UVB-induced Apoptosis Research Articles

Taurine Protects Lens Epithelial Cells Against Ultraviolet B-Induced Apoptosis

ABSTRACTBackground: The massive uptake of compatible osmolytes is a self-protective response shared by lens exposed to hypertonic stress and ultraviolet stress.Objective: This study aimed to investigate the protective effects of taurine against ultraviolet B-induced cytotoxicity in the lens epithelial cells.Methods: Real-time PCR was used to measure osmolytes transport. Radioimmunoassay was used to measure osmolytes uptake. Cell counting kit-8 assays were used to measure cellular viability. Flow cytometry analysis was used to measure apoptosis level.Results: Compared with normotonic stress, hypertonic stress-induced osmolytes uptake into the lens epithelial cells such as betaine, myoinositol and taurine. UVB exposure increased osmolytes transporter mRNA expression together with osmolytes uptake. Moreover, taurine suppressed UVB-induced cell apoptosis in the lens epithelial cells significantly.Conclusions: The effect of compatible osmolyte taurine on cell survival rate may play an important role in cell resistance and adaption to UVB exposure.

Blocking RhoA/ROCK inhibits the pathogenesis of pemphigus vulgaris by suppressing oxidative stress and apoptosis through TAK1/NOD2-mediated NF-κB pathway.

Oxidative stress and apoptosis play critical roles in pemphigus vulgaris (PV). The main aim of the present study was to investigate the effects of RhoA/ROCK signaling on UVB-induced oxidative damage, and to delineate the molecular mechanisms involved in the UVB-mediated inflammatory and apoptotic response. In HaCaT cells, we observed that blockage of RhoA/ROCK signaling with the inhibitor CT04 or Y27632 greatly inhibited the UVB-mediated increase in intracellular reactive oxygen species (ROS). Additionally, inhibition of RhoA/ROCK signaling reduced UVB-induced apoptosis, as exemplified by a reduction in DNA fragmentation, and also elevated anti-apoptotic Bcl-2 protein, concomitant with reduced levels of pro-apoptotic protein Bax, caspase-3 cleavage and decreased PARP-1 protein. The release of inflammatory mediators TNF-α, IL-1β, and IL-6 was also attenuated. Mechanically, we observed that blockage of RhoA/ROCK repressed the TAK1/NOD2-mediated NF-κB pathway in HaCaT cells exposed to UVB. Taken together, these data reveal that RhoA/ROCK signaling is one of the regulators contributing to oxidative damage and apoptosis in human keratinocytes, suggesting that RhoA/ROCK signaling has strong potential to be used as a useful therapeutic target in skin diseases including PV.

Nrf2 in keratinocytes modulates UVB-induced DNA damage and apoptosis in melanocytes through MAPK signaling

Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be influenced by their neighbouring keratinocytes (KC). We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC cocultured with primary human KC using a transwell co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Our findings showed that co-culture of MC with siNrf2-transfected KC enhanced UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation, together with phosphorylation of ERK, JNK and p38 in MC. Treatment of MC with conditioned medium (CM) from Nrf2-depleted KC also increased UVB-mediated MC damage, suggesting that KC modulated UVB-mediated MC responses via paracrine effects. Additionally, depletion of Nrf2 in KC suppressed UVB-induced α-MSH levels as early as 30min post-irradiation, although pretreatment with N-acetylcysteine (NAC) elevated its levels in CM from siNrf2-transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis and inflammatory response in MC. Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC damage, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of ROS levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate the paracrine effects of KC probably by affecting levels of the paracrine factor α-MSH via a ROS-dependent mechanism.

