Role of p38 MAPK in UVB-Induced Inflammatory Responses in the Skin of SKH-1 Hairless Mice
2005; Elsevier BV; Volume: 124; Issue: 6 Linguagem: Inglês
10.1111/j.0022-202x.2005.23747.x
ISSN1523-1747
AutoresArianna L. Kim, Jeffrey M. Labasi, Yucui Zhu, Xiuwei Tang, Kim F. McClure, Christopher A. Gabel, Mohammad Athar, David R. Bickers,
Tópico(s)melanin and skin pigmentation
ResumoThe p38 mitogen-activated protein kinase (MAPK) signaling pathway is activated by numerous inflammatory mediators and environmental stresses. We assessed the effects of ultraviolet B (UVB) on the p38 MAPK pathway and determined whether cyclooxygenase (COX)-2 expression is downstream of this kinase in the skin of UVB-irradiated SKH-1 mice. SKH-1 mice were irradiated with a single dose of UVB (360 mJ per cm2), and activation of the epidermal p38 MAPK pathway was assessed. UVB-induced phosphorylation of p38 MAPK occurred in a time-dependent manner. Phosphorylation of MAPK-activated protein kinase-2 (MAPKAPK-2) also was detected and correlated with an increase in its kinase activity. Phosphorylation of heat shock protein 27 (HSP27), a substrate for MAPKAPK-2, also was detected post-irradiation. Oral administration of the p38 inhibitor, SB242235, prior to UVB irradiation, blocked activation of the p38 MAPK cascade, and abolished MAPKAPK-2 kinase activity and phosphorylation of HSP27. Moreover, SB242235 inhibited expression of the pro-inflammatory cytokines interleukin (IL)-6 and KC (murine IL-8) and COX-2. Our data demonstrate that UVB irradiation of murine skin activates epidermal p38 MAPK signaling and induces a local pro-inflammatory response. Blockade of the p38 MAPK pathway may offer an effective approach to reducing or preventing skin damage resulting from acute solar radiation. The p38 mitogen-activated protein kinase (MAPK) signaling pathway is activated by numerous inflammatory mediators and environmental stresses. We assessed the effects of ultraviolet B (UVB) on the p38 MAPK pathway and determined whether cyclooxygenase (COX)-2 expression is downstream of this kinase in the skin of UVB-irradiated SKH-1 mice. SKH-1 mice were irradiated with a single dose of UVB (360 mJ per cm2), and activation of the epidermal p38 MAPK pathway was assessed. UVB-induced phosphorylation of p38 MAPK occurred in a time-dependent manner. Phosphorylation of MAPK-activated protein kinase-2 (MAPKAPK-2) also was detected and correlated with an increase in its kinase activity. Phosphorylation of heat shock protein 27 (HSP27), a substrate for MAPKAPK-2, also was detected post-irradiation. Oral administration of the p38 inhibitor, SB242235, prior to UVB irradiation, blocked activation of the p38 MAPK cascade, and abolished MAPKAPK-2 kinase activity and phosphorylation of HSP27. Moreover, SB242235 inhibited expression of the pro-inflammatory cytokines interleukin (IL)-6 and KC (murine IL-8) and COX-2. Our data demonstrate that UVB irradiation of murine skin activates epidermal p38 MAPK signaling and induces a local pro-inflammatory response. Blockade of the p38 MAPK pathway may offer an effective approach to reducing or preventing skin damage resulting from acute solar radiation. activating transcription factor 2 cyclooxygenase 2 extracellular signal-regulated kinase heat shock protein 27 interleukin c-Jun N-terminal kinase mouse KC/N51 originally identified as a PDGF (platelet-derived growth factor)-induced immediate early gene, known as the functional homolog of IL (interleukin)-8 mitogen-activated protein kinase MAPK-activated protein kinase-2 MAP kinase kinase mitogen and stress-activated kinase-1/2 nuclear factor-κB tumor necrosis factor-α ultraviolet B p38 mitogen-activated protein kinase (MAPK) belong to a highly conserved family of serine/threonine protein kinases that include extracellular signal-regulated protein kinases (ERK) and c-Jun N-terminal kinases (JNK). p38 MAPK is involved in numerous cellular processes, such as cell growth and survival, differentiation, development, cell cycle regulation, and cell death, depending on the cell type and stimulus (Juretic et al., 2001Juretic N. Santibanez J.F. Hurtado C. Martinez J. ERK 1,2 and p38 pathways are involved in the proliferative stimuli mediated by urokinase in osteoblastic SaOS-2 cell line.J Cell Biochem. 