Suramin sodium

ABSTRACT

UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogenactivated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1-ERK2) or JNK-SAPK (cJun NH^sub 2^-terminal kinase-stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signaling pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen-dependent activation of ligand-receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation.

INTRODUCTION

For a long time, UVA (320-400 nm) radiation was wrongly considered less noxious than UVB (290-320 nm), which can directly damage proteins and nucleic acids. However, UVA penetrates more deeply into the skin than UVB, reaching not only the epidermis but also the dermis. Moreover, after its absorption by endogenous chromophores, UVA leads to the formation of reactive oxygen species, which can alter protein and nucleic acid structure (1-3). UVA can thus modify cell-matrix interactions (4), disorganize the cytoskeleton (5) and alter different cellular functions (6,7). Among the cellular functions, UVA can activate transcription factors such as API, NF-kB or TCF and thus modulates the expression of various genes (6,8,9). However, how these UVA effects are transduced at the intracellular level is unclear.

Mitogen-activated protein kinase (MAPK) cascades are essential intracellular transduction pathways that are conserved from yeasts to mammalian cells. The extracellular-regulated kinase (ERK) cascade, involving ERK1 and ERK2, was the first to be discovered and is characterized by its ability to be strongly activated by various mitogenic signals such as growth factors and hormones (10,11). Two other MAPK cascades were subsequently identified, namely, the cJun NH^sub 2^-terminal kinase-stress-activated protein kinase (JNK-SAPK) (12) and the p38-reactivating kinase (p38 MAPK) (13). These two groups of MAPK arc also called SAPK because they are mainly activated by various cellular stresses such as inflammatory cytokines, UV radiation, osmotic shock and heat shock (14).

Most studies of the effects of UV radiation on MAPK activities have been carried out with UVC radiation (200-290 nm), which does not reach the earth's surface. UVC activates all the MAPK so far studied, albeit with a more marked effect on JNK-SAPK and p38 MAPK than on ERK1 and ERK2 (12,15,16). Moreover, these studies using UVC have provided a good deal of information on the different actors in MAPK cascades. Concerning UVA effects on skin cells, it can also stimulate ERK, JNK-SAPK and p38 MAPK activities in keratinocytes (8,17,18). Using foreskin fibroblasts, Klotz et al. (19) observed that high doses of UVA radiation activated JNK-SAPK and p38 MAPK but not ERK. However, high doses of UVA radiation can be toxic to fibroblasts, especially when cultured within a collagen matrix, an environment that confers characteristics close to those observed in vivo (20).

In this study we analyzed the effects of naturally occurring doses of UVA radiation on MAPK activities in human dermal fibroblasts in monolayer culture. We observed a selective activation of p38 MAPK by UVA and investigated parts of the UVA signaling pathway leading to this kinase activation. Our experiments demonstrated the involvement of different signaling pathways in p38 MAPK activation by UVA radiation.

MATERIALS AND METHODS

Cell culture and irradiation

Fibroblast cultures were established from skin explants obtained during breast plastic surgery from 18 to 45 year old healthy donors. Fibroblasts were grown in Earle modified Eagle medium (EMEM, Gibco Life Technologies, Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS, Gibco Life Technologies), 100 lU/mL penicillin (Boehringer Mannheim, Roche, Meylan, France), 100 ng/mL streptomycin (Boehringer Mannheim) and 2.5 [mu]g/mL amphotericin B (Gibco Life Technologies) in 5% C0^sub 2^-95% air at 37[degrees]C. Fibroblasts were further propagated in EMEM supplemented with 10% FCS but without antibiotics, amphotericin and phenol red.