745 REDD1 and AKT signaling in UVB-induced apoptosis

745 REDD1 and AKT signaling in UVB-induced apoptosis

BNIP3 upregulation via stimulation of ERK and JNK activity is required for the protection of keratinocytes from UVB-induced apoptosis

BNIP3 upregulation via stimulation of ERK and JNK activity is required for the protection of keratinocytes from UVB-induced apoptosis

Enhancement of UVB-induced apoptosis and elimination of DNA damages by irradiation of IPL does not depend on the repair of DNA damages

We have previously reported that repetitive irradiation of Intense Pulsed Light (IPL, 580–1200 nm) on hairless mice which harbor epidermal melanocytes producing only eumelanin (BHSCF mice) or pheomelanin (YHSCF mice), as well as no melanin (WHSCF mice) by back-crossing the K14-SCF mice, recessive yellow mice, and albino hairless mice, does not promote UVB-induced carcinogenesis nor wrinkle formation because pre-irradiation of I PL accelerated removal of cyclobutane pyrimidine dimer (CPD)s and enhanced UVB-induced apoptotic changes. To clarify whether the effect of IPL on these phenomena depends on the repair of DNA damages, we generated mice which are deficient in DNA repair by backcrossing BHSCF, YHSCF and WHSCF mice with XPA knockout mice. Pre-irradiation of IPL at doses of 0.90 J/cm2 (BHSCF mice) and 6.30 J/cm2 (YHSCF mice and WHSCF mice) on XPA-KO mice before or after UVB irradiation at doses of 1 J/cm2 for BHSCF, 0.1 J/cm2 for YHSCF, and 0.06 J/cm2 for WHSCF, accelerated removal of CPDs and enhanced apoptotic changes which were evaluated by TUNEL staining. These results indicate that the enhancement of UVB-induced apoptosis and acceleration of removal of CPDs by pre- or post-irradiation of IPL does not depend on the repair of DNA damages.

Effect of sericin concentration and ethanol content on gelation behavior, rheological properties, and sponge characteristics of silk sericin

Sericin gels and sponges have attracted attention in the biomedical and cosmetic fields because sericin has useful properties including a high UV and oxidation resistance, inhibition of UVB-induced apoptosis in human skin keratinocyte, wound healing, and wrinkle resistance effects. In this study, the effects of sericin concentration and ethanol content on the gelation behavior, rheological properties, and sponge characteristics of sericin were examined. As the sericin concentration increased, the rate of gelation of sericin accelerated and gel strength increased significantly. This occurred up to an ethanol content of 15%. Above that, the rate of gelation and the gel strength decreased. There was no change in the gel-sol transition temperature of sericin with the sericin concentration, although more ethanol addition resulted in a decrease in the starting temperature of thermal gel disruption. The crystallinity index of the frozen and thawed sericin sponge did not change with the sericin concentration, whereas it increased with increasing ethanol content up to 15% and decreased above 15%. As the sericin concentration was increased, the porosity and swelling ratio of the sericin sponge decreased and the mechanical properties of sericin sponge were remarkably improved.

The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation.

Prohibitin (PHB) plays a role in regulation of ultraviolet B light (UVB)-induced apoptosis of human keratinocytes, HaCaT cells. The regulatory function of PHB appears to be associated with its lipid raft translocation. However, the detailed mechanism for PHB-mediated apoptosis of these keratinocytes upon UVB irradiation is not clear. In this report, we determined the role of lipid raft translocation of PHB in regulation of UVB-induced apoptosis. Our data show that upon UVB irradiation PHB is translocated from the non-raft membrane to the lipid rafts, which is correlated with a release of both Akt and Raf from membrane. Overexpression of Akt and/or Raf impedes UVB-induced lipid raft translocation of PHB. Immunoprecipitation analysis indicates that UVB alters the interactions among PHB, Akt, and Raf. Reduced expression of PHB leads to a decreased phosphorylation of Akt and ERK, as well as a decreased activity of Akt, and increased apoptosis of the cells upon UVB irradiation. These results suggest that PHB regulates UVB-induced apoptosis of keratinocytes via a mechanism that involves detachment from Akt and Raf on the plasma membrane, and sequential lipid raft translocation.

Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes

UVB light induces generation of reactive oxygen species, ultimately leading to skin cell damage. Mitochondria are a major source of reactive oxygen species in UVB-irradiated skin cells, with increased levels of mitochondrial reactive oxygen species having been implicated in keratinocyte apoptosis. Peroxiredoxin III (PrxIII) is the most abundant and potent H2O2-removing enzyme in the mitochondria of most cell types. Here, the protective role of PrxIII against UVB-induced apoptosis of epidermal keratinocytes was investigated. Mitochondrial H2O2 levels were differentiated from other types of ROS using mitochondria-specific fluorescent H2O2 indicators. Upon UVB irradiation, PrxIII-knockdown HaCaT human keratinocytes and PrxIII-deficient (PrxIII-/-) mouse primary keratinocytes exhibited enhanced accumulation of mitochondrial H2O2 compared with PrxIII-expressing controls. Keratinocytes lacking PrxIII were subsequently sensitized to apoptosis through mitochondrial membrane potential loss, cardiolipin oxidation, cytochrome c release, and caspase activation. Increased UVB-induced epidermal tissue damage in PrxIII-/- mice was attributable to increased caspase-dependent keratinocyte apoptosis. Our findings show that mitochondrial H2O2 is a key mediator in UVB-induced apoptosis of keratinocytes and that PrxIII plays a critical role in protecting epidermal keratinocytes against UVB-induced apoptosis through eliminating mitochondrial H2O2. These findings support the concept that reinforcing mitochondrial PrxIII defenses may help prevent UVB-induced skin damage such as inflammation, sunburn, and photoaging.

BNIP3 upregulation via stimulation of ERK and JNK activity is required for the protection of keratinocytes from UVB-induced apoptosis

The human skin has an important role in barrier function. Ultraviolet rays (UV) from sunlight exposure can cause cell apoptosis in the skin epidermis, resulting in the disruption of the barrier. Previously, we have demonstrated that BNIP3 stimulates autophagy in epidermal keratinocytes and has a protective effect in these cells upon UVB irradiation. In this study, we found that the accumulation of reactive oxygen species (ROS) by UVB irradiation was sufficient to trigger the activation of JNK and ERK mitogen-activated protein kinase (MAPK) in human primary epidermal keratinocytes. In turn, activated JNK and ERK MAPK mediated the upregulation of BNIP3 expression. Treatment with an antioxidant reagent or a specific inhibitor of MAPK, U0126, and a JNK inhibitor significantly attenuated the expression of BNIP3 triggered by UVB, followed by the induction of cell death by apoptosis. Furthermore, UVB-induced apoptosis was significantly stimulated by chloroquine or bafilomycin A1, an inhibitor of autophagy. Moreover, BNIP3 was required for the degradation of dysfunctional mitochondria upon UVB irradiation. These data clearly indicated that BNIP3-induced autophagy, which occurs via UVB-generated ROS-mediated JNK and ERK MAPK activation, has a crucial role in the protection of the skin epidermis against UVB irradiation.

Photoaging protective effects of BIOGF1K, a compound-K-rich fraction prepared from Panax ginseng

