RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling

Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations, and the onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that lead to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB, encoding a small GTPase has been identified as a DNA damage-inducible gene. RhoB is involved in epidermal growth factor (EGF) receptor trafficking, cytoskeletal organization, cell transformation, and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP-bound form of RhoB is increased rapidly within 5 min of exposure to UVB, and then RhoB protein levels increased concomitantly with EGF receptor (EGFR) activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific small interfering RNAs inhibits AKT and glycogen synthase kinase-3beta phosphorylation through inhibition of EGFR expression. Moreover, down-regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicated that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.     

Cytokine polymorphisms play a role in susceptibility to ultraviolet B-induced modulation of immune responses after hepatitis B vaccination

UVB exposure can alter immune responses in experimental animals and humans. In an earlier human volunteer study, we demonstrated that hepatitis B-specific humoral and cellular immunity after vaccination on average were not significantly affected by UVB exposure. However, it is known that individuals differ in their susceptibility to UVB-induced immunomodulation, and it was hypothesized that polymorphisms in specific cytokines may play a role in this susceptibility. In this respect, we previously demonstrated that immune responses after hepatitis B vaccination are influenced by the minor allelic variant of IL-1 beta in the general population. For all volunteers, single nucleotide polymorphisms were determined for the following UV response-related cytokines: IL-1 receptor antagonist (+2018), IL-1 alpha (+4845), IL-1 beta (+3953), TNF-alpha (-308), and TNF-alpha (-238). Exposure to UVB significantly suppressed Ab responses to hepatitis B in individuals with the minor variant for the IL-1 beta polymorphism. Increased minimal erythema dose values (just perceptible), which resulted in higher absolute UVB exposures, were observed in the same individuals. There were no associations observed between UVB-induced immunomodulation and the other cytokine polymorphisms examined. This study indicates that individual susceptibility to UVB radiation needs to be considered when studying the effects of UVB in humans.     

Mutagenic DNA repair potential in Pseudomonas spp., and characterization of the rulABPc operon from the highly mutable strain Pseudomonas cichorii 302959

We assessed the tolerance to ultraviolet B (UVB; 290-320 nm) radiation and UVB-induced mutability in 28 Pseudomonas spp. and four Burkholderia cepacia strains. The UVB survival of 23 (72%) of the strains was elevated (>46% survival following irradiation with a 2250 J m-2 dose), and 17 (53%) strains were defined as mutable by UVB. A mutagenic DNA repair determinant was cloned and characterized from the highly mutable strain P. cichorii 302959 and shown by sequence analysis to be an allele of rulAB, a mutagenic DNA repair determinant previously characterized from Pseudomonas syringae. Phylogenetic analyses of RulA- and RulB-related sequences indicated that the sequences identified in environmental bacteria shared a common ancestor with UmuDC-like sequences from enteric bacteria but were considerably diverged. The dynamics of UVB-induced mutability to nalidixic acid resistance (NalR) and rifampicin resistance (RifR) were studied in replicate populations of P. cichorii 302959 subjected to a daily UVB dose of 2250 J m-2 for 14 consecutive days. While there was an initial spike in the frequency of NalR and RifR mutants recovered on Days 1 and 2 of two separate experiments, the frequencies were sharply reduced and then fluctuated throughout the duration of both experiments. These experimental results are intriguing because they point to the possibility that P. cichorii possesses additional mechanisms to curtail the induction of spontaneous mutants following repeated episodes of UVB irradiation.     

Mitogen- and stress-activated protein kinase 1 mediates activation of Akt by ultraviolet B irradiation

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

Photoprotective effects of some quinoxaline 1,4-dioxides in hairless mice

2-benzoyl-3-phenylquinoxaline 1,4-dioxide (BPQ) and other substituted quinoxaline 1,4-dioxides (QdO) were tested for their ability to inhibit the stimulations of ornithine decarboxylase (ODC) enzyme activity and DNA synthesis, two biochemical markers linked to skin tumour promotion by ultraviolet B (UVB) radiation. Topical application of BPQ on the dorsal skin of hairless mice was found to inhibit in a dose-dependent manner UVB-induced ODC activity and DNA synthesis. When applied 20 min before UVB radiation, a dose of 17 mg BPQ applied in 0.4 ml of vehicle inhibited UVB-induced ODC activity and DNA synthesis by 95% and 85%, respectively. This inhibitory effect is dependent on the time of administration of BPQ relative to UVB radiation, with a generally greater inhibition observed when this compound is applied before rather than after UVB treatment. The inhibitory abilities of the other QdO on the ODC and DNA responses induced by UVB radiation greatly varied and appear to be dependent on the structure of the compounds and their metabolic activation in the skin following irradiation. The remarkable effectiveness of BPQ against the ODC and DNA markers of UVB promotion is also observed following multiple applications of this agent. These results suggest that QdO, in particular BPQ and certain derivatives of it, may be useful in protecting the skin against UVB-induced skin damage.     

