The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts

The effects of ultraviolet A (UVA) radiation and reactive oxygen species (ROS), generated with a xanthine and xanthine oxidase (XOD) system, on collagen enzymatic degradation involving the matrix metalloproteinase (MMP) and its tissue inhibitor of metalloproteinase (TIMP) were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using cDNA clones for human interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2) and TIMP-2. UVA irradiation resulted in an increase in MMP-1 mRNA up to 2.3-fold, but did not stimulate MMP-2 or TIMP-2 mRNA expression. In contrast, ROS induced by the xanthine and XOD system resulted in a dose-related increase in the level of MMP-2 mRNA up to 2.1-fold and a decrease in the level of TIMP-2 mRNA by 49% in the same fibroblasts. Catalase, used as scavenger, essentially prevented the ROS-induced alterations in MMP-2 and TIMP-2 mRNA levels. These results suggest that ROS produced in the dermis may contribute to biological changes in the connective tissue matrix observed in photoaging skin by accelerating the MMP-2-related matrix degradation system. Received: 6 September 1994     

The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts

The effects of ultraviolet A (UVA) radiation and reactive oxygen species (ROS), generated with a xanthine and xanthine oxidase (XOD) system, on collagen enzymatic degradation involving the matrix metalloproteinase (MMP) and its tissue inhibitor of metalloproteinase (TIMP) were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using cDNA clones for human interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2) and TIMP-2. UVA irradiation resulted in an increase in MMP-1 mRNA up to 2.3-fold, but did not stimulate MMP-2 or TIMP-2 mRNA expression. In contrast, ROS induced by the xanthine and XOD system resulted in a dose-related increase in the level of MMP-2 mRNA up to 2.1-fold and a decrease in the level of TIMP-2 mRNA by 49% in the same fibroblasts. Catalase, used as scavenger, essentially prevented the ROS-induced alterations in MMP-2 and TIMP-2 mRNA levels. These results suggest that ROS produced in the dermis may contribute to biological changes in the connective tissue matrix observed in photoaging skin by accelerating the MMP-2-related matrix degradation system.     

Immunoprotective UVA (320-400 nm) irradiation upregulates heme oxygenase-1 in the dermis and epidermis of hairless mouse skin

The induction of heme oxygenase-1 (HO-1) by ultraviolet A (UVA) (320-400 nm) radiation provides a protective cellular defence against oxidative stress, and has been well demonstrated in cultured human skin fibroblasts, although keratinocytes were unreactive. The UVA responsiveness of HO-1 however, has not been confirmed in intact skin. Previously, we reported that UVA-inducible HO enzyme activity in mouse skin is protective against UVB-induced immunosuppression. This study identifies the induced HO isoform and its localization in mouse skin irradiated in vivo with such an immunoprotective UVA dose. We found that HO-1 mRNA was expressed in UVA-irradiated skin, but not in normal or UVB-irradiated skin, whereas constitutive HO-2 was always present. UVA-irradiated skin had increased HO enzyme activity and bilirubin content, and decreased heme content, consistent with HO-1 induction. In situ hybridization and immunohistochemical staining localized HO-1 mRNA and protein to both epidermis and dermis, with strongest expression in basal keratinocytes and weaker expression in dermal fibroblast-like and other cells, in contrast with UVA-induced HO-1 in cultured human skin fibroblasts. This suggests that cultured skin cells may not fully represent skin functions in vivo, or that there may be inherent differences between human and hairless mouse skin HO-1 responses.     

紫外线照射后生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用

目的：明确对UVA及UVB照射后皮肤成纤维细胞生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用。方法：紫外线照射人皮肤成纤维细胞,提取细胞上清液中的微囊泡,利用光散射分析技术鉴定分析微囊泡的大小及数量。将紫外线照射后生成的微囊泡与正常成纤维细胞共孵育,荧光酶标仪定量检测活性氧含量,流式细胞仪检测细胞凋亡率。结果：UVA及UVB照射后皮肤成纤维细胞释放的微囊泡数量及大小明显高于正常成纤维细胞释放的微囊泡。正常纤维细胞、UVA和UVB照射后的成纤维细胞与微囊泡共孵育后活性氧荧光值分别为（52.76±1.4347）、（82.60±4.082）和（85.94±6.264）,凋亡率分别为（3.260±1.732）%,（28.94±2.430）%和（34.48±2.718）%,细胞的氧化损伤和凋亡可被抗氧化剂逆转。结论：急性中长波紫外线照射可诱导皮肤成纤维细胞释放微囊泡进一步介导细胞的氧化损伤和凋亡。     

