Proteome analysis of ultraviolet-B-induced protein expression in vitro human dermal fibroblasts

Background: Ultraviolet‐B (UVB) radiation can result in acute photodamage, photoaging and skin cancer through the induction of reactive oxygen species, DNA damage, activation of signaling pathways, and regulation of gene expression. In this study, we investigated UVB‐induced alterations in protein expression in human dermal fibroblasts. Methods: Skin fibroblasts were irradiated with 100 mJ/cm² UVB, and cell viability was monitored by the 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐diphenytetrazoliumromide assay. Two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy were used to identify differentially expressed proteins. The mRNA and levels of identified proteins were detected using a quantitative real‐time polymerase chain reaction assay and Western blot. Results: UVB decreased the viability of skin fibroblasts. In UVB‐treated cells, eighteen differentially expressed proteins were identified. Among these proteins, the amounts of receptor‐interacting protein (RIP) and vimentin were significantly up‐regulated. However, their mRNA levels decreased and remained relatively stable, respectively. Conclusions: The differential expression of RIP and vimentin was validated in UVB‐irradiated fibroblasts. RIP may promote cell injury, and vimentin may contribute to the resistance of cells to UVB‐induced damage.     

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1‐Ca‐ERK pathway in ultraviolet B–irradiated human dermal fibroblasts

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca²⁺ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca²⁺ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca²⁺ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca²⁺ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca²⁺ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca²⁺ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin.     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

Astaxanthin,a xanthophyll carotenoid,inhibits ultraviolet‐induced apoptosis in keratinocytes

Intra‐cellular reactive nitrogen/oxygen species and apoptosis play important roles in ultraviolet (UV)‐induced inflammatory responses in the skin. Astaxanthin (AST), a xanthophyll carotenoid, exhibits diverse clinical benefits. The protective effects of AST against UV‐induced apoptosis were investigated in the present study. Astaxanthin (5 μm ) caused a significant decrease in the protein content and the mRNA levels of inducible nitric oxide (iNOS) and cyclooxygenase (COX)‐2, and decreased the release of prostaglandin E2 from HaCaT keratinocytes after UVB (20 mJ/cm²) or UVC (5 mJ/cm²) irradiation. No significant protective effects against UV‐induced reactive oxygen species (ROS) were observed in AST‐pretreated cells. Astaxanthin caused a significant inhibition of UV‐irradiation‐induced apoptosis, as evidence by a DNA fragmentation assay. Furthermore, we found that the treatment with AST caused a reduction in the UVB‐ or UVC‐induced protein and mRNA expression of macrophage migration inhibitory factor (MIF), IL‐1β and TNF‐α in HaCaT keratinocytes. These results suggest that AST effectively protects against UV‐induced inflammation by decreasing iNOS and COX‐2, and thereby inhibiting the apoptosis of keratinocytes.     

Intercellular pathway through hyaluronic acid in UVB‐induced inflammation

Ultraviolet B (UVB) radiation induces inflammation in the skin specifically at the site of exposure. We unexpectedly found that UVB‐induced inflammation was not induced in gp91phox‐depleted mice. To test whether gp91phox is directly involved in UVB‐induced inflammation, neutrophil‐ and hyaluronic acid–depleted mice were also irradiated and examined for their response. Hyaluronic acid–depleted mice showed strongly inhibited UVB‐induced inflammation, but the neutrophil‐depleted mice did not exhibit any suppressed UVB‐induced inflammation. To elucidate the pathway by which UVB irradiation induced inflammation, we examined the expression of nucleotide‐binding domain, leucine‐rich‐containing family, pyrin domain‐containing‐3 (NLRP3) and caspase‐1 in the mouse skin. An increase in the expression of NLRP3 and caspase‐1 was seen following the UVB irradiation of C57BL mice; however, the UVB‐irradiated gp91phox‐knockout (gp91phox^?/?) mice did not have this increase in expression. Furthermore, the plasma IL‐1β level increased after the UVB irradiation in C57BL mice, but there was no change in the gp91phox^?/? mice. These results clearly indicate that nicotinamide adenine dinucleotide phosphate oxidase is activated by gp91phox, which is expressed on the surface in response to the increased expression of hyaluronic acid induced by UVB irradiation, and as result, the generation of reactive oxygen species (ROS) increases. This ROS activate NLRP3, and NLRP3 leads to the production of caspase‐1, which subsequently increases IL‐1β, thereby finally inducing inflammation. It is thought that this system may play an important role in the damage and ageing of skin, and further studies are necessary to confirm these finding.     

