Stress-mediated signaling in PC12 cells - the role of the small heat shock protein,Hsp27, and Akt in protecting cells from heat stress and nerve growth factor withdrawal

We have investigated the role of stress-activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal-induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen-activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both non-stimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24-72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.     

A short lived protein involved in the heat shock sensing mechanism responsible for stress-activated protein kinase 2 (SAPK2/p38) activation

The stress-activated protein kinase 2 (SAPK2/p38) is activated by various environmental stresses and also by a vast array of agonists including growth factors and cytokines. This implies the existence of multiple proximal signaling pathways converging to the SAPK2/p38 activation cascade. Here, we show that there is a sensing mechanism highly specific to heat shock for activation of SAPK2/p38. After mild heat shock, cells became refractory to reinduction of the SAPK2/p38 pathway by a second heat shock. This was not the result of a toxic effect because the cells remained fully responsive to reinduction by other stresses, cytokines, or growth factors. Neither the activity of SAPK2/p38 itself nor the accumulation of the heat shock proteins was essential in the desensitization process. The cells were not desensitized to heat shock by other treatments that activated SAPK2/p38. Moreover, inhibiting SAPK2/p38 activity during heat shock did not block desensitization. Also, overexpression of HSP70, HSP27, or HSP90 by gene transfection did not cause desensitization, and inhibiting their synthesis after heat shock did not prevent desensitization. Desensitization rather appeared to be linked closely to the turnover of a putative upstream activator of SAPK2/p38. Cycloheximide induced a progressive and eventually complete desensitization. The effect was specific to heat shock and minimally affected activation by other stress inducers. Inhibiting protein degradation with MG132 caused the constitutive activation of SAPK2/p38, which was blocked by a pretreatment with either cycloheximide or heat shock. The results thus indicate that there is a sensing pathway highly specific to heat shock upstream of SAPK2/p38 activation. The pathway appears to involve a short lived protein that is the target of rapid successive up- and down-regulation by heat shock.     

Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus

Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-alpha, IL-15, IL-12) cytokines, hsp27, a small hsp, has been primarily identified as a substrate of mitogen-activated protein kinase-activated protein kinase-2 involved in the p38 signaling pathway and activated during monocyte IL-10 production. Hsp27 can also act as an endogenous protein circulating in the serum of breast cancer patients and a protein whose induction correlates to protection from LPS shock. However, the cytokine-stimulating properties of hsp27 have been unexplored. In this study, exogenous hsp27 is demonstrated for the first time as a potent activator of human monocyte IL-10 production, but only a modest inducer of TNF-alpha. Although exogenous hsp27 stimulation activated all three monocyte mitogen-activated protein kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38), only p38 activation was sustained and required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and p38 activation were required for induction of TNF-alpha when using the p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27's transient activation of the c-Jun N-terminal kinase pathway, which can down-regulate IL-10, may contribute to its potent IL-10 induction. Hsp27's ERK 1/2 activation was also less sustained than activation by stimuli like LPS, possibly contributing to its modest TNF-alpha induction. The failure of either PD98059 or anti-TNF-alpha Ab to substantially inhibit IL-10 induction implied that hsp27 induces IL-10 via activation of p38 signaling independently of TNF-alpha activation and may be predominantly an antiinflammatory monokine stimulus.     

Drosophila Heat Shock Response Requires the JNK Pathway and Phosphorylation of Mixed Lineage Kinase at a Conserved Serine-Proline Motif

Defining context specific requirements for proteins and pathways is a major challenge in the study of signal transduction. For example, the stress-activated protein kinase (SAPK) pathways are comprised of families of closely related transducers that are activated in a variety of tissues and contexts during development and organismal homeostasis. Consequently, redundant and pleiotropic effects have hampered a complete understanding of the individual contributions of transducers in distinct contexts. Here, we report on the function of a context-specific regulatory phosphorylation site, PXSP, in the Drosophila mixed lineage kinase protein, Slpr, a mitogen-activated protein kinase kinase kinase (MAP3K) in the Jun Kinase (JNK) pathway. Genetic analysis of the function of non-phosphorylatable (PXAP) and phosphomimetic mutant (PXEP) Slpr transgenes in several distinct contexts revealed minimal effects in JNK-dependent tissue closure processes but differential requirements in heat stress response. In particular, PXAP expression resulted in sensitivity of adults to sustained heat shock, like p38 and JNK pathway mutants. In contrast, PXEP overexpression conferred some resistance. Indeed, phosphorylation of the PXSP motif is enriched under heat shock conditions and requires in part, the p38 kinases for the enrichment. These data suggest that coordination of signaling between p38 and Slpr serves to maintain JNK signaling during heat stress. In sum, we demonstrate a novel role for JNK signaling in the heat shock response in flies and identify a posttranslational modification on Slpr, at a conserved site among MAP3K mixed lineage kinase family members, which bolsters stress resistance with negligible effects on JNK-dependent developmental processes.     

