Influence of extremely low frequency magnetic fields on Ca2+ signaling and NMDA receptor functions in rat hippocampus.

Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.

Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest.

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.

Activation of NF-kappa B and p38 MAP kinase is not sufficient for triggering efficient HIV gene expression in response to stress.

Recent studies have established an essential role for p38 MAP kinase in UV activation of human immunodeficiency virus (HIV) gene expression. However, p38 MAP kinase is not involved in activation of NF-kappa B, a key transcriptional activator of HIV gene expression, in response to UV, suggesting that NF-kappa B acts independently of p38 MAP kinase. In this study, we have investigated whether activation of HIV gene expression occurs when p38 MAP kinase and NF-kappa B are activated by separate stress-causing treatments, each relatively specific for activating only one of the factors. Treatment of cells with sorbitol (hyperosmotic shock) strongly activates p38 MAP kinase, whereas the cytokine TNF-alpha is a poor activator of p38 MAP kinase. On the other hand, TNF-alpha is a strong activator of NF-kappa B whereas sorbitol is not. Sorbitol, however, activates AP-1 DNA binding activity in a manner similar to that of UV. Most importantly, both sorbitol and TNF-alpha are poor activators of HIV gene expression in HeLa cells stably transfected with an HIVcat reporter gene, whereas UV elicits a strong response. The combined treatment with UV and hyperosmotic shock produces an additive effect on HIV gene expression, suggesting that these agents activate at least in part by different mechanisms. The combined treatment with sorbitol and TNF-alpha activates p38 and NF-kappa B to levels similar to those with UV, yet only results in 25-30% of the CAT levels elicited by UV. Inhibition of NF-kappa B activation by the protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) prevents UV activation of HIV gene expression, but does not inhibit p38 MAP kinase activation. We conclude that whereas both p38 MAP kinase and NF-kappa B are important for UV activation of HIV gene expression they act independently from each other and activation of both factors is not sufficient for triggering a full HIV gene expression response. Activation of HIV gene expression by UV must therefore involve additional cellular processes, such as those triggered by DNA damage, for generation of a full gene expression response.

UVB/UVA radiation activates a 48 kDa myelin basic protein kinase and potentiates wound signaling in tomato leaves.

We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet-C (< 280 nm) and UVB/UVA (280-390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen-activated protein kinase family. Ultraviolet-C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826-829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA-irradiated plants that were subsequently wounded accumulated 2.5-4-fold higher levels of proteinase inhibitor I when compared to wounded non-irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12-oxo-phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.

The extracellular-signal-regulated protein kinases (Erks) are required for UV-induced AP-1 activation in JB6 cells.

Mitogen activated protein (MAP) kinase belongs to a large family of serine/threonine protein kinases, including extracellular-signal-regulated protein kinases (Erks), P38 kinase and c-Jun N-terminal kinases (JNKs). Although previous work has shown that both Erks and JNKs are activated in cells in response to ultraviolet (UV) irradiation, most studies have focused only on the role of JNKs in UV-induced AP-1 activation. Hence, the role of Erks in UV-induced AP-1 activity is not well defined. We here have investigated this issue by using MAP kinase kinase (MEK1) inhibitor PD098059 and a dominant negative Erk2, as well as wild-type Erk2, in a JB6 cell model. PD098059 inhibited UVB- or UVC-induced AP-1 activity and phosphorylation of MEK1 and Erks, but not JNKs, in JB6 Cl 41 cells. Overexpression of wild-type Erk2 in Cl 30.7b cells that contain small amounts of Erks caused a 46.6- or 138.1-fold increase of AP-1 activity by UVB and UVC, respectively; introduction of a dominant negative Erk2 into Cl 41 cells significantly blocked the UV-induced Erks activation as well as the AP-1 activation. In contrast, overexpression of wild-type Erk2 in Cl 30.7b cells and dominant negative Erk2 in Cl 41 cells did not show a marked influence on the phosphorylation of JNKs. These results demonstrate that activation of Erks, in addition to the previously reported JNKs, is required for UV-induced AP-1 activation.

Transforming growth factor-beta enhances the ultraviolet-mediated stress response in p53-/- keratinocytes.