Predominant effects of Polypodium leucotomos on membrane integrity,lipid peroxidation,and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts,and keratinocytes

BACKGROUND: Polypodium leucotomos has been reported to have antioxidant, anti-inflammatory and photoprotective properties. Exposure of skin to ultraviolet (UV) radiation can lead to deposition of excessive elastotic material, reduction in collagen, and increased expression of matrix metalloproteinases (MMPs). OBJECTIVE: The goal of this research was to determine the effects of P. leucotomos in the absence or presence of UVA or UVB radiation on membrane damage, lipid peroxidation, and expression of elastin and MMP-1 in fibroblasts and keratinocytes, respectively. METHODS: Fibroblasts and keratinocytes, respectively, were irradiated by a single exposure to UVA (0.6, 1.8 or 3.6 J) or UVB radiation (0.75, 2.5 or 7.5 mJ), and then incubated with, or without, P. leucotomos (0.01, 0.1 and 1%) and examined for membrane damage, lipid peroxidation, expression of elastin (protein levels) and MMP-1 (protein levels or MMP-1 promoter activity). RESULTS: UV radiation did not significantly alter membrane integrity, lipid peroxidation or MMP-1 expression, but increased elastin expression. P. leucotomos significantly improved membrane integrity, inhibited lipid peroxidation, increased elastin expression, and inhibited MMP-1 expression in both fibroblasts, and keratinocytes. The effects of P. leucotomos predominated in the presence of UVA or UVB in both fibroblasts and keratinocytes, respectively, with the exception of inhibition of MMP-1 protein levels in fibroblasts only in combination with UV radiation. CONCLUSION: Lower concentration of P. leucotomos (lower than 0.1%), may be beneficial in preventing photoaging by improving membrane integrity and inhibiting MMP-1, without increasing elastin expression. Higher concentration (greater than 0.1%) of P. leucotomos may reverse the loss of normal elastic fibers associated with intrinsic aging.     

Attachment and chemotaxis of melanocytes after ultraviolet irradiation in vitro

Because ultraviolet (UV) radiation is able to influence the spatial distribution of melanocytes in melanocytic naevi in vivo, we investigated the influence of UV radiation on the ability of melanocytes to adhere to the extracellular matrix proteins fibronectin, laminin and collagen type IV in vitro. In addition, chemotaxis of melanocytes was studied using both fibronectin and the supernatants from irradiated, as well as non-irradiated, keratinocytes and fibroblasts as attractants. Melanocyte attachment to fibronectin was significantly increased 48 h after a single UV irradiation at 30 mJ/cm2 in comparison with that of non-irradiated melanocytes, whereas attachment to laminin and collagen type IV showed only minor changes after UV exposure. The UV-induced increase in attachment to fibronectin was suppressed by preincubation with antibodies against alpha5beta1 or alphavbeta3 integrin. Both immunohistochemistry and flow cytometric analysis showed an increase in alpha5beta1 integrin expression on melanocytes after UV exposure. The chemotaxis of melanocytes to fibronectin was not influenced by UV exposure. A decreasing migration rate of melanocytes towards the supernatants of UVA-irradiated fibroblasts was observed with increasing UVA doses. The chemotactic effects of conditioned medium of keratinocytes towards melanocytes was not influenced either by UVB or by UVA. The results indicate that UV radiation may alter the ability of melanocytes to adhere to certain substrates by modification of integrin expression. Because fibronectin, as the major target protein of UV-altered attachment, is located in the dermis, the UV-induced morphological changes in melanocytic lesions, with an increase in suprabasally located melanocytes within the epidermis, may be due to other changes in the adhesive properties of melanocytes.     

