Free Radical Biology & Medicine 43 (2007) 128–135www.elsevier.com/locate/freeradbiomed

Original Contribution

Cellular response to infrared radiation involves retrogrademitochondrial signaling

Peter Schroeder, Corinna Pohl, Christian Calles, Corinna Marks, Susanne Wild, Jean Krutmann ⁎

Institut für Umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University Düsseldorf gGmbH, Auf ’m Hennekamp 50,D-40225 Duesseldorf, Germany

Received 11 October 2006; revised 1 April 2007; accepted 2 April 2007Available online 10 April 2007

Abstract

Infrared A radiation (IRA) is a major component of sunlight. Similar to ultraviolet (UV) B and UVA, IRA induces gene transcription. Incontrast to the UV response very little is known about the IRA response. In the present study, IRA-induced expression of matrixmetalloproteinase-1 (MMP-1) was found to be mediated by the formation of intracellular reactive oxygen species (ROS). Staining of IRA-irradiated cells with MitoSox revealed an increase in mitochondrial superoxide anion production and treatment of fibroblasts with themitochondrial targeted antioxidant MitoQ completely abrogated the IRA, but not the UVB or UVA1, response. ROS relevant for IRA-inducedsignaling originated from the mt electron transport chain, because (i) chemical inhibition of the electron transport chain prevented IRA, but notUVB or UVA1, radiation-induced MMP-1 expression, (ii) rho0 fibroblasts specifically failed to increase MMP-1 expression in response to IRA,and (iii) peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) overexpressing fibroblasts with increased electron transportchain content were hypersensitive to IRA radiation-induced gene expression. Thus, IRA, in contrast to UV, elicits a retrograde signaling responsein human skin.© 2007 Elsevier Inc. All rights reserved.

Keywords: Infrared; Retrograde signaling; Mitochondria

Introduction

In addition to ultraviolet (UV) B (290–315 nm) and UVA(315–400 nm) radiation, human skin is exposed to infraredradiation (IR) from natural sunlight as well as artificial UV andinfrared irradiation devices [1]. While the photon energy ofinfrared radiation is lower than that of UV radiation, the totalamount of energy transferred by the sun is approximately 54%from IR radiation while UV radiation accounts only for 7% [2].Most of the IR radiation lies within the IRA (760–1440 nm)range (approximately 30% of total solar energy), which in

Abbreviations: DCF, 2’,7-dichlorofluorescein; DCFDA, 2’,7’-dichlorofluor-escein diacetate; ETC, electron transport chain; MAPKs, mitogen-activatedprotein kinases; MMP-1, matrix metalloproteinase-1; PBS, phosphate-bufferedsaline; PGC-1, peroxisome proliferator-activated receptor gamma coactivator-1;ROS, reactive oxygen species; RT-PCR, reverse transcriptase-polymerase chainreaction.⁎ Corresponding author. Fax: +49 211 312976.E-mail address: krutmann@rz.uni-duesseldorf.de (J. Krutmann).

0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2007.04.002

contrast to IRB (1440–3000 nm) or IRC (3000 nm–1 mm)deeply penetrates human skin. It is therefore not surprising thatepidemiological data and clinical observations suggest that IRAis not innocuous to human skin, but can induce biologicaleffects [3]. Indeed, recent studies demonstrate that in humandermal fibroblasts IRA radiation, similar to UV radiation, is ableto activate mitogen-activated protein kinases (MAPKs) and toinduce the expression of matrix metalloproteinase-1 (MMP-1)[4–6].

In previous years, the molecular mechanisms involved inUVA- or UVB radiation-induced gene expression have beenanalyzed in great detail [7–9]. In contrast, the molecularconsequences resulting from IRA exposure are virtuallyunknown. In order to elicit a biological effect, any type ofradiation must be absorbed by a chromophore. For IRAradiation, components of the mitochondrial (mt) respiratorychain, e.g., cytochrome c oxidase, have been suggested aspossible photoreceptors [10]. There is increasing evidence thatmitochondria may play a critical role in intracellular signaling

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[11]. The general term for mitochondrial signaling is retrograderegulation and is broadly defined as cellular responses tochanges in the functional state of mitochondria.

In the present study we provide evidence that IRA radiationexposure of human dermal fibroblasts leads to the generation ofsuperoxide anions originating from the respiratory chain andthereby initiates a retrograde mitochondrial signaling responsewhich is responsible for IRA radiation-induced MMP-1expression.

Materials and methods

Materials

MitoSox was from Invitrogen (Karlsruhe, Germany). MitoQwas a kind gift from Michael P. Murphy, Cambridge, UnitedKingdom. All other chemicals were purchased from Sigma(Taufkirchen, Germany).

Cell culture

Primary human normal skin fibroblasts were derived fromforeskins from healthy donors aged 3–8 years and were used forexperiments in passages between 4 and 14. Cells were culturedin Eagle's minimum essential medium (Life TechnologiesGmbH, Eggenstein, Germany) containing 10% fetal calf serum(Greiner, Frickenhausen, Germany), 0.1% L-glutamine, 2.5%NaHCO3, and 1% streptomycin/amphotericin B in a humidifiedatmosphere containing 5% CO2. Cells were kept in culturedishes (diameter 10 cm) for culture and irradiation.

Ethidium bromide treatment

For induced depletion of electron transport chain activityhuman fibroblasts which had been immortalized through stabletransfection with human telomerase (hTert [12]; kindlyprovided by O. Toussaint, Namur, Belgium) were cultured inDulbecco's modified Eagle's minimum essential mediumcontaining 4.5 g/L glucose, 10% fetal calf serum, 50 μg/mLuridine, 1 mM sodium pyruvate, and 50 ng/mL ethidiumbromide at 37°C and 5% CO2. Fresh medium was applied everysecond day. The content of mtDNAwas monitored by real-timePCR based on [13] and oxygen consumption by Clark electrodemeasurements as previously described [14].

Cell transfection

For generation of cells with increased electron transportchain activity, primary human skin fibroblasts were transientlytransfected (Lipofectamine 2000, Invitrogen, Karlsruhe Ger-many) with a two plasmid system (Rheoswitch mammalianinducible expression system, New England Biolabs, Beverly,MA) as previously described [15]: plasmid A, pNEBX-1 vectorcontaining the PPARγ coactivator 1 (PGC-1), transferred fromthe construct used by [16], kindly provided by T. Finkel(Bethesda, MD); plasmid B coded the activator for PGC-1expression on plasmid A. Control transfected cells were only

transfected with plasmid A. To induce gene expression thesynthetic inductor RSL-1 (500 nM) was applied 24 h prior toIRA treatments.

Western blot

Analysis employing the clone 1D6 anti-COX-1 antibody(Molecular Probes, Eugene, OR); clone AC-15 anti-β-actinantibody (Sigma, St. Louis, MO); phosphospecific anti-ERK1/2(pTpY185/187, Biosource, Camarillo, CA), or anti-GAPDH(ab8245, Abcam, Cambridge, UK) was performed as recom-mended by the manufacturer and expression was quantified bymeans of a Alpha Innotech FluorChem 8900 with AlphaEaseFCsoftware (Biozym, Oldendorf, Germany).

Irradiations

For irradiations, medium was replaced by phosphate-buffered saline buffer (PBS), lids were removed, and cellswere exposed to radiation from a Hydrosun 500 IRA device, ora UVASUN 5000 Biomed UVA1 metal halogenide irradiationdevice, or a bank of four Philipps TL20W/12RS UVBfluorescent bulbs (Phillips, Hamburg, Germany). The IRAradiation device was water-filtered and equipped with a blackfilter and emits wavelengths between 760 and 1440 nm aspreviously described [5]. The IRA output was determined with aHydrosun HBM1 measuring device and found to be 105 mW/cm2 at a distance of 40 cm. The UVA1 output (340–400 nm)was determined with a UVAMETER (Mutzhas, Munich,Germany) and found to be approximately 70 mW/cm2 UVA1at a lamp to target distance of 30 cm. The UVB output wasmeasured with a UV-Dosimeter Type II equipped with a UV6sensor (Waldmann Medizintechnik, Villingen-Schwenningen,Germany) and found to be 0.66 mW/cm2 at a tube to targetdistance of 30 cm. The IRA, UVA1, and UVB doses employedin this study are of physiological relevance and reflect the ratiosrelevant in the dermis of human skin [2,17].

ROS measurements

For measurement of cytosolic ROS level, cells wereincubated with 100 μM 2,7-dichlorofluorescein diacetate(DCFDA) in PBS immediately after exposure to IRA radiation.Five minutes later, 2,7-dichlorofluorescein (DCF) fluorescence(excitation, 485 nm; emission, 538 nm) was measured spectro-fluorometrically by means of a Fluoroskan Ascent (Labsystems,Helsinki, Finland). Mitochondrial superoxide levels weremonitored using MitoSox (Molecular Probes). After IRAradiation exposure, cells were incubated in PBS containing5 μM MitoSox for 10 min and afterward MitoSox fluorescence(excitation, 485 nm; emission, 538 nm) was measuredspectrofluorometrically.

Glutathione assay

Determination of total glutathione content was done usingthe 5,5-dithiobis-(2-nitrobenzoic acid)-glutathione disulfide

Fig. 1. Infrared A irradiation leads to formation of reactive oxygen species incultured human dermal fibroblasts. (A) The ROS-sensitive probe DCF wasadded immediately after irradiation (IRA, 30 J/cm2; UVA1, 6 J/cm2; UVB, 25 J/m2). Fluorescence of the formed oxidized product was measured in a microplatereader. Data are means±SD of 3–6 independent experiments. (B) Immediatelyafter the irradiation cells were harvested, GSH and GSSG content was measuredutilizing the glutathione recycling assay. Data are mean±SD of 6 independentexperiments. *Significantly different to respective sham (Pb0.05).

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(GSSG) reductase recycling assay for GSSG as previouslydescribed [18,19]. All samples including standards wereassayed in duplicate. A graph of the rate of change ofabsorbance over 15 min at 415 nm (Multiskan Ascent,Labsystems. Finland) versus the amount of GSSG was drawn,and the values for the samples were calculated using a linearregression.

Real-time RT-PCR

Total RNA was isolated using RNeasy Total RNA kits(Qiagen, Hilden, Germany). Expression of MMP-1 wasmeasured by quantitative RT-PCR based on [20] using theRT-PCR core kit (Applied Biosystems, Darmstadt, Germany)and a specific primer pair for MMP-1 (5′-CATGAAAGGTG-GACCAACAATTT-3′; 5′-CCAAGAGAATGGCCGAGTTC-3′). Semiquantitative analysis of the RT-PCR products wasdone using theΔΔct method utilizing 18S RNA (primer pair: 5-GCCGCTAGAGGTGAAATTCTTG-3′; 5′-CATTCTTGG-CAAATGCTTTCG-3′) for normalization.

Statistical analysis

Results are given as means + standard deviation. Resultswere compared using the unpaired Student t test assumingunequal variances. When normal distribution was not given aMann-Whitney rank-sum test was applied. A significance levelof Pb0.05 was considered to be statistically significant.

Results

ROS generation by IRA radiation

Infrared A, UVB, and UVA radiation have previously beenshown to resemble each other in their capacity to activatemitogen activate protein kinases and to induce transcriptionalexpression of MMP-1 in human dermal fibroblasts [3,9]. ForUVB and UVA radiation, MAPKs activation and MMP-1induction critically involve the generation of ROS [9,21]. Wetherefore asked whether IRA radiation induces an oxidativestress response as well. Formation of ROS was assessed in thesecells by fluorometric analysis after staining with the ROSsensitive fluorescent probe 2,7-dichlorofluorescein [22]. As seenin Fig. 1A, IRA irradiation caused a significant increase in DCFfluorescence immediately after exposure. Note that the IRAradiation dose is 1/12 of the dose used for gene regulation studies[5]. Higher doses would require longer irradiation periods(N30 min) and IRA-induced ROS production, which is a directevent, could, probably due to compensatory cellular responses,no longer be detected then. These results indicate that IRAirradiation leads to increased intracellular ROS productionwhich as one consequence could affect the redox state of thecells. Indeed, IRA radiation significantly shifted the equilibriumof reduced and oxidized glutathione of cells to the oxidized form(Fig. 1B). The total glutathione concentration was not altered bythe irradiation (9.9±1.3 nmol/mgProtein in sham-treated cells and10.2±1.9 nmol/mgProtein in IRA-treated cells).

Functional relevance of IRA radiation-induced ROSgeneration

In order to assess whether IRA radiation-induced ROSformation was of functional relevance for IRA radiation-induced ERK1/2 activation and MMP-1 expression [5], we nextincreased the antioxidant defense of human dermal fibroblastsby incubating them for 6 h prior to irradiation with N-acetylcysteine which has been shown to increase cellularglutathione [23]. This treatment led to a significant increase ofcellular glutathione (supporting information, Fig. S-1) and didnot alter basal MMP-1 expression (Fig. 2B). As seen in Fig. 2,N-acetylcysteine pretreatment significantly reduced IRA radia-tion-induced ERK1/2 activation (Fig. 2A) and MMP-1 induc-tion (Fig. 2B).