Opposing effects of ultraviolet B irradiation on interleukin-1 receptor antagonist and interleukin-1α messenger RNA expression in a human epidermoid carcinoma cell line

Interleukin-1 receptor antagonist (IL-1RA) is a cytokine that acts to antagonize IL-1 activity without agonist function. The expression of IL-1RA has been reported in many cell types, including the keratinocyte that covers the outer most part of the skin. However the modulation of IL-1RA by ultraviolet B (UVB), which is the most biologically active UV, has not been reported yet. We therefore selected a keratinocyte cell line with a cytokine-producing profile similar to that of keratinocytes and tested the effect of UVB on its ability to produce IL-1RA mRNA. IL-1RA mRNA was constitutively expressed in the cell line and began to be suppressed by 3 h after the UVB irradiation with 100 mJ/cm². The level of IL-1RA expression became lowest by 16 h after the irradiation with 100 mJ/cm². Simultaneously, IL-1α mRNA started to increase by 1 h and peaked by 3–16 h after the irradiation with 10–100 mJ/cm². The differential expression of IL-1α and IL-1RA mRNA following exposure to a high dose (100 mJ/cm²) of UVB may markedly potentiate the role of IL-1 in UV-induced inflammation.     

UV irradiation induces downregulation of bcl-2 expression in vitro and in vivo

Abstract Recently, the proto-oncogenes bcl-2 and bax have emerged as important regulators of the apoptotic form of cell death. We examined UV irradiation-elicited apoptosis and regulation of bcl-2 and bax expression both in vivo in human skin and in vitro in HeLa cells. Using flow cytometric analysis, HeLa cells were found to undergo apoptosis at the 12-h time-point after exposure to UVB irradiation (100 mJ/cm²). The expression of bcl-2 mRNA was found to decrease after a single dose of UVB radiation (doses 10–200 mJ/ cm²). In contrast, the expression of bax mRNA was not significantly changed. When human skin was irradiated with a single dose of solar-simulated radiation (40 mJ/cm²), Bcl-2-positive cells were significantly reduced in the epidermis at the 3- and 6-h time-points. Our results suggest that UV irradiation downregulates bcl-2 expression both in vitro at the mRNA level and in vivo at the protein level, and that downregulation of bcl-2 constitutes a mechanism of potential importance in UV-induced apoptosis in human epidermis. Received: 1 July 1998 / Received after revision: 12 October1998 / Accepted: 2 November 1998     

UVB induces epidermal 11β‐hydroxysteroid dehydrogenase type 1 activity in vivo

Detrimental consequences of ultraviolet radiation (UVR) in skin include photoageing, immunosuppression and photocarcinogenesis, processes also significantly regulated by local glucocorticoid (GC) availability. In man, the enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) generates the active GC cortisol from cortisone (or corticosterone from 11‐dehydrocorticosterone in rodents). 11β‐HSD1 oxo‐reductase activity requires the cofactor NADPH, generated by hexose‐6‐phosphate dehydrogenase. We previously demonstrated increased 11β‐HSD1 levels in skin obtained from photoexposed versus photoprotected anatomical regions. However, the direct effect of UVR on 11β‐HSD1 expression remains to be elucidated. To investigate the cutaneous regulation of 11β‐HSD1 following UVR in vivo, the dorsal skin of female SKH1 mice was irradiated with 50, 100, 200 and 400 mJ/cm² UVB. Measurement of transepidermal water loss, 11β‐HSD1 activity, mRNA/protein expression and histological studies was taken at 1, 3 and 7 days postexposure. 11β‐HSD1 and hexose‐6‐phosphate dehydrogenase mRNA expression peaked 1 day postexposure to 400 mJ/cm² UVB before subsequently declining (days 3 and 7). Corresponding increases in 11β‐HSD1 protein and enzyme activity were observed 3 days postexposure coinciding with reduced GC receptor mRNA expression. Immunofluorescence studies revealed 11β‐HSD1 localization to hyperproliferative epidermal keratinocytes in UVB‐exposed skin. 11β‐HSD1 expression and activity were also induced by 200 and 100 (but not 50) mJ/cm² UVB and correlated with increased transepidermal water loss (indicative of barrier disruption). UVB‐induced 11β‐HSD1 activation represents a novel mechanism that may contribute to the regulation of cutaneous responses to UVR exposure.     