BackgroundBIOGF1K, a compound-K-rich fraction, has been shown to display anti-inflammatory activity. Although Panax ginseng is widely used for the prevention of photoaging events induced by UVB irradiation, the effect of BIOGF1K on photoaging has not yet been examined. In this study, we investigated the effects of BIOGF1K on UVB-induced photoaging events. MethodsWe analyzed the ability of BIOGF1K to prevent UVB-induced apoptosis, enhance matrix metalloproteinase (MMP) expression, upregulate anti-inflammatory activity, reduce sirtuin 1 expression, and melanin production using reverse transcription-polymerase chain reaction, melanin content assay, tyrosinase assay, and flow cytometry. We also evaluated the effects of BIOGF1K on the activator protein-1 signaling pathway, which plays an important role in photoaging, by immunoblot analysis and luciferase reporter gene assays. ResultsTreatment of UVB-irradiated NIH3T3 fibroblasts with BIOGF1K prevented UVB-induced cell death, inhibited apoptosis, suppressed morphological changes, reduced melanin secretion, restored the levels of type I procollagen and sirtuin 1, and prevented mRNA upregulation of MMP-1, MMP-2, and cyclo-oxygenase-2; these effects all occurred in a dose-dependent manner. In addition, BIOGF1K markedly reduced activator-protein-1-mediated luciferase activity and decreased the activity of mitogen-activated protein kinases (extracellular response kinase, p38, and C-Jun N-terminal kinase). ConclusionOur results strongly suggest that BIOGF1K has anti-photoaging activity and that BIOGF1K could be used in anti-aging cosmeceutical preparations.

Lycium barbarum Polysaccharides Protect Rat Corneal Epithelial Cells against Ultraviolet B-Induced Apoptosis by Attenuating the Mitochondrial Pathway and Inhibiting JNK Phosphorylation.

Lycium barbarum polysaccharides (LBPs) have been shown to play a key role in protecting the eyes by reducing the apoptosis induced by certain types of damage. However, it is not known whether LBPs can protect damaged corneal cells from apoptosis. Moreover, no reports have focused on the role of LBPs in guarding against ultraviolet B- (UVB-) induced apoptosis. The present study aimed to investigate the protective effect and underlying mechanism of LBPs against UVB-induced apoptosis in rat corneal epithelial (RCE) cells. The results showed that LBPs significantly prevented the loss of cell viability and inhibited cell apoptosis induced by UVB in RCE cells. LBPs also inhibited UVB-induced loss of mitochondrial membrane potential, downregulation of Bcl-2, and upregulation of Bax and caspase-3. Finally, LBPs attenuated the phosphorylation of c-Jun NH2-terminal kinase (JNK) triggered by UVB. In summary, LBPs protect RCE cells against UVB-induced damage and apoptosis, and the underlying mechanism involves the attenuation of the mitochondrial apoptosis pathway and the inhibition of JNK phosphorylation.

MiR-26a Mediates Ultraviolet B-Induced Apoptosis by Targeting Histone Methyltransferase EZH2 Depending on Myc Expression

Background/Aims: Ultraviolet B (UVB) damage is the most essential etiological factor in skin carcinogenesis, and apoptosis leads to the efficient elimination of UVB-damaged cells. However, the mechanisms underlying resistance to UVB-induced apoptosis remain unclear. Methods: HaCaT and A431 cells were used in the present study. Quantitative real-time PCR, single cell PCR, and western blotting were used to examine cancer-related gene expression at the mRNA and protein levels. Results: We report that miR-26a, which is upregulated upon UVB irradiation, promotes UVB-induced apoptosis in HaCaT cells by targeting the histone methyltransferase EZH2. Moreover, the UVB/miR-26a/EZH2 regulatory axis largely depends on the MYC expression level. Interestingly, treatment with EZH2 inhibitors significantly enhanced UVB-induced apoptosis. Conclusion: miR-26a/EZH2 might be potential targets for skin cancer prevention and therapy.

Dual Protective Effects of Flavonoids from Petasites japonicus against UVB-Induced Apoptosis Mediated via HSF-1 Activated Heat Shock Proteins and Nrf2-Activated Heme Oxygenase-1 Pathways.