Fibroblasts between passages 4 and 6 were seeded at 4 x 10^sup 5^ cells per 60 mm tissue culture dish and grown for 3 days. Confluent cultures were then rendered quiescent by incubation for 24 h in EMEM containing 0.5% FCS but without antibiotics, amphotericin and phenol red. Just before irradiation or treatment, cells were rinsed once in Hanks balanced salt solution (HBSS, Gibco Life Technologies) and further incubated in HBSS for 45 min. Cultures, placed on a 37[degrees]C regulated plate, were irradiated with UVA doses ranging from 0.5 to 50 J/cm^sup 2^ (fluence rate equal to 4 mW/cm^sup 2^) or UVC of 0.01 J/cm^sup 2^. As previously described, cells were UVA-irradiated with an UV-365 illuminating table (Vilbert Lourmat, Marne la Vallee, France) equipped with two additional 4 mm thick sheets of glass to fully remove the UVB component, which is incompletely eliminated by the built-in filter (7). UVC irradiation (254 nm) was performed using an ultraviolet cross-linker (Amersham Life Science, Orsay, France). Controls consisted of sham-irradiated cells processed in the same way but kept in the dark. Experiments were repeated at least three times with fibroblasts from different donors.

Pharmacological treatments

When indicated, fibroblast cultures were treated with various compounds added during the last HBSS incubation step before irradiation.

Inhibition of p38 MAPK activity. SB203580 (Alexis Corporation, Paris, France), a p38 MAPK inhibitor, was used at 5 [mu]M for 20 min before irradiation.

Analysis of reactive oxygen species involvement in p38 MAPK activation. Fibroblast cultures were treated for 45 min with 200 [mu]M H^sub 2^O^sub 2^. We also pretreated fibroblasts before UVA radiation with the following antioxidants: 50 mM L-histidine, 20 mM sodium azide, 100 [mu]M o-phenanthroline, 100 [mu]M ascorbic acid, 100 mM mannitol or 30 mM N-acetylcysteine for 45 min and 50 [mu]M vitamin E for 24 h.

Studies of interactions between growth factor and UVA signaling pathways. To examine growth factor downregulation (desensitization), cells were pretreated for 2 h with either 50 ng/mL epidermal growth factor (EGF) or 20 ng/mL platelet-derived growth factor (PDGF); they were then treated again with 50 ng/mL EGF or 20 ng/mL PDGF for 5 min or were irradiated with 10 J/cm^sup 2^ UVA.

To inhibit ligand-receptor interactions, fibroblasts were pretreated with 0.3 mM suramin for 45 min before irradiation with 10 J/cm^sup 2^ UVA or 0.01 J/cm^sup 2^ UVC. We also studied the influence of suramin prelreatment on the effect of 50 ng/mL anisomycin (Sigma, Saint Quentin Fallavier, France) or 0.5 M NaCl for 45 min and 50 ng/mL EGF, 20 ng/mL PDGF, 100 nM insulin or 20% FCS for 5 min.

Evaluation of the role of ribotoxic stress in UVA-induced p38 MAPK activation. To evaluate the consequences of altered ribosome functioning, fibroblasts were pretreated with 100 [mu]g/mL emetine for 5 min before irradiation with 10 J/cm^sup 2^ UVA or 0.01 J/cm^sup 2^ UVC. Emetine-pretreated fibroblasts were also treated with 50 ng/mL anisomycin, 0.5 M NaCl or 200 [mu]W H^sub 2^O^sub 2^ for 45 min or 50 ng/mL EGF for 5 min.

Cell lysis

Fibroblast cultures were scraped in 100 [mu]L of lysis buffer 45 min after UVC irradiation and immediately after UVA irradiation together with cultures under pharmacological treatments. The lysis buffer contained 25 mM N-2-hydroxythylpiperazine-N'-2-ethane-sulfonic acid (HEPES) (pH 8), 0.2 mM ethylenediamine tetraacetic acid (EDTA), 1.5 mM MgCl^sub 2^, 0.3 M NaCl, 0.1% Triton X-100, 20 mM. [beta]-glycerophosphate, 1 [mu]M microcystine-LR, 0.1 mM orthovanadate and protease inhibitors (21) (all products were from Sigma unless otherwise stated). Cell suspensions were rotated at 4[degrees]C for 30 min and cleared by centrifugation at 13 000 g for 10 min at 4[degrees]C.

Supernatant fractions containing equal amounts of protein were processed according to the MAPK of interest.