Cystatin A suppresses ultraviolet B-induced apoptosis of keratinocytes

BACKGROUND: Cystatin A is a cysteine proteinase inhibitor abundantly expressed in keratinocytes. Although cystatin A is one of the cornified cell envelope constituents and expressed in the upper epidermis, its precise function is still unknown. Ultraviolet B irradiation (UVB) induces apoptosis accompanied with the activation of cysteine proteinases, caspases. OBJECTIVE: We investigated the effect of cystatin A on UVB-induced apoptosis of keratinocytes. METHODS: We assessed the caspase activities and apoptotic cell numbers induced by UVB ittadiation in cystatin A gene transfected keratinocytes. RESULTS: UVB-induced pro-caspase 3 cleavage and caspase 3 activation were suppressed in cystatin A expression vector-transfected SV40-transformed human keratinocytes (SVHK). Furthermore, the transfected SVHK cells were resistant to UVB-induced apoptosis. In contrast neither caspase 8 nor caspase 9 activities were affected by UVB irradiation in cystatin A-transfected SVHK cells. The effects were also observed in cystatin A expression adenovirus vector-transfected cultured normal human keratinocytes (NHK). Conversely knockdown of cystatin A by si-RNA induced marked apoptosis of NHK cells following UVB irradiation accompanied with increased caspase 3 activity. In order to confirm the antiapoptotic effect of cystatin A in vivo UVB irradiation was performed on cystatin A transgenic mice (cystatin A-tg). The epidermis from cystatin A-tg was resistant to UVB-induced apoptosis compared to control mice epidermis. CONCLUSION: These results indicate that cystatin A suppresses UVB-induced apoptosis of keratinocytes by the inhibition of caspase 3 activation.     

Ultraviolet B radiation regulates cysteine‐rich protein 1 in human keratinocytes

Background: Cysteine‐rich protein 1 (CRP1) is a growth‐inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. Methods: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A‐431) by immunoblotting. UVB‐induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV‐damaged skin and squamous cell carcinoma (SCC), a UV‐induced skin cancer. Results: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis‐inducing doses of UV radiation, CRP1 was cleaved in a caspase‐dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. Conclusion: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.     

Narrowband ultraviolet B radiation suppresses contact hypersensitivity

Background/purpose: A main mechanism responsible for the efficacy of narrowband ultraviolet (UV)B is thought to be the induction of apoptosis in pathogenetically relevant cells. Narrowband UVB therapy, however, generally induces a relatively long remission period. Recently, evidence that UVB radiation induces regulatory T (Treg) cells was reported. Based on these findings, we examined whether narrowband UVB suppresses contact hypersensitivity (CHS) by inducing Treg cells. Methods: The shaved abdomens of C3H/HeN mice were irradiated with broadband or narrowband UVB. CHS was defined as an ear‐swelling response. To examine whether tolerance can be induced by adoptive transfer, lymph node cells from UVB‐irradiated mice were injected into naïve mice before sensitization and CHS challenge. Results: Narrowband UVB exposure dose dependently suppressed CHS. Significant suppression was observed at doses between 1000 and 3000 mJ/cm² (P<0.05). The suppressive effect achieved with 1000 mJ/cm² narrowband UVB was very similar to the effect achieved with 100 mJ/cm² broadband UVB. The suppressive effects on CHS were transferred to naïve mice by the injection of lymph node cells from tolerant mice. Conclusion: Narrowband UVB induced local and systemic suppression of CHS. In addition, narrowband UVB induces tolerance to CHS and the suppressive effects were transferable to naïve mice.     