ER signaling is activated to protect human HaCaT keratinocytes from ER stress induced by environmental doses of UVB

Proteins are folded properly in the endoplasmic reticulum (ER). Various stress such as hypoxia, ischemia and starvation interfere with the ER function, causing ER stress, which is defined by the accumulation of unfolded protein (UP) in the ER. ER stress is prevented by the UP response (UPR) and ER-associated degradation (ERAD). These signaling pathways are activated by three major ER molecules, ATF6, IRE-1 and PERK. Using HaCaT cells, we investigated ER signaling in human keratinocytes irradiated by environmental doses of ultraviolet B (UVB). The expression of Ero1-Lα, an upstream signaling molecule of ER stress, decreased at 1-4 h after 10 mJ/cm² irradiation, indicating that the environmental dose of UVB-induced ER stress in HaCaT cells, without growth retardation. Furthermore, expression of intact ATF6 was decreased and it was translocated to the nuclei. The expression of XBP-1, a downstream molecule of IRE-1, which is an ER chaperone whose expression is regulated by XBP-1, and UP ubiquitination were induced by 10 mJ/cm² UVB at 4 h. PERK, which regulates apoptosis, was not phosphorylated. Our results demonstrate that UVB irradiation generates UP in HaCaT cells and that the UPR and ERAD systems are activated to protect cells from UVB-induced ER stress. This is the first report to show ER signaling in UVB-irradiated keratinocytes.     

Genetic and molecular analysis of light-regulated plant development

Light regulates many physiological and developmental events in plants through the action of multiple sensory pigment systems. Although our understanding of the regulatory photoreceptors, including phytochromes (that principally absorb red and far-red energy) and blue light receptors, has advanced considerably in the recent past, the mechanisms of light signal transduction in higher plants are poorly understood. To unravel the molecular events associated with light-regulated plant development, a large number of photomorphogenic mutants have been isolated in several different plant species, including Arabidopsis, cucumber, tomato, pea, Brassica and Sorghum, which are either impaired in normal perception of light signal (photoreceptor mutants) or are affected in some specific or a sub-set of phenotypic traits (signal transduction mutants). Their physiological and molecular analysis is proving to be valuable in (1) assigning specific function to discrete phytochrome species, (2) elucidation of elements that constitute the transduction pathway downstream of signal perception, and (3) determining how different photosensory systems regulate many diverse responses. The progress made in the analysis of photomorphogenic mutants, as reviewed in this article, clearly indicates that multiple photoreceptors, either of the same or different class, interact through an intricate network of signal transduction pathways to finally determine the light-dependent phenotype of both monocots and dicots.     

Protein oxidation,UVA and human DNA repair

Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency – with particular reference to NER and skin cancer risk.     

Mast cells,neuropeptides, histamine,and prostaglandins in UV-induced systemic immunosuppression

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.     

Involvement of UVB-induced reactive oxygen species in TGF-beta biosynthesis and activation in keratinocytes

TGF-beta produced by keratinocytes in response to UVB (290-320 nm) is a potential mediator for effects of acute and chronic solar radiation on skin. This study was designed to determine whether reactive oxygen species (ROS) mediate UVB-induced TGF-beta biosynthesis in keratinocytes and the subsequent activation of the latent TGF-beta complex. UVB irradiation elevated both total (latent plus active) and active TGF-beta in the keratinocyte supernatants, with a greater increase in the active form. UVB irradiation induced up to a 30% increase in ROS, and the ROS were detected up to 90 min after irradiation. NAC and Trolox, cytoplasmic ROS scavengers, abolished the UVB-induced TGF-beta and intracellular ROS, suggesting that UVB-induced ROS are involved in TGF-beta regulation. Inhibitors of NADPH oxidase activity, DPI and apocynin, decreased UVB-induced ROS. The increase in NADPH oxidase activity was mediated by EGFR activation. UVB-induced ROS also activated latent TGF-beta complex by stimulating MMP-2 and -9 activities. In summary, physiological doses of UVB increase intracellular ROS, which upregulate TGF-beta biosynthesis and activation of TGF-beta through increased activity of MMPs.     