Western blot analysis of p38 MAPK and ERK1-ERK2

Supernatants were mixed with 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (40% glycerol, 8% SDS, 250 mM Tris-HCl (pH 6.8), 0.004% bromophenol blue and 20% [beta]-mercaptoethanol) to obtain a 1x final concentration. Samples were then separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Membranes were blocked for 1 h with 5% dried milk in Tris-buffered saline Tween-20 (TBST-20 mM Tris-HCl [pH 8], 150 mM NaCl and 0.05% Tween-20 [Bio-Rad, Marnes la Coquette, France]) and incubated overnight at 4[degrees]C with p38 MAPK antibody, phospho-specific p38 MAPK antibody or phospho-specific ERK1-ERK2 antibody (1:2000, New England Biolabs, Ozyme, Saint Quentin en Yvelines, France). After three washes for 5-10 min in TBST, membranes were incubated for 1 h with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (1:5000, Amersham Life Science). Membranes were finally washed three times for 5-10 min in TBST before detection of the immune signal with the enhanced chemiluminescence detection system (NEN Research Products, Paris, France).

JNK-SAPK activation analysis by solid-phase assays with glutathione S-transferase fusion protein

Kinase assays were performed according to the method of Hibi et al. (12) with a few modifications. Supernatants obtained after cell lysis were diluted with three volumes of dilution buffer containing 20 mM HEPES (pH 8), 0.1 mM EDTA, 2.5 mM MgCl^sub 2^, 0.05% Triton X-100, 20 mM [beta]-glycerophosphate, 0.1 mM orthovanadate and protease inhibitors as described above. The diluted cell extracts were rotated at 4[degrees]C for 30 min and centrifuged at 13 000 g for 10 min at 4[degrees]C. Supernatants were mixed with 10-20 [mu]L of GSH-agarose suspension containing 10-20 [mu]g of glutathione S-transferase (GST)-cJun 1-79 (kindly provided by Dr. Louis Mahadevan; Nuclear Signalling Laboratory, Oxford, UK). The mixtures were rotated overnight at 4[degrees]C and then washed four times in HEPES binding buffer (HBIB) (20 mM HEPES [pH 8], 50 mM NaCl, 0.1 mM EDTA, 25 mM MgCl^sub 2^ and 0.05% Triton X-100) and once in kinase buffer (20 mM HEPES [pH 8], 2 mM dithiotreitol [DTT], 20 mM [beta]-glycerophosphate, 20 mM MgCl^sub 2^ and 0.1 mM orthovanadate).

Pellets were finally suspended in 30 [mu]L of kinase buffer containing 20 [mu]M unlabeled adenosine triphosphate (ATP) and 3 [mu]Ci of [[gamma]-32P]ATP (NEN Research Products). After 40 min at 30[degrees]C the reaction was terminated by two washes with HBIB. Phosphorylated proteins were eluted in 30 [mu]L of boiling 2x SDS-PAGE sample buffer, resolved on 10% polyacrylamide gels and detected by autoradiography with Kodak X-Omat films.

RESULTS

p38 MAPK is selectively activated by UVA

Confluent quiescent human libroblasts were irradiated with UVA at doses from 1 to 50 J/cm^sup 2^. No activation of JNK-SAPK or ERK kinases was observed. However, both MAPK groups could be activated in our experimental conditions. As expected, GST-cJun 1-79 phosphorylation by JNK-SAPK was stimulated by UVC (positive control) (Fig. 1a). Concerning ERK1 and ERK2, 5 min of EGF treatment (positive control) further stimulated their phosphorylation, which was already induced in HBSS-incubated control cells (Fig. 1b). However, no additional stimulation was observed after UVA and UVC irradiation. On the contrary, increasing doses of UVA irradiation led to a decrease in ERK1 and ERK2 phosphorylation, and over 20 J/cm^sup 2^ the phosphorylation was no longer observed. This could be due to UVA-induced expression of a phosphatase. Indeed, UV radiation and oxidative stress (22) can induce the expression of CL100, a phosphatase with dual specificity for ERK1 and ERK2. In contrast, we observed a dose-dependent increase in p38 MAPK phosphorylation, starting at 2 or 5 J/cm^sup 2^ UVA according to the experiment. As expected, p38 MAPK was phosphorylated after UVC irradiation (Fig. 1c).