Skin cancer is the most common tumor type in Caucasians, with an incidence that approaches the lifetime risk for all other cancer subtypes combined. The most common predisposing factor in the development of non-melanoma skin cancer is exposure to ultraviolet (UV) radiation in sun-light. UV radiation activates c-Jun amino-terminal kinases (JNK); this kinase pathway is involved in UV-mediated apoptosis and phosphorylation of c-Jun, all of which are part of the cellular stress response. Transforming growth factor-beta1 (TGF-beta1) is an important negative regulator of keratinocyte proliferation and has other pleiotropic effects in these cells. The purpose of these investigations was to decide whether TGF-beta1 activated c-Jun amino-terminal kinases in a spontaneously immortalized human keratinocyte cell line, HaCaT, and if TGF-beta1 modulated the activation of JNK in keratinocytes exposed to ultraviolet C (UVC) radiation. Results from these investigations showed that TGF-beta1 (10 ng/ml) activated JNK within 5 min. Pretreatment with TGF-beta1 enhanced UV-mediated JNK activation and was time- and UV-dose-dependent. Pretreatment with TGF-beta1 also enhanced activity of the c-Jun promoter-reporter construct, TRE(x5)-CAT. These results suggested that TGF-beta1 modulates the response of keratinocytes to ultraviolet radiation and implicates TGF-beta1 as a potential mediator the cellular of stress response in keratinocytes.

Calcium-dependent stimulation of mitogen-activated protein kinase activity in A431 cells by low doses of ionizing radiation.

Ionizing radiation at 2 Gy activates the epidermal growth factor receptor (EGFR) kinase activity in A431 squamous carcinoma cells and as a consequence transiently activates a downstream effector, mitogen-activated protein kinase (MAPK). A dose-response analysis shows fourfold activation 3-5 min after irradiation at 0.5 Gy with no additional activation after doses up to 4 Gy. Activation is independent of protein kinase C as defined by marginal effects of protein kinase C down-regulation and the protein kinase C inhibitor, chelerythrine. In contrast, an intracellular Ca2+ chelator (BAPTA/AM), a Ca2+ antagonist (TMB-8) and a phospholipase C inhibitor (U73223), which inhibits radiation-induced Ca2+ oscillations, all block MAPK stimulation. The upstream component, Raf-1, is also activated through a mechanism that is dependent on EGFR and Ca2+. Activation of Raf-1, monitored by tyrosine phosphorylation and co-immunoprecipitation with Ras, was inhibited by BAPTA/AM and TMB-8, indicating that the Ca2+-dependent step occurs at or before the interaction of Ras and Raf-1. Neither the Ras guanosine triphosphate exchange protein, SOS, nor Ca2+-activated tyrosine kinases linked to the MAPK pathway, focal adhesion kinase and PYK2, were stimulated by radiation. In contrast, EGF activated SOS as shown by the enhanced association of SOS with EGFR in co-immunoprecipitation experiments. These results suggest that activation of EGFR-dependent downstream signaling induced by radiation differs from that induced by the natural ligands of EGFR.

Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo.

Human skin is exposed daily to solar ultraviolet (UV) radiation. UV induces the matrix metalloproteinases collagenase, 92-kD gelatinase, and stromelysin, which degrade skin connective tissue and may contribute to premature skin aging (photoaging). Pretreatment of skin with all-trans retinoic acid (tRA) inhibits UV induction of matrix metalloproteinases. We investigated upstream signal transduction pathways and the mechanism of tRA inhibition of UV induction of matrix metalloproteinases in human skin in vivo. Exposure of human skin in vivo to low doses of UV activated EGF receptors, the GTP-binding regulatory protein p21Ras, and stimulated mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38. Both JNK and p38 phosphorylated, and thereby activated transcription factors c-Jun and activating transcription factor 2 (ATF-2), which bound to the c-Jun promoter and upregulated c-Jun gene expression. Elevated c-Jun, in association with constitutively expressed c-Fos, formed increased levels of transcription factor activator protein (AP) 1, which is required for transcription of matrix metalloproteinases. Pretreatment of human skin with tRA inhibited UV induction of c-Jun protein and, consequently, AP-1. c-Jun protein inhibition occurred via a posttranscriptional mechanism, since tRA did not inhibit UV induction of c-Jun mRNA. These data demonstrate, for the first time, activation of MAP kinase pathways in humans in vivo, and reveal a novel posttranscriptional mechanism by which tRA antagonizes UV activation of AP-1 by inhibiting c-Jun protein induction. Inhibition of c-Jun induction likely contributes to the previously reported prevention by tRA of UV induction of AP-1-regulated matrix-degrading metalloproteinases in human skin.

Solar ultraviolet light activates extracellular signal-regulated kinases and the ternary complex factor in human normal keratinocytes.