The contributions of UVA and UVB to connective tissue damage in hairless mice

UVA, in high-dose single exposures, can, like UVB, be deleterious to skin. Dermal damage resulting from chronic exposure to UVA has not been studied. To investigate the long-term effects, we irradiated albino hairless mice for 30-34 weeks with UVA radiation, alone, from two sources with differing spectral qualities, and in combination with UVB as solar-simulating radiation. The results were compared to UVB alone. Like UVB, the UVA waveband, especially that with a spectral distribution similar to solar UVA, caused elastic fiber damage, increased glycosaminoglycan levels, and produced hypertrophy of deep dermal tissues. There were, however, striking differences between UVB- and UVA-irradiated skin. A combination of UVA and UVB summated the effects of both wavebands. Substantial protection against these effects was afforded by a broad-spectrum sunscreen.     

Sunscreens with low sun protection factor inhibit ultraviolet B and A photoaging in the skin of the hairless albino mouse.

We examined the chronic effect of long daily suberythemal, fluorescent solar-stimulated radiation (FSSR; ultraviolet B (UVB)+A(UVA)) and UVA alone on female Skh-1 hairless albino mouse skin. Mice were dorsally irradiated 8 h every weekday for 16 weeks with FSSR or UVA, or 32 weeks with UVA alone. Various topical, low concentration, UVB and/or UVA sunscreens were applied before irradiation. Damage was assessed by skin-fold thickness, histology and biochemically by changes in the proportion of type III collagen. All FSSR-exposed mice showed increased skin thickening, elastic fibre hyperplasia, collagen damage and an increased proportion of type III collagen. Application of the UVB sunscreen (2.00%) resulted in marked protection for all nonbiochemical endpoints. There was no obvious advantage of adding 0.75% UVA sunscreen to the UVB sunscreen, but adding 2.00% UVA sunscreen reduced biochemical changes and connective tissue damage. Sixteen weeks of UVA irradiation caused skin thickening and laxity but the histology and biochemistry were indistinguishable from the controls. The mice irradiated with UVA for 32 weeks showed slight elastic fibre hyperplasia and collagen damage histologically, and increased skin thickening and laxity; these changes were unmodified by the 0.75% UVA sunscreen. These mice showed a significant increase in the proportion of type III collagen against which the UVA sunscreen offered protection. Our data suggest that UVA may be important in photoaging and that the use of low sun protection factor UVB+ UVA sunscreens on a day-to-day basis may offer some protection from solar photoaging.     

Matrix metalloproteinases as mediators of tissue injury in different forms of cutaneous lupus erythematosus

Background Matrix metalloproteinases (MMPs) contribute to tissue destruction, regeneration, inflammation and apoptosis and several of them are upregulated by ultraviolet (UV) radiation in skin. Although some MMPs associate with organ manifestations of systemic lupus erythematosus (SLE), their role in cutaneous lupus erythematosus (LE) is elusive. Objectives Our aim was to evaluate the expression of MMPs in SLE, subacute cutaneous LE (SCLE) and discoid LE (DLE) skin lesions and their relation to apoptosis and epidermal changes. Methods Lesional skin biopsies from 20 patients with SLE, 20 with DLE and 17 with SCLE, and from UVA/UVB‐photoprovoked skin of healthy volunteers were immunostained using antibodies to multiple MMPs and tissue inhibitors of metalloproteinases (TIMPs). The TUNEL (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labelling) method was used for detection of apoptosis. Results MMP‐3, ‐10, ‐19 and ‐26 were abundantly expressed by keratinocytes in SLE, DLE and SCLE skin samples. MMP‐7 was detected in keratinocytes in regions of oedema and vacuolization especially in SLE and SCLE, while MMP‐14 was only occasionally observed in keratinocytes. Photoprovocation did not induce MMP‐10 or ‐26 expression in skin of healthy volunteers. Epithelial TIMP‐1 expression was low while occasional positive fibroblasts were seen in the dermis. TIMP‐3 was abundantly expressed in the epidermis, endothelial cells and macrophages. Conclusions Different subtypes of cutaneous LE are fairly similar in their MMP expression profile. MMP‐3 and ‐10 mediate both epidermal changes and dermal tissue remodelling but are not present in lymphocytes. Low expression of TIMP‐1 suggests that lupus skin is characterized by proteolytic events, and targeted action using selective MMP inhibitors may reduce lupus‐induced damage in inflamed tissues.     