We also did a time-course analysis to evaluate the activation state of the three types of MAPK after UVA radiation. Fibroblasts were irradiated with UVA (20 J/cm^sup 2^), and the activation level of MAPK was studied at different times for 60 min after irradiation. We could not see any activation of ERK1, ERK2 and JNK-SAPK, although p38 MAPK activation by UVA lasted up to 60 min (data not shown).

We also observed that UVA-induced p38 MAPK activation was completely inhibited by a pretreatment with 5 [mu]M SB203580, a specific p38 MAPK inhibitor (23), whereas UVC-induced phosphorylation of p38 MAPK was only partially inhibited (Fig. 2). The decrease in p38 MAPK phosphorylation could be due to the inhibition of its ability to autophosphorylate (24,25) or to a direct effect of SB203580 on p38 activation (26).

This selective activation of p38 MAPK suggests that this kinase plays a critical role in transducing the effects of UVA at the intracellular level.

Singlet oxygen is involved in UVA activation of p38 MAPK

To examine the involvement of reactive oxygen species in p38 MAPK activation in response to UVA in human dermal fibroblasts, we first treated fibroblast cultures with H^sub 2^O^sub 2^; like UVA and UVC, H^sub 2^O^sub 2^ stimulated p38 MAPK phosphorylation (Fig. 3a). H^sub 2^O^sub 2^ also stimulated JNK-SAPK activity but had no effect on ERK1-ERK2 activity (data not shown).

We also studied the effects of antioxidants on UVA activation of p38 MAPK. p38 MAPK was still activated by UVA in the presence of an iron chelator (o-phenanthroline), free radical scavengers (ascorbic acid, manitol and N-acetylcysteine) or an inhibitor of lipid peroxidation (vitamin E) (data not shown). p38 MAPK phosphorylation in response to UVA (10 J/cm^sup 2^) was clearly inhibited by the singlet oxygen quenchers, L-histidine (50 mM) and sodium azide (20 mM), whereas these reagents had no significant effect in sham-irradiated fibroblasts (Fig. 3b).

The convergent results obtained with L-histidine and sodium azide support a particular role of singlet oxygen in UVA-induced p38 MAPK activation.

Growth factor signaling pathways are involved in UVA-induced p38 MAPK activation

To further analyze the mechanism of p38 MAPK activation by UVA, we investigated whether the UVA signaling pathway shared components with growth factor signaling pathways. To test this hypothesis, we arbitrarily used EGF and PDGF among the large growth factor family. A single short treatment of fibroblasts with EGF (50 ng/mL) or PDGF (20 ng/mL) stimulated p38 MAPK phosphorylation (Fig. 4). As expected, both growth factors were able to desensitize their own signaling pathways: after a prolonged treatment for 2 h with EGF or PDGF (desensitized cells), p38 MAPK recovered a basal level of phosphorylation, but fibroblasts were no longer able to respond to a second short treatment with EGF or PDGF (Fig. 4). Relative to nondesensitized fibroblasts, p38 MAPK activation in EGF- or PDGF-desensitized cells was partially inhibited in response to UVA. These observations from several experiments reveal interactions between the UVA and EGF-PDGF signaling pathways (Fig. 4).

We also used suramin, a nonselective inhibitor of ligand-receptor interactions (27-29), to determine whether growth factor receptors were involved in p38 MAPK activation by UVA. Pretreatment of fibroblasts with suramin blocked p38 MAPK activation in response to UVA and UVC but not in response to NaCl or anisomycin (Fig. 5a). Moreover, in suramin-pretreated cells, p38 MAPK activation was strongly inhibited by growth factors such as PDGF and insulin and partially inhibited by FCS. However, suramin did not block the effects of EGF on p38 MAPK activation (Fig. 5b).