2001; 83: 92-98Crossref PubMed Scopus (51) Google Scholar; Yosimichi et al., 2001Yosimichi G. Nakanishi T. Nishida T. Hattori T. Takano-Yamamoto T. Takigawa M. CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK).Eur J Biochem. 2001; 268: 6058-6065Crossref PubMed Scopus (135) Google Scholar). The ERK pathway is generally activated by mitogenic stimuli, whereas the JNK and p38 pathways are activated by pro-inflammatory or stressful stimuli. Various stressors, such as ultraviolet (UV) radiation, oxidative injury, heat shock, cytokines, and other pro-inflammatory stimuli, are known to trigger induction of the p38 MAPK-dependent signaling cascade. p38 MAPK exists as four isoforms (α, β, γ, and δ) and is activated by several upstream kinases in vitro, including MAP kinase kinases (MKK) 3, 4, and 6 (Kyriakis and Avruch, 2001Kyriakis J.M. Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation.Physiol Rev. 2001; 81: 807-869Crossref PubMed Scopus (2752) Google Scholar). Phosphorylation of p38 MAPK in turn activates numerous downstream substrates, including p90 MAPK-activated protein kinase-2 (MAPKAPK-2) (Maizels et al., 2001Maizels E.T. Mukherjee A. Sithanandam G. Peters C.A. Cottom J. Mayo K.E. Hunzicker-Dunn M. Developmental regulation of mitogen-activated protein kinase-activated kinases-2 and-3 (MAPKAPK-2/-3) in vivo during corpus luteum formation in the rat.Mol Endocrinol. 2001; 15: 716-733Crossref PubMed Scopus (41) Google Scholar) and mitogen and stress-activated kinase-1/2 (MSK1/2) (Deak et al., 1998Deak M. Clifton A.D. Lucocq L.M. Alessi D.R. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB.EMBO J. 1998; 17: 4426-4441Crossref PubMed Scopus (822) Google Scholar). p90 MAPKAPK-2 and MSK1/2 function to phosphorylate heat shock protein 27 (HSP27) and cAMP-response element binding protein transcriptional factor, respectively (Kato et al., 2001Kato K. Ito H. Iwamoto I. Lida K. Inaguma Y. Protein kinase inhibitors can suppress stress-induced dissociation of Hsp27.Cell Stress Chaperones. 2001; 6: 16-20Crossref PubMed Scopus (42) Google Scholar; Wiggin et al., 2002Wiggin G.R. Soloaga A. Foster J.M. 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In addition to its direct mutagenic effects, UVB induces inflammatory responses that are critical for tumor induction (Wilgus et al., 2002Wilgus T.A. Parrett M.L. Ross M.S. Tober K.L. Robertson F.M. Oberyszyn T.M. Inhibition of ultraviolet light B-induced cutaneous inflammation by a specific cyclooxygenase-2 inhibitor.Adv Exp Med Biol. 2002; 507: 85-92Crossref PubMed Scopus (44) Google Scholar). The UVB-induced inflammatory response is characterized by the acute development of edema and erythema, and increases in dermal inflammatory cell infiltrates and augmented prostaglandin synthesis (Hruza and Pentland, 1993Hruza L.L. Pentland A.P. Mechanisms of UV-induced inflammation.J Invest Dermatol. 1993; 100: 35S-41SCrossref PubMed Scopus (268) Google Scholar; Terui and Tagami, 2000Terui T. Tagami H. Mediators of inflammation involved in UVB erythema.J Dermatol Sci. 2000; 23: S1-S5Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Cyclooxygenases (COX) convert free arachidonic acid into a series of pro-inflammatory eicosanoids including prostaglandins. The COX-1 isoform serves as a housekeeping enzyme and is expressed in most tissues, whereas COX-2 is an inducible isoform generated in response to pro-inflammatory stimuli, including cytokines. The role of p38 MAPK in UVB-induced COX-2 expression has been documented by several laboratories using a human keratinocyte cell line, HaCaT. In this cell system, p38 MAPK, but not ERK, was required for UVB-induced COX-2 expression (Chen et al., 2001Chen W. Tang Q. Gonzales M.S. Bowden G.T. Role of p38 MAP kinases and ERK in mediating ultraviolet-B induced cyclooxygenase-2 gene expression in human keratinocytes.Oncogene. 