Repeated exposure of human fibroblasts to UVR induces secretion of stem cell factor and senescence

Background Some of chronic hyperpigmentary diseases, such as melasma, induced by multiple factors including chronic sunlight exposure, can recur even after chemical epidermal removal. Dermal factors may be involved in the pathogenesis of melasma. Changes in dermal fibroblasts resulting from chronic sun exposure might cause melanocytes to synthesize melanin in the epidermis. Objective This study aimed at determining the effects of repetitive ultraviolet (UV) radiation on cultured fibroblasts and the secretion of melanogenic factors. Methods Cultured human fibroblasts were exposed to ultraviolet A (UVA) or ultraviolet B (UVB) for five consecutive days. After each irradiation, the supernatant medium was isolated from each dish and measured for levels of stem cell factor (SCF) and hepatocyte growth factor using an ELISA kit assay. To assess the effect of the keratinocyte-derived factors on fibroblast-secretion of SCF and hepatocyte growth factor, we added supernatants of the UV-irradiated keratinocytes to the non-irradiated fibroblasts. Finally, the irradiated fibroblasts were stained with senescence associated-β-galactosidase to assess their senescent change. Results Fibroblasts irradiated with UVA or UVB for five consecutive days, secreted SCF at levels that increased with repeated UVA or UVB exposure. Conditioned culture medium from UV-irradiated keratinocytes also induced SCF release from fibroblasts, depending on the number of UV exposures. UVA- or UVB-irradiated fibroblasts stained positive for senescence associated-β-galactosidase, and the staining intensity increased with repeated exposure. Conclusion These results suggest that fibroblast senescence and increased SCF secretion after repeated UV irradiation may be related to the pathogenesis of recurring hyperpigmentation disorders induced by chronic sun exposure.     

GM-CSF production by epithelial cell line: Upregulation by ultraviolet A

It was demonstrated that UVB increases synthesis and expression of IL-1α and GM-CSF by keratinocytes. Upregulation of GM-CSF by UVB is reported to be mediated by IL-1α. However, regulation of IL-1α and GM-CSF by UVA is not well-known. The purpose of the present study was to evaluate the effects of UVA on IL-1α and GM-CSF production. Here we used a competitive RT-PCR for measuring cytokine gene expression in an epidermal cell line after UVA irradiation. IL-1α and GM-CSF mRNA did not show any change at 1 h and 6 h following exposure to UVA. After UVA irradiation, however, IL-1α mRNA decreased and GM-CSF mRNA increased at 24 h and the level of GM-CSF in culture supernatant increased at 24 h and 48 h. Addition of antihuman IL-1α neutralizing antibody to UVA irradiated cells did not prevent the increase of GM-CSF mRNA expression. These results suggest that UVA radiation may induce GM-CSF production through an IL-1α independent pathway.     

Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA radiation

BACKGROUND: Apoptosis has been proposed to act as an important mechanism for eliminating keratinocytes that have been irreversibly damaged by ultraviolet (UV) irradiation. One way to induce apoptosis in keratinocytes is through activation of the cell surface receptor Fas (CD95), either with the ligand (FasL) or directly with UV radiation. OBJECTIVES: To investigate the regulation of Fas and FasL expression in human skin and the formation of apoptotic cells after in vivo exposure to UVB or long-wave UVA radiation. METHODS: Volunteers were irradiated with either 3 minimal erythema doses (MED) of UVB (n = 6) or 3 MED of long-wave UVA (n = 6) on buttock skin 12, 24 and 72 h before skin punch biopsies were taken. Expression of Fas and FasL was demonstrated by immunohistochemistry on cryostat sections. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate nick-end labelling reaction. RESULTS: In five of six subjects, exposure to UVB radiation resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 24 and 72 h after irradiation. In all subjects, exposure to long-wave UVA resulted in increased homogeneous expression of Fas on epidermal cells, with greatest expression at 12 h after irradiation. In five of six subjects, exposure to UVB radiation resulted in temporarily decreased expression of FasL, but after 72 h the expression of FasL had returned to the preirradiation level. The expression of FasL on epidermal cells after exposure to long-wave UVA showed considerable variation. UVB irradiation was a stronger inducer of epidermal apoptosis than was UVA irradiation. The number of apoptotic epidermal cells did not correlate with expression of Fas or FasL. CONCLUSIONS: In human skin the expression of Fas on epidermal cells increases after in vivo exposure to UVB or long-wave UVA. Exposure to UVB causes a temporary decrease in the expression of FasL on epidermal cells.     