Our observations imply that components of growth factor signaling pathways are involved in the transduction of UVA effects. Furthermore, the results of suramin pretreatment suggest that binding of growth factors to their receptor, likely different from the EGF receptor, could be involved in p38 MAPK activation by UVA as for the UVC activation.

Active ribosomes are involved in UVA-induced p38 MAPK activation

To examine possible interactions between the UVA signaling pathway involved in p38 MAPK activation and ribotoxic stress, we used emetine to alter ribosome functioning (30,31). As expected, emetine partially inhibited p38 MAPK phosphorylation induced by UVC, H^sub 2^O^sub 2^ and anisomycin (30,31) but not that induced by NaCl or EGF (Fig. 6). Emetine also markedly inhibited the effects of UVA on p38 MAPK activation (Fig. 6a), suggesting that UVA triggers the ribotoxic stress response, leading to modulation of p38 MAPK activation.

DISCUSSION

Selective activation of p38 MAPK by UVA

We examined the effects of naturally occurring doses and fluence rate of UVA radiation on MAPK activities in normal human adult dermal fibroblasts. MAPK are known to be involved in the transduction of many cellular stresses (14) and their activities after UVC radiation has been widely studied. In contrast, few studies have analyzed the effects of UVA on MAPK.

In this study we observed a dose-dependent increase in p38 MAPK phosphorylation in fibroblast monolayers, starting at a UVA dose of 2 or 5 J/cm^sup 2^. In contrast, JNK-SAPK and ERK kinases were not activated by UVA, whereas they were activated by UVC and EGF, respectively. These observations show a selective activation of p38 MAPK compared with JNK-SAPK, whereas usually p38 MAPK and JNK-SAPK are activated together (32).

Klotz et al. (19) observed UVA activation of JNK-SAPK and p38 MAPK but not of ERK, but these results were obtained with foreskin fibroblasts and high UVA doses (30 J/cm^sup 2^) with a fluence rate 10 times higher than our values (40 mW/cm^sup 2^). We suggest that low UVA doses or fluence rate (or both) may selectively trigger p38 MAPK activation, whereas high doses or fluence rate (or both) would also lead to JNK-SAPK activation because of nonspecific cellular stresses. However, in response to UVA the behavior of MAPK is different according to the skin cell type studied. In keratinocytes, UVA activates the three groups of MAPK even at doses as low as 0.3 J/cm^sup 2^ (8,17,18), whereas in melanocytes UVA (1 J/cm^sup 2^) activates only ERK1 and ERK2 (33).

SB203580 is a selective inhibitor of MAPK isoforms (23), p38[alpha] and p38[beta] MAPK. We observed a decrease of UVA-induced p38 MAPK phosphorylation in SB203580-treated fibroblasts. This could be due to an inhibition of the ability of p38 MAPK to autophosphorylate (24,25). However, Frantz et al. (26) demonstrated that SB203580 could also inhibit inducible phosphorylation of p38 MAPK. Whatever the mechanism involved in the inhibition of UVA-induced phosphorylation of p38 MAPK by SB203580, our results suggest the involvement of p38a or p38[beta] MAPK (or both) in the effects of UVA. Other p38 MAPK isoforms are probably involved in p38 MAPK phosphorylation by UVC because SB203580 pretreatment only slightly inhibited UVC-induced p38 MAPK phosphorylation under our experimental conditions.

Selective activation of p38 MAPK by naturally occurring doses and fluence rate of UVA suggests that this kinase could play a key role in transducing the effects of UVA at the intracellular level. Interestingly, MAPK-activated protein kinase-2 (MAPKAP kinase-2), which is specifically activated by p38 MAPK in vivo, leads to the phosphorylation of the small heat shock protein 27 (Hsp27), which is known to promote actin polymerization and to facilitate recovery from the cytoskeleton disruption by cellular stresses (13). Nevertheless, the regulation of actin dynamics via p38 MAPK activation depends on the level of Hsp27 expression, which is low in fibroblasts (34). This could explain why UVA radiation lends to disorganize actin polymerization in human dermal fibroblasts (5). In addition, p53, the tumor suppressor protein that plays a major role in cell defense against DNA-damaging agents (35,36), is also a p38 MAPK substrate. p38 MAPK activation by UVB has been shown to enhance resistance of keratinocytes to apoptosis by stabilizing cytoplasmic p53 (37). Thus, p38 MAPK activation and downstream effectors could be important for protecting cells against UVA radiation.