Isoflavones in aglycone solution enhance ultraviolet B‐induced DNA damage repair efficiency

The isoflavones daidzein and genistein are natural compounds which have anti‐inflammatory and photoprotective activities, and may be effective in the repair of ultraviolet (UV)‐induced photodamage. In this study, an alcoholic solution of aglycone isoflavones with a genistein:daidzein ratio of 1:4 [Rottapharm (RPH)‐aglycone] was examined for its effects on the repair of DNA damage induced by a single dose of UVB irradiation (20 mJ/cm²). For this purpose, human skin cells were first UVB‐irradiated and then treated with RPH‐aglycone. Comet assay analysis was used to estimate the UVB‐induced DNA damage at different time points after treatment by measuring the tail moment parameter. We found that treatment with 10 μmol/L RPH‐aglycone solution resulted in a significantly reduced tail moment at 1 h after treatment, and 34–35% enhancement of damage repair at 4 h after treatment. These results suggest that isoflavone aglycones are protective against UVB‐induced DNA damage.     

NB‐UVB irradiation attenuates inflammatory response in psoriasis

Psoriasis is a chronic inflammatory disease characterized by immunological imbalance and vasodilation. Many triggering factors for psoriasis initiate inflammation via the activation of NF‐κB. Narrow‐band ultraviolet B (NB‐UVB) irradiation can be used as a general treatment for psoriasis, although the molecular mechanism has not yet been determined. The aim of this study was to elucidate the potential molecular mechanism of NB‐UVB irradiation therapy on psoriasis. We collected serum samples from patients with psoriasis and healthy control, and detected the expression of inflammatory factors by ELISA. In addition, we established mouse model of psoriasis. After different doses of NB‐UVB irradiation, the proportion of CD4⁺, CD8⁺, and CD11c⁺ cells in mouse spleen was detected by flow cytometry. Meanwhile, the expression of inflammatory factors in the damaged skin of mice was detected by RT‐PCR and Western blot analysis, and mouse serum levels of inflammatory factors were detected by ELISA. Our results showed that NB‐UVB irradiation regulated the expression of inflammatory factors in psoriasis patients. In mice, high‐dose NB‐UVB irradiation effectively eliminated IMQ‐induced psoriasis‐like dermatitis and inhibited the expression of pro‐inflammatory factors. In conclusion, our results indicate that NB‐UVB irradiation could regulate the expression of inflammatory factors and attenuate psoriasis plaques.     

Ultraviolet B-induced LGI3 secretion protects human keratinocytes

Leucine‐rich glioma inactivated 3 (LGI3) is known to be expressed mainly in the brain. However, the expression and physiological roles of LGI3 in skin cells remain unknown. In this study, it was found for the first time that LGI3 is expressed mostly by normal human keratinocytes. Furthermore, ELISA analysis showed that HaCaT human keratinocytes increased LGI3 secretion after exposure to ultraviolet B (UVB) in a time‐ and dose‐dependent manner. We next investigated the possible role of LGI3 in keratinocytes. LGI3 (50 ng/ml) increased survival of HaCaT cells by 20% after UVB irradiation (150 mJ/cm²). It was also found that LGI3 stimulates the phosphorylation of Akt, which is involved in the cell survival‐signalling cascade. Furthermore, LGI3 led to the phosphorylation of MDM2 and subsequent p53 degradation. Taken together, the data suggest that LGI3 may regulate p53 levels and that keratinocyte‐derived LGI3 may act as a novel cytokine for skin homoeostasis.     

Effect of infrared radiation A on photoaged hairless mice harboring eumelanin and pheomelanin in the epidermis