2001; 20: 3921-3926Crossref PubMed Scopus (157) Google Scholar). In contrast,Ashida et al., 2003Ashida M. Bito T. Budiyanto A. Ichihashi M. Ueda M. Involvement of EGF receptor activation in the induction of cyclooxygenase-2 in HaCaT keratinocytes after UVB.Exp Dermatol. 2003; 12: 445-452Crossref PubMed Scopus (52) Google Scholar recently reported that UVB induction of COX-2 is associated with the activation of EGFR, ERK, p38 MAPK, and PI (phosphatidylinositol) 3-kinase. p38 MAPK transcriptionally regulates COX-2 in HaCat cells following UVB irradiation (Chen et al., 2001Chen W. Tang Q. Gonzales M.S. Bowden G.T. Role of p38 MAP kinases and ERK in mediating ultraviolet-B induced cyclooxygenase-2 gene expression in human keratinocytes.Oncogene. 2001; 20: 3921-3926Crossref PubMed Scopus (157) Google Scholar; Tang et al., 2001Tang Q. Gonzales M. Inoue H. Bowden G.T. Roles of Akt and glycogen synthase kinase 3beta in the ultraviolet B induction of cyclooxygenase-2 transcription in human keratinocytes.Cancer Res. 2001; 61: 4329-4332PubMed Google Scholar). The involvement of p38 MAPK in regulation of the stability of the COX-2 message was also demonstrated in interleukin (IL)-1-treated HeLa cells and lipopolysaccharide-treated primary human monocytes (Ridley et al., 1998Ridley S.H. Dean J.L. Sarsfield S.J. Brook M. Clark A.R. Saklatvala J. A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA.FEBS Lett. 1998; 439: 75-80Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar; Dean et al., 1999Dean J.L. Brook M. Clark A.R. Saklatvala J. p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes.J Biol Chem. 1999; 274: 264-269Crossref PubMed Scopus (455) Google Scholar; Lasa et al., 2000Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade.Mol Cell Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (363) Google Scholar). These data suggest that UVB-induced p38 MAPK activity and its attendant pro-inflammatory signaling are involved in the pathogenesis of skin disorders including SCC. In this study, we have assessed the in vivo effects of UVB irradiation on acute inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Our data indicate that acute UVB exposure induces the rapid activation of the p38 kinase signaling cascade, leading to COX-2 and pro-inflammatory cytokine production. Furthermore, oral administration of a p38 MAPK inhibitor abrogated these UVB-mediated inflammatory responses. Irradiation of SKH-1 mice with a single dose of 360 mJ per cm2 of UVB-induced consistent visible erythema and edema, and this dose was used throughout this study. To demonstrate that UVB-induced activation of p38 MAPK signaling in vivo, mice were irradiated and sacrificed at various time points thereafter (0.5, 2, 8, and 24 h). Dorsal skin patches were excised and the dermis was removed by scraping, and extracts of the epidermal samples were prepared as described in Materials and Methods. Rapid phosphorylation of p38 MAPK was documented following analysis of these extracts by western blotting using a phospho-specific antibody (Figure 1). p38 MAPK phosphorylation was detected as early as 30 min post-irradiation, and was maximal at 24 h. No phosphorylation was detected in non-irradiated skin. Phosphorylation of MAPKAPK-2, a downstream substrate of p38 MAPK, was detected 30 min post-irradiation and gradually increased to its maximum level by 24 h. The total endogenous p38 MAPK and MAPKAPK-2 levels were not altered by UVB irradiation (Figure 1). Western blot analysis also demonstrated that kinases upstream of p38 (MKK3/6) were phosphorylated immediately