Involvement of singlet oxygen in UVA activation of p38 MAPK

UVA is known for its photooxidative effects generating reactive oxygen species, which can affect several cellular functions (1). When we treated fibroblast cultures with H^sub 2^O^sub 2^, which can penetrate the plasma membrane and mimic the effects of endogenous H^sub 2^O^sub 2^ produced in response to stress (38), both p38 MAPK phosphorylation and INK-SAPK activity were induced. Consequently, the specificity of p38 MAPK activation by UVA points to the involvement of reactive oxygen species other than H^sub 2^O^sub 2^.

The lack of effects of an iron chelator, free radical scavengers and a lipid peroxidation inhibitor suggests that these pathways are not involved in UVA-induced p38 MAPK phosphorylation. In contrast, the inhibition of UVA-induced p38 MAPK phosphorylation by singlet oxygen quenchers, L-histidine and sodium azide confirms the key role of singlet oxygen in p38 MAPK response to UVA, as already reported by Klotz et al. (19).

Involvement of growth factor signaling pathways in UVA aetivation of p38 MAPK

The H^sub 2^O^sub 2^ UVB and UVC signaling pathways share components with growth factor signaling pathways (32,39-42). This was demonstrated by exploiting the property of growth factors to temporarily block subsequent stimulation with the same growth factor (by desensitizing components] of its signaling pathway: homologous desensitization) and also with any agent using the same desensitized component(s) (heterologous desensitization) (32). This phenomenon is due to degradation of the receptor or of a signaling component or to a negative feedback mechanism within the signaling pathway (43-46).

For our experiments, among the large family of growth factors, we arbitrary used EGP and PDGF. In EGF- or PDGF-desensitized fibroblasts we found that p38 MAPK activation was slightly inhibited in response to UVA compared with nondesensitized fibroblasts.

In parallel, suramin, a nonselective inhibitor of ligand-receptor interactions (27-29), blocked the activation of p38 MAPK in response to UVA and UVC but had no effect on p38 MAPK activation by NaCl or anisomycin, which act independently of membrane receptors. The mechanism by which suramin blocks growth factor activity is not fully understood. Some authors have concluded that suramin directly "poisons" plasma membrane receptors (28) and others that it induces conformational changes in the growth factor-receptor complexes but only with certain growth factors such as PDGF, FGF, IGF1 and not with EGF (27,29), in keeping with our observations.

The inhibition of p38 MAPK activation by UVA after the signaling pathway downregulation of growth factors such as PDGF and EGF first indicates that UVA and growth factor signaling pathways could share intracellular component(s). Second, the effects of suramin pretreatment of fibroblasts strongly suggest the intervention of growth factor receptors in the transduction of these UVA effects. Different observations support the conclusion that the activation of growth factor receptors could be due to reactive oxygen species. First, H^sub 2^O^sub 2^ mimics the actions of insulin and EGF by activating their receptors (40,41). Second, Huang et al. (47) demonstrated that UVC activates growth factor receptors via reactive oxygen intermediates. Consequently, UVA could trigger growth factor receptor activation via singlet oxygen, a highly reactive oxygen species, which reacts directly with amino acid residues on proteins (2). However, it is noteworthy that growth factor receptor activation by UVA should also lead to the activation of ERK1 and ERK2 because they are strongly activated by mitogens (10,11). We observed no ERK1 and ERK2 activation by UVA. Because the effects of suramin are not fully elucidated, we cannot exclude the possibility that the component shared by the UVA and growth factor signaling pathways could be downstream of receptors in the p38 MAPK cascade. Indeed, Gro[beta] et al. (48) showed that UV-induced signal transduction could be the consequence of the inactivation of some tyrosine phosphatases that normally dephosphorylate and inactivate growth factor receptors.