Exposure to ultraviolet radiation of solar light is responsible for inflammation, premature skin aging and is the main cause of human skin carcinogenesis. While the noxious consequences of U.V. exposure are known, the molecular events triggered by this radiation are poorly understood. We observed that U.V.-A and U.V.-B irradiation of human keratinocytes induces the activation of tyrosine kinase pathways leading to the tyrosine phosphorylation of several cellular proteins. We also observed a stimulation of the Stress Activated Protein kinases (SAPKs), p38 and JNK, and an activation of the transcription factors AP-1 in response to U.V.-A and U.V.-B radiation. Furthermore, we clearly demonstrated that physiological U.V. doses are able to activate the Extracellular signal-Regulated Kinases, ERK1 and ERK2, which could explain the activation of the Ternary Complex Factor. Thus, in human keratinocytes, solar U.V. light activates multiple signalling pathways that could be involved in skin inflammation following U.V.-induced skin injury or in U.V.-induced skin carcinogenesis.

Ionizing radiation activates c-Jun NH2-terminal kinase (JNK/SAPK) via a PKC-dependent pathway in human thyroid cells.

Thyroid gland is known to be higher sensitive to carcinogenic effects of external ionizing radiation (IR) than other tissues. To clarify the cell-specific response following irradiation, activations of c-Jun NH2-terminal kinases (JNKs), which is one of mitogen-activated protein kinases (MAPKs) family members, and extracellular signal-regulated kinase (ERK) were examined in primary cultured human thyroid cells in comparison with human diploid fibroblast cells, WI-38. Although UV exposure strikingly induced JNK activity in both cells, the dose-response increase following IR exposure was observed in thyroid cells with the maximal JNK activity (3.5 fold induction) obtained at 10 Gy exposure, but no increase in WI-38 cells. The JNK activity was reached a maximum of 2.2 fold induction at 30 min after 5 Gy exposure and then sustained for at least 12 hr. On the other hand, ERK activity was not stimulated in thyroid cells following irradiation. The effects of 12-O-tetradecanoylphorbol beta-acetate (TPA) mimicked those of radiation on JNK cascade and 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine 2HCl (H7) and pretreatment with TPA blocked JNK activation following irradiation. Our results demonstrate that IR stimulates JNK activity in cultured human thyroid cells but not in fibroblasts indicating distinct activation and regulation mechanisms of JNK cascade. The JNK activation following IR exposure is mediated at least partially through a PKC-dependent pathway.

Involvement of protein kinase C in the activation of extracellular signal-regulated kinase 1/2 by UVC irradiation.

UVC irradiation activates mitogen-activated protein kinases (MAPKs), including ERK, JNK, and P38. This study examined the role of protein kinase C (PKC) in the regulation of UVC-stimulated MAPKs activation. Either the depletion of PKC by prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or the inhibition of PKC by a selective PKC inhibitor, UCN-01-ME, attenuated UVC-activation of ERK1/2, keeping the activation of JNK1/2 intact. However, K252a, a non-selective PKC inhibitor, inhibited the activation of both ERK1/2 and JNK1/2 by UVC. In three isoforms of PKC (alpha, delta, epsilon) examined, PKC epsilon shows the most evident translocation, a temporal association with cell membrane, upon the UVC irradiation of NIH 3T3 cells. These results suggest that PKC is acting in the UVC-dependent activation of ERK1/2, and PKC epsilon is one of the PKC isozymes playing such a role.

[Effect of x-irradiation on properties of Ca2+-calmodulin-dependent protein kinase from rat spleen lymphocytes]. / Vliianie rentgenovskogo oblucheniia na svoistva Ca2+-kal'modulinzavisimoi proteinkinazy limfotsitov selezenki krys.

Ca(2+)-calmodulin-dependent kinase has been isolated and purified from rat spleen lymphocytes cytosol in control and 12 h after the effect of X-ray radiation in a dose of 0.5 and 1 Gy. The isolated enzyme showed a remarkable similar substrate specificity and kinetic properties to those of rat brain and rat spleen calmodulin-dependent protein kinase II.

Dose rate and mode of exposure are key factors in JNK activation by UV irradiation.