following UVB irradiation (data not shown). MAPKAPK-2 activation by p38 MAPK requires the phosphorylation of two threonine sites at positions 222 and 334. We assessed the phosphorylation pattern of these two residues after UVB irradiation. Skin sections from UVB-irradiated and non-irradiated mice were subjected to immunohistochemical staining using Thr222- and Thr334-phospho-specific MAPKAPK-2 antibodies. Phosphorylated MAPKAP-2 proteins in both residues were detectable throughout the epidermis following UVB irradiation, and 24 h later, all epidermal cells stained positively for the phospho-MAPKAPK-2 protein (Figure 2). The staining was minimal in the non-irradiated control skin. To distinguish the basal layer staining, the skin section (1 h post-UVB) was stained for proliferating cell nuclear antigen (PCNA), which is an established marker of cellular proliferation in both normal and neoplastic tissues and is required for DNA replication. PCNA expression was noted to be more intense in the basal layer (Figure 2f). MAPKAPK-2 kinase activity was assessed in UVB-irradiated skin extracts by immunoprecipitating with an anti-MAPKAPK-2 antibody and assaying these complexes for kinase activity, using an MAPKAPK-2 substrate peptide; incorporation of 32P-ATP into the substrate peptide was measured by scintillation counting. Figure 3b compares MAPKAP kinase-2 activity in the non-irradiated control skin and the UVB-irradiated skin at 1, 3, and 24 h post-irradiation. Compared with the non-irradiated control, a 5-fold (p=0.018) increase in kinase activity was observed at 3 h, and increases of 2.5-fold (p=0.032) and 4.4-fold (p=0.003) were observed at 1 and 24 h, respectively. Moreover, activation of MAPKAPK-2 correlated with the appearance of phospho-HSP27 substrate (Figure 3a). These data indicate that a single skin exposure to UVB activates the p38 MAPK signaling cascade in the skin of SKH-1 hairless mice. This finding is consistent with studies demonstrating that p38 kinase is involved in responses to UVB in murine and human cells in vitro (Bulavin et al., 2001Bulavin D.V. Higashimoto Y. Popoff I.J. et al.Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase.Nature. 2001; 411: 102-107Crossref PubMed Scopus (448) Google Scholar). To further explore the role of p38 in mediating UVB-induced inflammatory responses, we administered the p38 MAPK inhibitor SB242235 (Badger et al., 2000aBadger A.M. Griswold D.E. Kapadia R. Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis.Arthritis Rheum. 2000; 43: 175-183Crossref PubMed Scopus (200) Google Scholar, Badger et al., 2000bBadger A.M. Roshak A.K. Cook M.N. et al.Differential effects of SB 242235, a selective p38 mitogen-activated protein kinase inhibitor, on IL-1 treated bovine and human cartilage/chondrocyte cultures.Osteoarthritis Cartilage. 2000; 8: 434-443Abstract Full Text PDF PubMed Scopus (46) Google Scholar) orally to mice 30 min prior to UVB irradiation. Epidermal extracts from these mice were analyzed for HSP27 phosphorylation by western blotting (Figure 4a) and for MAPKAPK-2 kinase activity (Figure 4b). As shown in Figure 4a, HSP27 phosphorylation induced by UVB was abolished at 1 h (lanes 3–6) and significantly reduced at 3 h (lanes 7–10) and 24 h (lanes 11–14) in mice treated with SB242235, relative to vehicle-treated controls. The level of total endogenous HSP27 is not altered by UVB irradiation (Figure 4a). SB242235 treatment completely abolished MAPKAPK-2 kinase activity at the earlier time points (1 and 3 h), which was returned to baseline at 24 h (Figure 4b). Figure 4c compares the level of MAPKAPK-2 kinase activity observed in the skin of SB242235-treated animals (dotted line) with plasma concentration of SB242235 (solid line) at 1, 3, and 24 h post-UVB irradiation. Plasma concentrations of SB242235 (4.5 μg per mL) were maximal at 1 h post-dose and gradually decreased; at 24 h, no plasma-associated drug was detectable. We have previously shown that COX-2 expression is enhanced by UVB irradiation and that COX-2 is overexpressed in UVB-induced SCC in SKH-1 mice (Athar et al., 2001Athar M. An K.P. Morel K.D. et al.Ultraviolet B(UVB)-induced cox-2 expression in murine skin: An immunohistochemical study.Biochem Biophys Res Commun. 