Involvement of ribotoxic stress in UVA activation of p38 MAPK

Active ribosomes have been shown to be necessary for transducing the effects of UVC and UVB on JNK-SAPK activity and also for transducing the effects of anisomycin on JNK-SAPK and p38 MAPK. This recently identified stress signaling pathway, largely described by lordanov et al. (30,31), has been called ribotoxic stress and depends on a modification of the 3'-end of the large (23S-28S) ribosomal RNA subunit. Emetine rapidly inhibits translation by altering ribosome functionality and blocks some cellular signaling pathways. Moreover, contrary to other translation inhibitors such as anisomycin and cycloheximide, it does not stimulate JNK-SAPK and p38 MAPK activities (30,31).

As expected, in our experiments emetine partially inhibited p38 MAPK phosphorylation induced by UVC, H^sub 2^O^sub 2^ and anisomycin and had no effect on NaCl or EGF stimulation of p38 MAPK, neither of which involves ribotoxic stress (30,31). Our observation that the effects of UVA are strongly inhibited by emetine suggests that, like UVC and UVB, UVA triggers the ribotoxic stress response leading to p38 MAPK activation but probably via a different mechanism. Indeed, contrary to UVC and UVB, which can cause cell damage through direct absorption by proteins and nucleic acids (1), UVA is poorly absorbed by these cellular components. However, reactive oxygen species generated by UVA radiation, particularly singlet oxygen, can also damage nucleic acids (3). Consequently, UVA could trigger the ribotoxic stress response by damaging ribosomal RNA and subsequently affecting ribosome functioning. However, lordanov and Magun (49) concluded that distinct pathways are involved in JNK-SAPK activation by UVB: one involving reactive oxygen species and another involving ribotoxic stress.

CONCLUSIONS

In this study we examined the effects of naturally occurring doses of UVA radiation on MAPK activities in human dermal fibroblasts and observed the selective activation of p38 MAPK compared with JNK-SAPK, ERK1 and ERK2. We also obtained evidence that singlet oxygen plays a key role in transducing this effect of UVA. In addition, our results implicate two different signaling pathways in p38 MAPK activation by UVA, one involving growth factor signaling component(s) and the other involving ribosome functioning. The involvement of these two distinct pathways could be due to singlet oxygen, which could alter the structure of component(s) of growth factor signaling pathways or ribosome structure (or both). Despite the involvement of different signaling pathways, the selective activation of p38 MAPK suggests a key role of this protein kinase in the effects of UVA radiation.

Acknowledgements-This work was supported by grants from INSERM, European Community Biotech BIO-CT-960036, DGA/DRET 95-127 and DGA 00-34-004. We are extremely grateful to Dr. Patrice Morliere for his advice, to Corinne Lebreton-De Coster and Armelle Tupet for their technical assistance and to David Young for checking the English.

[para]Posted on the website on 5 June 2003

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Rozen Le Panse, Louis Dubertret and Bernard Coulomb*

INSERM Unite 532, Institut de Recherche sur la Peau, Pavillon Bazin, Hopital Saint-Louis, Paris Cedex, France

Received 26 September 2002; accepted 15 May 2003

*To whom correspondence should be addressed at: INSERM Unite 532, Institut de Recherche sur la Peau, Hopital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France. Fax: 33-1-5372-2051; e-mail: bcoulomb@chu-stlouis.fr

Abbreviations: ATP, adenosine triphosphate; EDTA, ethylenediamine tetraacetic acid; EGF, epidermal growth factor; EMEM, Earle modified Eagle medium; ERK, extracellular regulated Kinase; FCS, fetal calf serum; HBIB, HEPES binding buffer; HBSS, Hanks balanced salt solution; HEPES, hydroxythylpiperazine-N'-2-ethane-sulfonic acid; GST, glutathione S-transferase; Hsp27, heat shock protein 27; JNK-SAPK, c-Jun NH^sub 2^-terminal kinase-stress-activated protein kinase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; TBST, Tris-buffered saline Tween-20.

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