DNA repair elicited by UVB during PUVA therapy for psoriasis

Summary The skin of patients receiving psoralen and UVA (PUVA) therapy for psoriasis is exposed to trace amounts of UVB radiation emitted by PUVA irradiators in addition to UVA. DNA repair activity was measured using autoradiography in the uninvolved skin of PUVA-treated patients in order to determine whether 8-methoxypsoralen (8-MOP) plus UVA elicits repair, inhibits the skin repair response to UVB, or protects epidermal-cell DNA from UVB damage by promoting a tan. Epidermal-DNA repair activity was observed in 27 out of 37 patients following the first PUVA treatment. Phototesting with multiples of the initial UV dose elicited a linear increase in repair activity. Glass-filtered radiation failed to stimulate repair, indicating that the reaction was due to UVB, not to 8-MOP plus UVA. The same amount of repair activity was observed in the skin of patients irradiated either before or after 8-MOP ingestion, demonstrating that the drug did not interfere with the response of the skin to UVB. At clearing, however, the repair activity was never greater than that elicited at the initial treatment and was often undetectable despite a tenfold increase in UV exposure. It is proposed that DNA damage should be measured to determine whether epidermal cells are entirely protected from UVB radiation at the completion of therapy.     

Impact of ultraviolet radiation on the expression of marker proteins of gap and adhesion junctions in human epidermis

Aims/background: We aimed to investigate the impact of ultraviolet B (UVB) as well as UVA1 on the epidermal expression of specific markers of gap and adhesion junctions. Methods: Twelve healthy subjects were enrolled in the study. The back of the subjects was irradiated with three MED‐UVB as well as three MED‐UVA1. Twenty‐four hours later, punch biopsies were taken from irradiated and non‐irradiated skin. Immunohistochemical procedures were used for the detection of connexin 43, E‐cadherin, involucrin, Ki‐67 using specific antibodies. Results: Staining intensity of connexin 43 in UVB‐exposed skin was significantly increased when compared with non‐exposed and UVA1‐exposed sites. By contrast, staining intensity of E‐cadherin in UVB‐exposed skin was significantly decreased when compared with non‐exposed and UVA1‐exposed sites. Involucrin and Ki‐67 staining of keratinocytes was significantly increased in UVB‐exposed sites as compared with non‐exposed and UVA1‐irradiated sites. Conclusions: UVB significantly alters the epidermal expression of gap and adhesion junction proteins possibly indicating a role of these proteins in the regulation of UV‐induced inflammation and development and progression of skin cancer.     

皮肤光老化机制研究进展

光老化指皮肤由于反复光暴露引起的提前老化,其发生机制涉及细胞内信号转导通路、细胞表面细胞因子和表皮生长因子受体的活化,活性氧族是激发细胞信号转导过程中的重要介质,其结果导致基质金属蛋白酶合成增加,从而使真皮纤维结缔组织过度降解和弹性纤维变性,同时真皮胶原的合成亦减少,角质形成细胞、成纤维细胞及炎症浸润细胞等多种细胞参与光老化过程.     

皮肤光老化机制研究进展

光老化指皮肤由于反复光暴露引起的提前老化,其发生机制涉及细胞内信号转导通路、细胞表面细胞因子和表皮生长因子受体的活化,活性氧族是激发细胞信号转导过程中的重要介质,其结果导致基质金属蛋白酶合成增加,从而使真皮纤维结缔组织过度降解和弹性纤维变性,同时真皮胶原的合成亦减少,角质形成细胞、成纤维细胞及炎症浸润细胞等多种细胞参与光老化过程.