy-related genes and the induction of an autophagic response. One consequence of autophagy induction caused by JNK deficiency is improved neuronal survival (Xu, 2011).
FoxO transcription factors are implicated in the induction of both cell death (apoptosis) and cell survival (autophagy) responses. The results of this study identify JNK as a signaling molecule that may contribute to the coordination of these divergent responses to FoxO transcription factor activation (Xu, 2011).
FoxO activation in neurons leads to the expression of the target gene Bim, a proapoptotic BH3-only protein, and causes cell death. JNK activation in neurons promotes expression of Bim, most likely because JNK-dependent AP-1 activity is required for Bim expression. Moreover, JNK phosphorylates Bim on an activating site, and also causes the release of Bim from complexes with the anti-apoptotic Bcl2 family protein Mcl-1. Together, these processes initiate JNK-dependent apoptosis. JNK inhibition can therefore prevent neuronal cell death. Indeed, small molecule inhibitors of JNK cause neuroprotection in models of neurodegenerative disease (Xu, 2011).
Activation of FoxO transcription factors can also cause increased expression of autophagy-related genes, including Atg8/Lc3b, Atg12, and Bnip3. While JNK cooperates with FoxO to increase proapoptotic Bim expression, JNK deficiency prevents induction of Bim expression and promotes a survival response that is mediated by increased FoxO-dependent expression of the autophagy-related target genes Atg8/Lc3b, Atg12, and Bnip3. Indeed, inhibition of autophagy in JNK-deficient neurons causes rapid death. This neuronal survival response is relevant to stroke models in which neuronal death is mediated by a JNK-dependent mechanism (Xu, 2011).
Together, these data demonstrate that cross-talk between the FoxO and JNK signaling pathways leads to neuronal death. In contrast, loss of JNK promotes FoxO-induced survival mediated by increased autophagy. JNK therefore acts as a molecular switch that defines the physiological consequence of FoxO activation in neurons (Xu, 2011).
Thus, JNK is implicated in the induction of autophagy in nonneuronal cells. However, JNK1 is constitutively activated in neurons, and these cells are refractory to JNK-induced autophagy. Instead, JNK acts to suppress autophagy in neurons by inhibiting FoxO-induced expression of autophagy-related genes (e.g., Atg8/Lc3b, Atg12, and Bnip3) and increasing the expression of proapoptotic genes (e.g., Bim). JNK inhibition causes neuroprotection that is mediated by loss of proapoptotic gene expression and increased autophagy (Xu, 2011).
Many of the actions of serine/threonine kinase receptors for the transforming growth factor-beta (TGFbeta) are mediated by DPC4, a human MAD-related protein identified as a tumor suppressor gene in pancreatic carcinoma. Overexpression of DPC4 is sufficient to induce the activation of gene expression and cell cycle arrest, characteristic of the TGFbeta response. The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is also one of the downstream targets required for TGFbeta-mediated signaling. Expression of the dominant-interfering mutant of various components of the SAPK/JNK cascade specifically block both TGFbeta and DPC4-induced gene expression. These dominant-interfering mutants also inhibited TGFbeta-stimulated DPC4 transcriptional activity. Overexpression of DPC4 causes transfected cells to undergo the morphological changes typical of apoptosis. These findings define a mechanism whereby TGFbeta signals mediated by DPC4 and SAPK/JNK cascade are integrated in the nucleus to activate gene expression and identify a new cellular function for DPC4 (Atfi, 1997b).
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).
Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5-/- and p35-/- mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival have been unknown. This study shows that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis (Li, 2002).
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