2001; 280: 1042-1047Crossref PubMed Scopus (101) Google Scholar; An et al., 2002An K.P. Athar M. Tang X. et al.Cyclooxygenase-2 expression in murine and human nonmelanoma skin cancers: Implications for therapeutic approaches.Photochem Photobiol. 2002; 76: 73-80Crossref PubMed Scopus (172) Google Scholar). We therefore determined whether p38 MAPK could be involved in UVB-induced COX-2 expression. Mice were irradiated with UVB and COX-2 protein detected by western blotting of epidermal extracts. COX-2 protein was detectable 3 h following UVB irradiation (lanes 7–10) and increased further by 24 h (lanes 11–14) (Figure 5a). Non-irradiated epidermal samples (two mice: lanes 1 and 2) and the irradiated epidermis at 1 h following UVB (lanes 3–6) showed no detectable COX-2 protein. SB242235 administration significantly reduced the level of COX-2 protein observed 3 h after UVB irradiation. COX-2 expression, however, was observed in some animals at the 24 h time point. Since COX-2-derived metabolites are components of inflammatory responses, we asked whether the p38 MAPK inhibitor influenced the development of UVB erythema. UVB-induced erythema in these mice generally peaks 5 d after exposure. Figure 5b shows a representative picture of animals exposed to 360 mJ per cm2 on day 5 (left panel: a). Variable erythema was seen in these four mice, likely reflecting the fact that the animals are free to move around the cage during irradiation. Typically, four mice in a cage are irradiated simultaneously. Although the degree of redness varied among the four animals, each showed clear evidence of erythema. A single oral dose of SB242235 administered 30 min prior to UVB irradiation virtually eliminated the development of erythema in these mice (right panel: b). Keratinocytes are major contributors to epidermal cytokine production either constitutively or in response to various stimuli involved in the inflammatory responses, as well as cell proliferation and differentiation processes (Grone, 2002Grone A. Keratinocytes and cytokines.Vet Immunol Immunopathol. 2002; 88: 1-12Crossref PubMed Scopus (308) Google Scholar). IL-6 and IL-8 are among the best-characterized keratinocyte-derived pro-inflammatory cytokines (Ansel et al., 1990Ansel J. Perry P. Brown J. et al.Cytokine modulation of keratinocyte cytokines.J Invest Dermatol. 1990; 94: 101S-107SAbstract Full Text PDF PubMed Google Scholar; Luger et al., 1990Luger T.A. Schwarz T. Krutmann J. Kock A. Urbanski A. Kirnbauer R. Cytokines and the skin.Curr Probl Dermatol. 1990; 19: 35-49Crossref PubMed Google Scholar; McKenzie et al., 1994McKenzie R.C. Venner T.J. Sauder D.N. Farkas-Himsley H. 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We assessed the production of IL-6 and KC in UVB-irradiated murine epidermis and determined whether p38 MAPK was involved in their production. The levels of both KC and IL-6 increased substantially in response to UVB, showing a 9-fold (p=0.0007) and 3-fold (p=0.001) induction, respectively, at 24 h after irradiation, as compared with the non-irradiated control (Figure 6). A significant reduction in the levels of KC and IL-6 was observed with SB242235 treatment. These results demonstrate that the p38 MAP kinase signaling pathway is necessary for UVB-induced cytokine expression in murine skin. Sunburn is a visible cutaneous manifestation of activation of inflammatory signaling pathways resulting from environmental exposure to solar radiation, particularly UVB. Furthermore, sun exposure is clearly the most important factor in the development of skin