(-)-epigallocatechin gallate inhibits prostaglandin D2-stimulated HSP27 induction via suppression of the p44/p42 MAP kinase pathway in osteoblasts

We previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and the mechanism. EGCG significantly reduced the HSP27 induction stimulated by PGD2 without affecting the levels of HSP70. The PGD2-induced phosphorylation of p38 MAP kinase or SAPK/JNK was not affected by EGCG. On the contrary, EGCG markedly suppressed the PGD2-induced phosphorylation of p44/p42 MAP kinase and MEK1/2. However, the PGD2-induced phosphorylation of Raf-1 was not inhibited by EGCG. These results strongly suggest that EGCG suppresses the PGD2-stimulated induction of HSP27 at the point between Raf-1 and MEK1/2 in osteoblasts.     

A 2-Cys peroxiredoxin regulates peroxide-induced oxidation and activation of a stress-activated MAP kinase

Oxidative stress-induced cell damage is an important component of many diseases and ageing. In eukaryotes, activation of JNK/p38 stress-activated protein kinase (SAPK) signaling pathways is critical for the cellular response to stress. 2-Cys peroxiredoxins (2-Cys Prx) are highly conserved, extremely abundant antioxidant enzymes that catalyze the breakdown of peroxides to protect cells from oxidative stress. Here we reveal that Tpx1, the single 2-Cys Prx in Schizosaccharomyces pombe, is required for the peroxide-induced activation of the p38/JNK homolog, Sty1. Tpx1 activates Sty1, downstream of previously identified redox sensors, by a mechanism that involves formation of a peroxide-induced disulphide complex between Tpx1 and Sty1. We have identified conserved cysteines in Tpx1 and Sty1 that are essential for normal peroxide-induced Tpx1-Sty1 disulphide formation and Tpx1-dependent regulation of peroxide-induced Sty1 activation. Thus we provide new insight into the response of SAPKs to diverse stimuli by revealing a mechanism for SAPK activation specifically by oxidative stress.     

Teratogen-induced activation of ERK, JNK, and p38 MAP kinases in early postimplantation murine embryos

BACKGROUND: Although many teratogens are known to activate apoptotic pathways culminating in abnormal development, little is known about how the embryo transduces a teratogenic exposure into specific responses. Signal reception and transduction are regulated by a number of signal transduction pathways, including the extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the stress-activated protein kinase, p38. METHODS: To analyze the effects of teratogens on MAP kinases, we used whole embryo culture, Western blot analyses, and antibodies recognizing inactive or active MAP kinases, or both. RESULTS: We show that heat shock (HS) induces a rapid, strong, but transient activation of ERK, JNK, and p38 with maximal activation occurring within 30 min of the heat shock. By contrast, cyclophosphamide (CP) and staurosporine (ST) failed to activate ERK or JNK during the time period studied (7. 5 hr). ST and CP did induce a low but reproducible activation of p38 beginning at around 3 hr and 5 hr, respectively, after the initiation of exposure. Previous work has shown that heat shock induces elevated cell death in the embryo, primarily in the developing neuroepithelium, but not in the embryonic heart. Thus, we also compared the activation of these three MAP kinase pathways in heads, hearts, and trunks isolated from day 9 embryos exposed to 43 degrees C for 15 min. The results show that ERK, JNK, and p38 are activated in heads, hearts, and trunks. CONCLUSIONS: Our results show that day 9 embryos do activate MAP kinase signaling pathways in response to teratogenic exposures; however, activation of a particular pathway does not appear to be required for teratogen-induced apoptosis.     

Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with -754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38alpha, beta, and delta decreased and p38gamma increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (-668/-654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (-47/-42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates HO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.     

Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.     

Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.     

Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.     

Seasonal variations of cellular stress response in the heart and gastrocnemius muscle of the water frog (Pelophylax ridibundus)

The present study aimed to investigate the seasonal cellular stress response in the heart and the gastrocnemius muscle of the amphibian Pelophylax ridibundus (former name Rana ridibunda) during an 8 month acclimatization period in the field. Processes studied included heat shock protein expression and protein kinase activation. The cellular stress response was addressed through the expression of Hsp70 and Hsp90 and the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). Due to a general metabolic depression during winter hibernation, the induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs are retained at low levels of expression in the examined tissues of P. ridibundus. Recovery from hibernation induces increased levels of the specific proteins, probably providing stamina to the animals during their arousal.     

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

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