The leaves of Petasites japonicus are used for their anti-allergic properties in traditional Korean, Japanese, and Chinese medicine. This study aimed to identify bioactive compounds isolated from P. japonicus leaves. All compounds were assessed for their ability of transcriptional activation, induction of phase 2 enzymes and heat shock proteins (HSPs), as well as protection against the UVB-induced apoptotic cell death. Bioactive compounds were isolated from P. japonicus leaves. All compounds were evaluated for their protective effect using human dermal fibroblasts (HDF) and human epidermal keratinocyte cells (HEKC) treated with UVB radiation. Four flavonoids were isolated from the leaves of P. japonicus and identified as kaempferol-3-O-(6″-acetyl)-β-D-glucoside (1), quercetin-3-O-(6″-acetyl)-β-D-glucoside (2), kaempferol-3-O-β-D-glucoside (3), and quercetin-3-O-β-D-glucoside (4). These compounds activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heat-shock response transcription elements (HSE) that resulted in the induction of heme oxygenase-1 (HO-1) and HSP70, respectively. Activation of these pathways provided protection to the skin cells against UVB radiation. The isolated compounds activated the Nrf2 and HSE pathways and could protect against UVB-induced apoptosis.

Proteomic Analysis and Functional Studies of Baicalin on Proteins Associated with Skin Cancer.

Abundant evidence supports the key role of ultraviolet radiation (UVR) in skin cancer development. The human skin, especially the epidermal layer, is the main defense against UV radiation. Baicalin is a major bioactive component of Scutellaria baicalensis Georgi, a plant which has been found to exhibit antitumor activity. The anticarcinogenic mechanism of baicalin is not completely understood. We have reported that baicalin inhibited UVB-induced photo-damage and apoptosis in HaCaT cells (human skin keratinocytes). The aim of the present study is to investigate the cellular gene targets responsible for baicalin's antitumor activity by performing two-dimensional electrophoresis liquid chromatography-mass spectrometry/mass spectrometry (2-DE LC-MS/MS) with HaCaT cells following UVB and baicalin exposure. Two-DE for protein separation was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry and database searches. Nucleophosmin (NPM)-specific siRNA was designed and synthesized, and the small interfering RNA was transfected into skin squamous cancer A431 cells to knockdown the NPM expression. Proliferation and cell cycle status were assessed by CCK8 and flow cytometric analyses, respectively. We have identified 38 protein spots that are differentially expressed in HaCaT cells exposed to baicalin and/or UVB irradiation These proteins are involved in detoxification, proliferation, metabolism, cytoskeleton and motility. In particular, we found several proteins that have been linked to tumor progression and resistance, such as NPM.Baicalin treatment reduced the cellular proliferation rate and induced arrest during the S-phase of the cell cycle in A431 cells. NPM1 silencing significantly enhanced the effect of baicalin. Our data indicated that baicalin results in the significant inhibition of tumor growth in the A431 cell line, which may be associated with the regulation of the NPM gene expression.

Echinacoside Alleviates UVB Irradiation-Mediated Skin Damage via Inhibition of Oxidative Stress, DNA Damage, and Apoptosis.

Ultraviolet B (UVB) irradiation has been known to cause skin damage, which is associated with oxidative stress, DNA damage, and apoptosis. Echinacoside is a phenylethanoid glycoside isolated from Herba Cistanches, which exhibits strong antioxidant activity. In this study, we evaluate the photoprotective effect of echinacoside on UVB-induced skin damage and explore the potential molecular mechanism. BALB/c mice and HaCaT cells were treated with echinacoside before UVB exposure. Histopathological examination was used to evaluate the skin damage. Cell viability, lactate dehydrogenase (LDH) levels, antioxidant enzyme activities, reactive oxygen species (ROS) generation, DNA damage, and apoptosis were measured as well. Western blot was used to measure the expression of related proteins. The results revealed that pretreatment of echinacoside ameliorated the skin injury; attenuated oxidative stress, DNA damage, and apoptosis caused by UVB exposure; and normalized the protein levels of ATR, p53, PIAS3, hnRNP K, PARP, and XPA. To summarize, echinacoside is beneficial in the prevention of UVB-induced DNA damage and apoptosis of the skin in vivo and in vitro.

PTEN Reduced UVB-Mediated Apoptosis in Retinal Pigment Epithelium Cells.

Age-related macular degeneration (AMD) is a leading cause of blindness and progressive loss of central vision in the elderly population. The important factor of AMD pathogenesis is the degeneration of retinal pigment epithelial (RPE) cells by oxidative stress. Inactivation of PTEN can disrupt intercellular adhesion in the RPE cells, but the mechanism of oxidative stress is less known. Here we presented evidence that UVB-mediated oxidative stress induced apoptosis in ARPE-19 cells. Downregulation of the expression of PTEN in UVB-irradiative RPE cells triggered DNA damage and increased the level of UVB-induced apoptosis by activating p53-dependent pathway. However, overexpression of PTEN increased cell survival by suppressing p-H2A in response to DNA damage and apoptosis. When using Pifithrin-α (one of p53 inhibitors), the level of p53-dependent apoptosis was significantly lower than untreated, which suggested that p53 was possibly involved in PTEN-dependent apoptosis. Thus, it elucidated the molecular mechanisms of UVB-induced damage in RPE cells and may offer an alternative therapeutic target in dry AMD.

TNF-R1 and FADD mediate UVB-Induced activation of K+ channels in corneal epithelial cells

The goal of this study was to elucidate the role of Fas, TNF-R1, FADD and cytochrome c in UVB-induced K+ channel activation, an early step in UVB-induced apoptosis, in human corneal limbal epithelial (HCLE) cells. HCLE cells were treated with Fas, TNF-R1 or FADD siRNA and exposed to 80 or 150 mJ/cm2 UVB. K+ channel activation and loss of intracellular K+ were measured using whole-cell patch-clamp recording and ion chromatography, respectively. Cytochrome c was measured with an ELISA kit. Cells in which Fas was knocked down exhibited identical UVB-induced K+ channel activation and loss of intracellular K+ to control cells. Cells in which TNF-R1 or FADD were knocked down demonstrated reduced K+ channel activation and decreased loss of intracellular K+ following UVB, relative to control cells. Application of TNF-α, the natural ligand of TNF-R1, to HCLE cells induced K+ channel activation and loss of intracellular K+. Cytochrome c was translocated to the cytosol by 2 h after exposure to 150 mJ/cm2 UVB. However, there was no release by 10 min post-UVB. The data suggest that UVB activates TNF-R1, which in turn may activate K+ channels via FADD. This conclusion is supported by the observation that TNF-α also causes loss of intracellular K+. This signaling pathway appears to be integral to UVB-induced K+ efflux, since knockdown of TNF-R1 or FADD inhibits the UVB-induced K+ efflux. The lack of rapid cytochrome c translocation indicates cytochrome c does not play a role in UVB-induced K+ channel activation.

65 - Nrf2 in Keratinocytes Modulates UVB-Induced DNA Damage and Apoptosis in Melanocytes through ROS-Dependent Mechanism

Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be regulated by their neighbouring keratinocytes (KC). Aims We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC co-cultured with primary human KC using a trans-well co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Results Co-culture of MC with siNrf2-transfected KC enhanced UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation in correlation with phosphorylation of ERK, JNK and p38 in MC. MC damage induced by UVB was increased in MC treated with conditioned medium (CM) derived from Nrf2-depleted KC, suggesting that KC modulated UVB-mediated MC responses via paracrine effects. Additionally, at 30 min post-irradiation, depletion of Nrf2 in KC suppressed UVB-induced α-MSH levels, although pre-treatment with N-acetylcysteine (NAC) elevated its levels in CM from siNrf2-transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis in MC. Conclusion Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC responses, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of reactive oxygen species (ROS) levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate paracrine effect of KC probably by affecting levels of the paracrine factor α-MSH via a ROS-dependent mechanism.

Enhancement of UVB-induced apoptosis and elimination of DNA damages by pre-irradiation of IRA does not depend on the repair of DNA damages

Enhancement of UVB-induced apoptosis and elimination of DNA damages by pre-irradiation of IRA does not depend on the repair of DNA damages