Infrared radiation A (IRA) is absorbed by melanin and generates heat. Therefore, the effect of IRA could be well analyzed using skin, which contains melanin in the epidermis. Hairless mice harboring epidermal melanocytes that produce eumelanin, pheomelanin, or non‐melanin were generated by backcrossing K14‐stem cell factor mice, recessive yellow mice, and then albino hairless mice. High‐dose IRA was irradiated over 18 weeks after the establishment of photoaged mice by irradiation with ultraviolet B (UVB) three times a week for 14 weeks. Tumor formation was assessed every week. The formation of cyclobutane pyrimidine dimer and apoptotic cells by the irradiation of IRA and UVB was evaluated. Repetitive irradiation of IRA did not promote tumor formation in all types of mice. Pre‐irradiation of IRA to UVB, but not post‐irradiation, accelerated the elimination of cyclobutane pyrimidine dimers and enhanced apoptosis; these effects were most obvious in eumelanin‐producing mice. Real‐time polymerase chain reaction analysis showed downregulation of FLICE (cellular caspase 8)‐like inhibitory protein and B‐cell lymphoma‐extra large and upregulation of Bcl‐2‐associated X protein by UVB, but further enhancement of these molecules by pre‐irradiation of IRA was not observed. These results indicate that IRA does not confer the promotion of UVB‐induced carcinogenesis in photoaged mice harboring epidermal melanocytes and that photochemical reaction between IRA and melanin might be involved in the induction of apoptosis and the elimination of cyclobutane pyrimidine dimers by UVB. The enhancement of apoptosis by pre‐irradiation of IRA to UVB might be induced by mechanisms other than the modification of the mRNA expression of FLICE (cellular caspase 8)‐like inhibitory protein, B‐cell lymphoma‐extra large, and Bcl‐2‐associated X.     

Caspase-3、Survivin在UVB诱导HaCaT凋亡细胞中的表达

目的探讨Caspase-3、Survivin在UVB诱导HaCaT凋亡细胞中的作用。方法研究对象为人角质形成细胞HaCaT细胞,实验分为正常对照组、10、20、40、80 m J/cm^2中波紫外线组(UVB)。四甲基偶氮唑蓝(MTT)比色法观察增殖能力;流式细胞仅(FCM)实验检测细胞凋亡;实时定量PCR、Western-blot检测细胞内的Survivin、Caspase-3表达水平。结果与正常对照组相比,NB-UVB照射组细胞增殖抑制作用及凋亡明显增强(P<0.05),并随着照射剂量的增加,其细胞凋亡作用增加。同时与正常对照组相比,不同UVB照射剂量组Caspase-3表达均增强,其表达增强程度随着照射剂量的增加而增加,与正常对照组相比,10 m J/cm^2照射组,细胞表达Survivin水平最高(P<0.05),20 m J/cm^2组Survivin表达水平下降,稍低于对照组水平,40、80 m J/cm^2组Survivin进一步下降,较对照组下降明显(P<0.05)。结论 Survivin、Caspase-3参与了UVB诱导HaCaT凋亡细胞。Survivin表达水平与UVB辐射剂量有关。     

Could colorimetric method replace the individual minimal erythemal dose (MED) measurements in determining the initial dose of narrow‐band UVB treatment for psoriasis patients with skin phototype III–V?

Background Assessment of minimal erythemal dose (MED) for individual patients has been used to guide the narrowband Ultraviolet B (NB‐UVB) phototherapy, which sometimes causes discomfort and additional time. The L* value (the lightness of color in Commission Internationlale de l’Eclairge L*a*b* color scale) measured by colorimeter was shown to be useful for predicting sensitivity to NB‐UVB irradiation. Objective To compare the efficacy and safety of NB‐UVB phototherapy between 50% of MED and colorimetric L* value starting dose regimens for skin phototype III–V Korean patients with psoriasis. Method Twenty seven patients determined starting doses based on colorimetric L* value, and 27 patients based on 50% of MED. Since correlation analysis showed that L* value had the most significant association with MED compared with skin phototypes, a*, and b* values, we designated starting doses of L* value regimen as follows: 300 mJ/cm² (L* >66), 400 mJ/cm² (62 < L*≤66), and 500 mJ/cm² (L*≤62). Results There was no significant difference between two groups in clinical efficacy including response rate, mean number of sessions, duration of treatment, maximum dose and cumulative dose until achieving the state of near clearance. The proportion of adverse effects was not also significantly different. Conclusions NB‐UVB starting dose determination based on colorimetric L* value was comparable with conventional MED based regimen in efficacy and safety for skin phototype III–V patients. Since it provides much convenience and ease for both patients and physicians, colorimetric L* value could partly substitute the MED checking methods in NB‐UVB phototherapy.