Single exposure of cells to UVC (254 nm for 30 s) or to UVB (300 nm for 10 min) was shown to activate jun-NH2 kinases which, in turn, phosphorylate their substrates ELK-1, c-jun and ATF-2. While UVC (40-80 J/m2) activates JNK up to 4 h, with maximal induction after 30 min, UVB (150-300 J/m2) activates JNK over a prolonged period, up to 24 h, with maximal induction after 6 h. UV-mediated activation of src-related tyrosine kinases and MAPK revealed different kinetics, with maximal induction after 24 h. As recent studies had indicated a role of a UVC component in mediating the ability of UVB to activate JNK, we have examined the effect of dose rate as well as of multiplicity of exposures on the activation of these kinases. The UVC portion found in 300 J/m2 UVB (5%, corresponding to 15 J/m2, administered within 10 s) did not activate JNK. However, when the same dose was administered at a lower rate (i.e. over 10 min, as needed for UVB irradiation) it was found capable of activating JNK, MAPK and src kinases, but to a lower degree and with different kinetics than found for UVB. Such differences point to cellular changes which are elicited by UVB, but not UVC. Although a single UVB exposure using a filter that blocks wavelengths below 300 nm prevented activation of JNK, multiple exposures of filtered UVB wavelengths (mimicking chronic exposure) were able to activate JNK. We conclude that the mode of UVB exposure (dose rate and multiplicity) is a crucial determinant for physiologically relevant activation of JNK.

Activation of c-Jun-NH2-kinase by UV irradiation is dependent on p21ras.

We have demonstrated previously that Jun-NH2-kinase (JNK) activation in vitro is potentiated by association with the p21(ras) protein. To determine if in vivo activation of JNK also depends on p21(ras), we have used M1311 cells that carry the cDNA for the neutralizing antibody to p21(ras), Y13-259, under a dexamethasone-inducible promoter. The ability of UV to activate JNK gradually decreased over a 4-day period of cell growth in dexamethasone. This decrease coincides with weaker transcriptional activation measured via gel shift and chloramphenicol acetyltransferase assays. Peptides corresponding to amino acids 96-110 on p21(ras), which were shown to block Ras-JNK association, inhibited UV-mediated JNK activation in mouse fibroblast 3T3-4A cells as well as in M1311 cells, further supporting the role of p21(ras) in UV-mediated JNK activation. Overall, the present studies provide in vivo confirmation of the role p21(ras) plays in JNK activation by UV irradiation.

UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells.

UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirement for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-alpha- and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis.

The stress response to ionizing radiation involoves c-Abl-dependent phosphorylation of SHPTP1.

c-Abl is a nonreceptor tyrosine kinase that is activated by certain DNA-damaging agents. The present studies demonstrate that nuclear c-Abl binds constitutively to the protein tyrosine phosphatase SHPTP1. Treatment with ionizing radiation is associated with c-Abl-dependent tyrosine phosphorylation of SHPTP1. The results demonstrate that the SH3 domain of c-Abl interacts with a WPDHGVPSEP motif (residues 417-426) in the catalytic domain of SHPTP1 and that c-Abl phosphorylates C terminal Y536 and Y564 sites. The functional significance of the c-Abl-SHPTP1 interaction is supported by the demonstration that, like c-Abl, SHPTP1 regulates the induction of Jun kinase activity following DNA damage. These findings indicate that SHPTP1 is involved in the response to genotoxic stress through a c-Abl-dependent mechanism.

Impaired jun-NH2-terminal kinase activation by ultraviolet irradiation in fibroblasts of patients with Cockayne syndrome complementation group B.

c-jun-NH2 kinases (JNK) are among the UV-activated protein kinases that play an important role in cellular stress response via the phosphorylation of c-jun, ATF2, and p53. Activation of JNK by UV irradiation requires cooperation between membrane and nuclear components, including DNA lesions per se. The role of DNA lesions in JNK activation led us to explore the inducibility of these kinases in cells of repair-deficient patients. Analyses of primary fibroblast cell lines from patients with Cockayne Syndrome of complementation group B (CS-B) revealed poor JNK activation after UV irradiation in four of five cases when compared with three repair-proficient, normal human fibroblast cell lines. Impaired ability to activate JNK persisted at various time points and with different doses of UV irradiation and coincided with failure of in vitro damaged DNA to activate these kinases. In contrast to UV irradiation, other forms of stress, such as H2O2 or heat shock were capable of inducing JNK activation in CS-B cells. Interestingly, when UV irradiation was administered after osmotic shock, it led to JNK activation in CS-B cells, indicating that alternate signal transduction pathways that are activated in response to other forms of stress can potentiate JNK activation by UV irradiation. Unlike CS-B cells, those of other repair-deficient cells, including xeroderma pigmentosum of different complementation groups, revealed proper activation of JNK by UV irradiation. Together, our findings point to deficiency of JNK activation by UV irradiation in CS-B cells, a phenomenon which may be associated with impaired CS-B, the mutant repair gene in these patients.

Cellular stresses and cytokines activate multiple mitogen-activated-protein kinase kinase homologues in PC12 and KB cells.