Increase in CPD photolyase activity functions effectively to prevent growth inhibition caused by UVB radiation

Rice cultivars vary widely in their sensitivity to ultraviolet B (UVB) and this has been correlated with cyclobutane pyrimidine dimer (CPD) photolyase mutations that alter the structure/function of this photorepair enzyme. Here, we tested whether CPD photolyase function determines the UVB sensitivity of rice (Oryza sativa) by generating transgenic rice plants bearing the CPD photolyase gene of the UV-resistant rice cultivar Sasanishiki in the sense orientation (S-B and S-C lines) or the antisense orientation (AS-D line). The S-B and S-C plants had 5.1- and 45.7-fold higher CPD photolyase activities than the wild-type, respectively, were significantly more resistant to UVB-induced growth damage, and maintained significantly lower CPD levels in their leaves during growth under elevated UVB radiation. Conversely, the AS-D plant had little photolyase activity, was severely damaged by elevated UVB radiation, and maintained higher CPD levels in its leaves during growth under UVB radiation. Notably, the S-C plant was not more resistant to UVB-induced growth inhibition than the S-B plant, even though it had much higher CPD photolyase activity. These results strongly indicate that UVB-induced CPDs are one of principal causes of UVB-induced growth inhibition in rice plants grown under supplementary UVB radiation, and that increasing CPD photolyase activity can significantly alleviate UVB-caused growth inhibition in rice. However, further protection from UVB-induced damage may require the genetic enhancement of other systems as well.     

Monochloramine inhibits ultraviolet B-induced p53 activation and DNA repair response in human fibroblasts

Monochloramine (NH2Cl) is one of the inflammation-derived oxidants, and has various effects on cell cycle, apoptosis and signal transduction. We studied the effects of NH2Cl on DNA repair response induced by ultraviolet B (UVB) irradiation in normal human diploid fibroblasts, TIG-1. TIG-1 irradiated with 20 mJ/cm2 UVB showed marked increase in thymine dimer, which decreased by about 50% after 24 h. This decrease in thymine dimer was significantly attenuated (P < 0.05) by the pretreatment of NH2Cl (200 microM), which indicated DNA repair inhibition. UVB induced p53 phosphorylation at Ser15, Ser20 and Ser37, and p53 accumulation, and NH2Cl also inhibited these changes. Consequently, UVB-induced increase in the downstream effectors of p53, namely p21Cip1 and Gadd45a, were almost completely inhibited by NH2Cl. Immunoprecipitation study indicated that the association of p53 and MDM2, an E3 ubiquitin ligase for p53, did not change substantially by NH2Cl and/or UVB. The phosphorylation of p53 (Ser15 and Ser37) by UVB is catalyzed by ATR (ataxia telangiectasia mutated and Rad3 related kinase), which works as DNA damage sensor, and ATR also phosphorylates checkpoint kinase 1(Chk1) at Ser345. NH2Cl also inhibited the phosphorylation of Chk1 (Ser345). As UVB-induced DNA damage is repaired by nucleotide excision repair (NER) in human cells, these findings indicated that NH2Cl inhibited NER through the inhibition of p53 phosphorylation and accumulation, and NH2Cl probably impaired DNA damage recognition and/or ATR activation. NH2Cl may facilitate carcinogenesis through the inhibition of NER that repairs DNA damages from various carcinogens.     

MHY884, a newly synthesized tyrosinase inhibitor,suppresses UVB-induced activation of NF-κB signaling pathway through the downregulation of oxidative stress

The skin is the primary target of prolonged and repeated ultraviolet (UVB) irradiation which induces cutaneous inflammation and pigmentation. Nuclear factor κB (NF-κB) is the major factor mediating UVB-induced inflammatory responses through the expression of various proinflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We have previously reported that the synthetic novel compound 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) strongly suppressed tyrosinase activity and melanin synthesis in B16F10 melanoma cells. In the present study, we investigated the effect of MHY884 on the inhibition of UVB-induced NF-κB activation and its proinflammatory downstream proteins through the suppression of oxidative stress in an in vivo model of photoaging. Generation of reactive oxygen species (ROS) and peroxynitrite was measured in vitro and in B16F10 melanoma cells to verify the scavenging activity of MHY884. MHY884 suppressed oxidative stress both in vitro and in the melanoma cells in a dose-dependent manner. Next, melanin-possessing hairless mice were pre-treated with MHY884 and then irradiated with UVB repeatedly. Topical application of MHY884 attenuated UVB-induced oxidative stress, resulting in reduced NF-κB activity. Pre-treatment with MHY884 inhibited Akt and IκB kinase α/β signaling pathways, leading to decreased translocation and phosphorylation of p65, a subunit of NF-κB. This result correlated with the expression levels of iNOS and COX-2 in the skin of MHY884-treated mice. Thus, the novel tyrosinase inhibitor MHY884 suppressed NF-κB activation signaling pathway by scavenging UVB-induced oxidative stress. The discovery of MHY884, a novel tyrosinase inhibitor that targets NF-κB signaling, is significant, because this compound is a promising protective agent against UVB-induced skin damage.     

Inhibitory mechanisms of tea polyphenols on the ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway

In this study, we investigated the effect of tea polyphenols, (-)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (-)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.     

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21^CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence.