Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.     

Distinct melanogenic response of human melanocytes in mono-culture, in co-culture with keratinocytes and in reconstructed epidermis, to UV exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co-cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm2), an increase in melanin synthesis whereas in melanocyte mono-cultures, UVB doses up to 50 mJ/cm2 had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono- and co-cultures. Removing certain keratinocyte growth factors from the co-culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte-melanocyte co-cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB-induced pigmentation, (ii) UVA-induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV-induced pigmentation in vitro.     

Involvement of ROS/ASMase/JNK signalling pathway in inhibiting UVA-induced apoptosis of HaCaT cells by polypeptide from Chlamys farreri

Polypeptide from Chlamys farreri (PCF), a novel marine active material isolated from gonochoric Chinese scallop C. farreri, has potential antioxidant activity and protective effect against ultraviolet (UV) irradiation. The aim was to investigate whether PCF protects HaCaT cells from apoptosis induced by UVA and explore related molecular mechanisms. The results showed that PCF significantly prevented UVA-induced apoptosis of HaCaT cells. PCF not only strongly reduced the intracellular reactive oxygen species (ROS) production, but also diminished expression of acid sphingomyelinase (ASMase) and phosphorylated JNK in HaCaT cells radiated by UVA in a dose-dependent manner. Pre-treatment with ROS scavenger NAC, ASMase inhibitor Desipramine or JNK inhibitor SP600125 was found to effectively prohibit UVA-induced apoptosis and Desipramine markedly blocked phosphorylation of JNK. So it is concluded that PCF obviously protects HaCaT cells from apoptosis induced by UVA and protective effects may attribute to decreasing intracellular ROS level and blocking ASMase/JNK apoptotic signalling pathway.     

UVA-induced calcium oscillations in rat mast cells

UVA is a major bio-active component in solar irradiation, and is shown to have immunomodulatory and anti-inflammatory effects. The detailed molecular mechanism of UVA action in regard to calcium signaling in mast cells, however, is not fully understood. In this study, it was found that UVA induced ROS formation and cytosolic calcium oscillations in individual rat mast cells. Exogenously added H2O2 and hypoxanthine/xanthine oxidase (HX/XOD) mimicked UVA effects on cytosolic calcium increases. Regular calcium oscillation induced by UVA irradiation was inhibited completely by the phosphatidylinositol-specific phospholipase C inhibitor U73122, but U73343 was without effect. Tetrandrine, a calcium entry blocker, or calcium-free buffer abolished UVA-induced calcium oscillations. L-type calcium channel blocker nifedipine and stores-operated calcium channel blocker SK&F96365 had no such inhibitory effect. ROS induction by UVA was abolished after pre-incubation with anti-oxidant NAC or with NAD(P)H oxidase inhibitor DPI; such treatment also made UVA-induced calcium oscillation to disappear. UVA irradiation did not increase mast cell diameter, but it made mast cell structure more granular. Spectral confocal imaging revealed that the emission spectrum of the endogenous fluorophore in single mast cell contained a sizable peak which corresponded to that of NAD(P)H. Taken together, these data suggest that UVA in rat mast cells could activate NAD(P)H oxidase, to produce ROS, which in turn activates phospholipase C signaling, to trigger regular cytosolic calcium oscillation.     

Ultraviolet A-Induced Cathepsin K Expression Is Mediated via MAPK/AP-1 Pathway in Human Dermal Fibroblasts

Background

Cathepsin K (CatK), a cysteine protease with the potent elastolytic activity, plays a predominant role in intracellular elastin degradation in human dermal fibroblasts (HDFs), and contributes to solar elastosis. In previous studies, CatK expression was downregulated in photoaged skin and fibroblasts, but upregulated in acute UVA-irradiated skin and fibroblasts. The underlying mechanisms regulating UVA-induced CatK expression remain elusive.

Objective

This study investigates mechanisms involved in the regulation of UVA-induced CatK expression in HDFs.

Methods

Primary HDFs were exposed to UVA. Cell proliferation was analyzed using a colorimetric assay of relative cell number. Quantitative real-time RT-PCR and Western blot were performed to detect CatK expression in HDFs on three consecutive days after 10 J/cm² UVA irradiation, or cells treated with increasing UVA doses. UVA-activated MAPK/AP-1 pathway was examined by Western blot. Effects of inhibition of MAPK pathway and knockdown of Jun and Fos on UVA-induced CatK expression were also measured by real-time RT-PCR and Western blot.

Results

UVA significantly increased CatK mRNA and protein expression in a dose-dependent manner. UVA-induced CatK expression occurred along with UVA-activated phosphorylation of JNK, p38 and Jun, UVA-increased expression of Fos. Inactivation of JNK and p38MAPK pathways both remarkably decreased UVA-induced CatK expression, which was suppressed more by inhibition of JNK pathway. Furthermore, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatK expression.

Conclusion

UVA is capable of increasing CatK expression in HDFs, most likely by activation of MAPK pathway and of AP-1, which has been shown to be the case for matrix metalloproteinases. As current strategies for selecting anti-photoaging agents focus on their ability to decrease MMPs'' expression through inhibiting UV- activated MAPK pathway, future strategies should also consider their effect on CatK expression.     

Involvement of ROS/ASMase/JNK signalling pathway in inhibiting UVA-induced apoptosis of HaCaT cells by polypeptide from Chlamys farreri

Polypeptide from Chlamys farreri (PCF), a novel marine active material isolated from gonochoric Chinese scallop C. farreri, has potential antioxidant activity and protective effect against ultraviolet (UV) irradiation. The aim was to investigate whether PCF protects HaCaT cells from apoptosis induced by UVA and explore related molecular mechanisms. The results showed that PCF significantly prevented UVA-induced apoptosis of HaCaT cells. PCF not only strongly reduced the intracellular reactive oxygen species (ROS) production, but also diminished expression of acid sphingomyelinase (ASMase) and phosphorylated JNK in HaCaT cells radiated by UVA in a dose-dependent manner. Pre-treatment with ROS scavenger NAC, ASMase inhibitor Desipramine or JNK inhibitor SP600125 was found to effectively prohibit UVA-induced apoptosis and Desipramine markedly blocked phosphorylation of JNK. So it is concluded that PCF obviously protects HaCaT cells from apoptosis induced by UVA and protective effects may attribute to decreasing intracellular ROS level and blocking ASMase/JNK apoptotic signalling pathway.     

UVA radiation stimulates ceramide production: relationship to oxidative stress and potential role in ERK, JNK, and p38 activation

Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4-9 J/cm(2). The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.     

Ultraviolet A induces apoptosis via reactive oxygen species in a model for Smith-Lemli-Opitz syndrome

Solar ultraviolet A (UVA) radiation induces many responses in skin including oxidative stress, DNA damage, inflammation, and skin cancer. Smith-Lemli-Opitz syndrome (SLO-S) patients show dramatically enhanced immediate (5 min) and extended (24-48 h) skin inflammation in response to low UVA doses compared to normal skin. Mutations in Delta7-dehydrocholesterol reductase, which converts 7-dehydrocholesterol to cholesterol, produces high levels of 7-dehydrocholesterol in SLO-S patient's serum. Since 7-dehydrocholesterol is more rapidly oxidized than cholesterol, we hypothesized that 7-dehydrocholesterol enhances UVA-induced oxidative stress leading to keratinocyte death and inflammation. When keratinocytes containing high 7-dehydrocholesterol and low cholesterol were exposed to UVA (10 J/cm2), eightfold greater reactive oxygen species (ROS) were produced than in normal keratinocytes after 15 min. UVA induced 7-dehydrocholesterol concentration-dependent cell death at 24 h. These responses were inhibited by antioxidants, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium) and a mitochondria-specific radical quencher. Cell death was characterized by activation of caspases-3, -8, and -9 and by phosphatidylserine translocation. Studies using antioxidants and specific caspase inhibitors indicated that activation of caspase-8, but not caspase-9, mediates ROS-dependent caspase-3 activation and suggested that ROS from NADPH oxidase activate caspase-8. These results support a ROS-mediated apoptotic mechanism for the enhanced UVA-induced inflammation in SLO-S patients.     

Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from <Emphasis Type="Italic">Chlamys farreri</Emphasis> in human HaCaT keratinocytes

Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes, including apoptosis. Polypeptide from Chlamys farreri (PCF) is a novel marine active material isolated from the gonochoric Chinese scallop C. farreri. In our previous studies, PCF was found to be an effective antioxidant inhibiting UVA-induced ROS production and a potential inhibitory agent for UVA-induced apoptosis in the human keratinocyte cell line HaCaT. The intracellular mechanisms of how PCF protects HaCaT cells from UVA-induced apoptosis are not understood. Thus, we here investigate the effect of PCF on UVA-induced intracellular signaling of apoptosis. Pretreatment with the ROS scavenger N-acetylcysteine (NAC), the p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor Ac-DEVD-CHO was found to effectively prevent UVA-induced apoptosis, indicating that ROS, p38 MAPK and caspase-3 play important roles in apoptosis. H₂O₂-induced apoptosis was attenuated by PCF, suggesting that PCF plays its anti-apoptotic role through its antioxidant activity. In addition, PCF treatment inhibited UVA-induced p38 MAPK activation and caspase-3 activation, as assayed by Western blot analysis and flow cytometry, respectively. Our results suggest that PCF attenuates UVA-induced apoptosis through a reduction of ROS generation and diminished p38 MAPK and caspase-3 activation.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Ultraviolet radiation-induced photodegradation and 1O2, O2-. production by riboflavin, lumichrome and lumiflavin

Although UVA (320-400 nm) is considered less harmful to skin as compared to UVB (290-320 nm) and UVC (200-290 nm) radiation, certain endogenous chromophores may enhance UVA-induced cutaneous reactions by largely O2-dependent photodynamic reactions. Photodegradation pattern and singlet oxygen (1O2), superoxide anion radical (O2-.) producing capacity of riboflavin (RF), lumiflavin (LF) and lumichrome (LC) were examined to assess their phototoxic potential under UVA. Photolysis of RF upon exposure to UVA, UVB or UVC revealed considerable degradation to LF and LC with a near identical spectral pattern of photodegradation between 250-500 nm. Both LF and LC were stable to UVA (3 J/cm2) and UVB (400 mJ/cm2), whereas RF was photodegraded by 30 and 20%, respectively, under similar irradiation conditions. UVA-sensitized LF and LC respectively, produced nearly 15% higher and 60% lower yield of 1O2 in comparison to RF, whereas, O2-. was generated predominently by RF. Both RF and LF thus appeared to be potential chromophores for evoking deleterious effects of UVA in normal human skin.     

Fate of photo-induced 8-methoxypsoralen mono-adducts in yeast. Evidence for bypass of these lesions in the absence of excision repair

A fraction of UVA-induced 8-methoxypsoralen (8-MOP) mono-adducts can be transformed by a second UVA (365 nm) irradiation procedure into lethal cross-links in Saccharomyces cerevisiae. To follow the fate of cross-linkable mono-adducts, cells were incubated in complete medium between the two UVA doses and survival was measured. The killing effect of the second UVA dose decreases rapidly in haploid wild-type as well as in strains blocked in mutagenic (RAD6+ type) or in recombinogenic (RAD52+ type) repair pathways. This is also true in the pso1-1 and pso2-1 strains selected for sensitivity to 8-MOP plus UVA treatment. In contrast, persistence of mono-adducts is observed in strains blocked in the excision-resynthesis repair pathway. In other words, cross-linkable mono-adducts are repaired by the excision process. The use of the cell-cycle conditional mutant strain (cdc14-1) permitted us to apply the second dose at a specific cell-cycle stage (post-G2 phase) after a 'priming' UVA treatment on stationary (G1) phase cells. Such experiments showed a bypass of mono-adducts in an excision-deficient context for at least one round of DNA replication.     

A photochemical study of cells loaded with 2',7'-dichlorofluorescin: implications for the detection of reactive oxygen species generated during UVA irradiation

There have been several attempts to implicate reactive oxygen species in UVA-induced damage by loading cells with 2',7'-dichlorofluorescin (DCFH) and following the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. However, both DCF and DCFH have significant absorption in the 300-400 nm range so it is possible that photochemical reactions will occur in cells containing these dyes when they are irradiated with UVA. HaCaT keratinocytes loaded with DCFH were irradiated with 0, 1, 2, or 4 J/cm(2) UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm(2) UVA exhibiting an almost 10-fold increase over dark controls. However, there was no difference in cell viability, as measured by the MTS assay or LDH release, between the dark and the 4 J/cm(2) UVA-exposed groups. Furthermore, a large increase in DCF fluorescence was observed when a cell-free system containing DCF, DCFH, and horseradish peroxidase was UVA irradiated. As a control, keratinocytes loaded with DCFH were incubated in the dark with either exogenously added H(2)O(2) or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H(2)O(2)). In both cases, the cells showed a concentration-dependent increase in DCF fluorescence and a concomitant decrease in viability. Our findings suggest that DCFH can not be used to detect the UVA-induced generation of reactive oxygen species in cells when the dye is present during exposure.     

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.     

L-arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide.

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-arginine (L-Arg) for producing nitric oxide (NO*). This report delineates the relations between NO* and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L-Arg just after irradiation. L-Arg (5 mM) supply led to an increase in UVA (25.3 J/cm(2)) cytotoxicity (% of viability 18 +/- 3%) whereas neither L-Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L-thiocitrulline (L-Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L-Thio and SOD prevented L-Arg-mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L-Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the 'comet assay' and quantified using the 'tail moment'. UVA alone was genotoxic (mean tail moment: 25.43 +/- 1.23, P<0.001 compared control cells). The addition of L-Arg potentiated DNA damage (mean tail moment: 41.05+/-3.9) whereas L-Thio prevented them (mean tail moment 9.86 +/- 0.98). We attempted to assess the effect of poly(ADP-ribose) polymerase (PARP) inhibition on cell death. Using the PARP inhibitor 3-aminobenzamide, we established that PARP determines both cell lysis and DNA damage induced by UVA and/or L-Arg. Our findings demonstrated that L-Arg was able to increase UVA-mediated deleterious effects in keratinocytes (both DNA damage and cytotoxicity) and that the ratio NO*/O2*- plays a key role in these processes.     

UVA irradiation induces L-isoaspartyl formation in melanoma cell proteins.

It has been reported that UVA effects are partly mediated by production of reactive oxygen species. Moreover, oxidative stress increases protein damage, involving the occurrence of isoaspartyl residues, a product of protein deamidation/isomerization reactions. This work was undertaken in order to study the effects of UVA irradiation, mediated by oxidation, on sensitive protein targets. Melanoma cells exposed to UVA rays have been chosen as a model for monitoring the occurrence of L-isoaspartyl sites. A dramatic increase of these abnormal residues, specifically recognized and methylated by the enzyme L-isoaspartate(D-aspartate) O-methyltransferase (PCMT; EC 2.1.1.77), can be detected after exposure of M14 cells to raising doses of UVA. The effect of UVA on NO and TBARS accumulation, as well as on DNA fragmentation, has also been investigated. NO formation parallels the increase in isoaspartyl formation, while lipid peroxidation occurs only at the highest UVA doses. No DNA fragmentation has been detected under the employed experimental conditions. These results, as a whole, indicate that protein damages are one of the early events on UVA-induced cell injury. The endogenous activity of PCMT remains remarkably stable under UVA treatment, suggesting that this enzyme might play a crucial role in the repair and/or disposal of damaged proteins in UVA-irradiated cells.