The identities of the upstream activators of the mitogen-activated protein (MAP) kinase homologues termed stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK) and SAP kinase-2 (also known as p38, RK and CSBP) were investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators, SAP kinase kinase-1 (SAPKK-1) and SAPKK-2 were activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 was capable of activating both SAP kinase-1 and SAP kinase-2 and was similar, if not identical, to the previously described MAP kinase kinase homologue MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activated SAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, whose appearance depended on the nature of the stimulus. SAPKK-3, which was strongly induced by every stimulus tested (osmotic shock, ultraviolet irradiation, anisomycin or IL-1), accounted for about 95% of the SAP kinase-2 activator activity in these cells, did not activate SAP kinase-1 and eluted from Mono S at a lower salt concentration than SAPKK-2. SAPKK-4 and SAPKK-5 were also eluted from Mono S at higher NaC1 concentrations than SAPKK-3 and these enzymes activated SAP kinase-1 but not SAP kinase-2. SAPKK-4 was the only SAP kinase-1 activator induced by interleukin-1 or ultraviolet irradiation, while two SAP kinase-1 activators, SAPKK-1 and SAPKK-5, were induced by osmotic shock or anisomycin. SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5, were not activated by MEK kinase in vitro, were separable from the major activator(s) of p42 MAP kinase, and were not recognised by anti-MKK4 antibodies. At least two of these enzymes are likely to be novel MAP kinase kinase homologues. Our results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type.

jun-NH2-terminal kinase activation mediated by UV-induced DNA lesions in melanoma and fibroblast cells.

jun-NH2-terminal kinase (JNK) belongs to a family of protein kinases that phosphorylates c-Jun, ATF2, and Elk1 in response to various forms of stress including UV irradiation and heat shock. Although in previous studies we have demonstrated the importance of membrane components for JNK activation by UV irradiation, here we have elucidated the role of DNA damage in this response. We show that in vitro-irradiated or sonicated DNA that is added to proteins prepared from UV-treated cells can further induce JNK activation in a dose-dependent manner. When compared with UV-B (300 nm), UV-C (254 nm), which is better absorbed by the DNA, is significantly more potent in activating JNK. Furthermore, when wavelengths lower than 300 nm were filtered out, UV-B was no longer able to activate JNK. With the aid of melanoma and fibroblast cells, which exhibit different resistances to irradiation and require different UV doses to generate the same number of DNA lesions, we demonstrate that above a threshold level of 0.45 lesions and up to 0.75 lesions per 1875 bp, the degree of JNK activation correlates with the amount of lesions induced by UV-C irradiation. Finally, to explore the role of nuclear and mitochondrial DNA (mtDNA) in mediating JNK activation after UV irradiation, we have used cells that lacks mtDNA. Although the lack of mtDNA did not impair the ability of UV to activate JNK, when enucleated, these cells had lost the ability to activate JNK in response to UV irradiation. Overall, our results suggest that DNA damage in the nuclear compartment is an essential component that acts in concert with membrane-anchored proteins to mediate c-Jun phosphorylation by JNK.

UV irradiation and heat shock mediate JNK activation via alternate pathways.

To elucidate cellular pathways involved in Jun-NH2-terminal kinase (JNK) activation by different forms of stress, we have compared the effects of UV irradiation, heat shock, and H2O2. Using mouse fibroblast cells (3T3-4A) we show that while H2O2 is ineffective, UV and heat shock (HS) are potent inducers of JNK. The cellular pathways that mediate JNK activation after HS or UV exposure are distinctly different as can be concluded from the following observations: (i) H2O2 is a potent inhibitor of HS-induced but not of UV-induced JNK activation; (ii) Triton X-100-treated cells abolish the ability of UV, but not HS, to activate JNK; (iii) the free radical scavenger N-acetylcysteine inhibits UV- but not HS-mediated JNK activation; (iv) N-acetylcysteine inhibition is blocked by H2O2 in a dose-dependent manner; (v) a Cockayne syndrome-derived cell line exhibits JNK activation upon UV exposure, but not upon HS treatment. The significance of Jun phosphorylation by JNK after treatment with UV, HS, or H2O2 was evaluated by measuring Jun phosphorylation in vivo and also its binding activity in gel shifts. HS and UV, which are potent inducers of JNK, increased the level of c-Jun phosphorylation when this was measured by [32P]orthophosphate labeling of 3T3-4A cultures. H2O2 had no such effect. Although H2O2 failed to activate JNK in vitro and to phosphorylate c-Jun in vivo, all three forms of stress were found to be potent inducers of binding to the AP1 target sequence. Overall, our data indicate that both membrane-associated components and oxidative damage are involved in JNK activation by UV irradiation, whereas HS-mediated JNK activation, which appears to be mitochondrial-related, utilizes cellular sensors.