cancer. In this study, we used SKH-1 hairless mice to determine the role of the p38 MAPK signaling pathway in the inflammatory response to UVB. Our results demonstrate that UVB activates the p38 MAPK pathway, inducing acute inflammatory responses and erythema. The time-dependent activation of p38 MAPK signaling correlates with UVB-mediated elaboration of the pro-inflammatory cytokines IL-6 and murine KC (IL-8), as well as COX-2. p38 MAPK pathway activation was confirmed by detecting phosphorylation of MAPKAPK-2 and HSP27 and by directly measuring the kinase activity of MAPKAPK-2. Phosphorylation of MAPKAPK-2 occurred rapidly (within 15 min) after UVB exposure, suggesting that p38 MAPK signaling is involved in the immediate response to this form of energy. Acute responses to UVB are driven by inflammatory pathways and may reflect the direct effects of absorbed photons on DNA (Tedesco et al., 1997Tedesco A.C. Martinez L. Gonzalez S. 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Deficiency of the stress kinase p38alpha results in embryonic lethality: Characterization of the kinase dependence of stress responses of enzyme-deficient embryonic stem cells.J Exp Med. 2000; 191: 859-870Crossref PubMed Scopus (250) Google Scholar). In A431 cells, for example, p38 MAPK mediates IL-6 synthesis in response to IL-4, and inhibition of p38 MAPK reduces the stability of IL-6 mRNA (Wery-Zennaro et al., 2000Wery-Zennaro S. Zugaza J.L. Letourneur M. Bertoglio J. Pierre J. IL-4 regulation of IL-6 production involves Rac/Cdc42-and p38 MAPK-dependent pathways in keratinocytes.Oncogene. 2000; 19: 1596-1604Crossref PubMed Scopus (53) Google Scholar). Our results show that IL-6 and KC (IL-8) are induced in SKH-1 murine skin following UVB irradiation. Furthermore, oral administration of the p38 MAPK inhibitor SB242235 completely inhibited the production of these cytokines as well as COX-2 expression, suggesting that regulation of COX-2 is dependent on p38 MAPK. Recently, MAPKAPK-2 was shown to contribute to increased COX-2 mRNA stability following taxane treatment, and a selective p38 MAPK inhibitor suppressed taxane-mediated COX-2 stimulation in human mammary epithelial cells (Subbaramaiah et al., 2003Subbaramaiah K. Marmo T.P. Dixon D.A. Dannenberg A.J. Regulation of cyclooxgenase-2 mRNA stability by taxanes: Evidence for involvement of p38, MAPKAPK-2, and HuR.J Biol Chem. 2003; 278: 37637-37647Crossref PubMed Scopus (176) Google Scholar). Following oral administration of SB242235, the UVB-induced inflammatory response was attenuated. This agent effectively suppressed activation of MAPKAPK-2 and phosphorylation of HSP27 following UVB irradiation. SB242235 also suppressed UVB-induced induction of COX-2 and the cytokines IL-6 and KC. These pharmacological effects were manifested within 1 and 3 h of irradiation, but returned to baseline by 24 h. SB242235 demonstrated a short plasma half-life, and at the dose used in this study, plasma levels of the compound were undetectable after 24 h, suggesting that its disappearance coincided with loss of its pharmacological effects. Moreover, a single oral dose of SB242235 effectively abrogated erythema development in UVB-irradiated murine skin. The spectral property of SB242235 showed that it absorbs UVR between 290 and 380 nm, with maximum absorption at 330 nm. In order to determine whether the oral administration of SB242235 exerts a photoprotective effect in the epidermis, we measured thymine dimers in epidermal DNA, which is a direct index for DNA damage following UV radiation of the skin (Antille et al., 2003Antille C. Tran C. Sorg O. Carraux P. Didierjean L. Saurat J.H. Vitamin A exerts a photoprotective action in skin by absorbing ultraviolet B radiation.J Invest Dermatol. 2003; 121: 1163-1167Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We found that UVB irradi
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