Mitochondria as a source for IRA-induced ROS production

We next sought to identify the intracellular source of IRAradiation-induced ROS formation. Components of the mito-chondrial respiratory chain have been suggested as chromo-phores for IRA radiation [10]. In order to assess whether IRAradiation-induced ROS are derived from mitochondria, humandermal fibroblasts were stained with the specific fluorescent dyeMitoSox [24], which preferentially detects mitochondrialsuperoxide anions. As seen in Fig. 3A, MitoSox fluorescencewas significantly increased on exposure of cells to IRA

Fig. 3. Infrared A irradiation leads to formation of mitochondrial superoxide,functionally relevant for MMP-1 upregulation. (A) Detection of mitochondrialsuperoxide with MitoSox immediately after irradiation (IRA, 30 J/cm2; UVA1,6 J/cm2; UVB, 25 J/m2). Data are mean±SD of 7 independent experiments.*Significantly different from unirradiated (sham) control (Pb0.05). (B)Measurement of MMP-1 mRNA 24 h after IRA irradiation (360 J/cm2) inlysate from cells not treated with antioxidants, 100 nM MitoQ, or 100 nMIdebenone, prior, during, and after irradiation.

Fig. 2. Infrared A-induced ROS production is functionally relevant for MMP-1upregulation. (A) Measurement of ERK1/2 phosphorylation 15 min after IRAirradiation (360 J/cm2) from cells incubated for 6 h with 2 mM N-acetylcysteineor the respective solvent control prior to the irradiation. Upper panel:densitometric analysis of Western blot, mean±SD of 3 independent experi-ments. Lower panel: Western blot, representative of three independentexperiments. (B) Measurement of MMP-1 mRNA expression 24 h after IRAirradiation (360 J/cm2) from cells either not pretreated or incubated for 6 h with2 mM N-acetylcysteine prior to IRA irradiation. Mean±SD of five independentexperiments. *Significantly different to NAC-pretreated cells (Pb0.05).

Fig. 4. Infrared A-induced MMP-1 upregulation needs a functional mitochon-drial respiratory chain. Measurement of MMP-1 mRNA 24 h after irradiation(IRA, 360 J/cm2; UVA, 30 J/cm2; UVB 100 J/m2) in lysate from cells pretreatedeither with DMSO, Rotenone, antimycin A, or TTFA prior to the irradiation.Data are mean±SD of 3–4 independent experiments. *Significantly differentfrom vehicle (DMSO)-treated cells (Pb0.05).

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radiation. UVA1 or UVB irradiation had only a slight or noeffect on MitoSox fluorescence. Continuing measurements upto 30 min after cessation of the irradiation confirmed thesefindings (supporting information, Fig. S-4). At later time points(6 h, 24 h) no difference in ROS production was observed.

Relevance of mitochondria for IRA radiation-induced MMP-1expression

In order to assess the functional relevance of mitochondria-derived ROS for IRA radiation-induced MMP-1 expression, wecompared the efficacy of mitochondria-targeted and untargetedantioxidants derived from coenzyme Q10 [25–27] in preventingMMP-1 expression. Treatment of human dermal fibroblastswith the mitochondrially targeted antioxidant MitoQ at aconcentration of 100 nM completely abrogated IRA radiation-induced MMP-1 expression, whereas the untargeted analogueIdebenone had no effect at this concentration (Fig. 3B). In fact,

for Idebenone, 50-fold higher concentrations were necessary toachieve at least partial inhibition of the IRA response (data notshown). Note that MitoQ specifically inhibits IRA radiation-

Fig. 5. Infrared A-mediated MMP-1 upregulation is abrogated in cells lacking afunctional ETC and enhanced in cells with stimulated mitogenesis. (A)Measurement of MMP-1 mRNA 24 h after IRA irradiation (360 J/cm2) ofhTert fibroblasts and corresponding ρ0 cells. Data are mean±SD of 3independent experiments. *Significantly different from ρ+ cells (PN0.05) (B)Induction of MMP-1 mRNA expression compared to respective sham controls24 h after IRA irradiation. Prior to the irradiation procedure cells had beentransfected and PGC-1 expression had been induced as described. *Significantlydifferent from respective sham irradiated control (Pb0.05). #Significantlydifferent from control transfected cells (Pb0.05).

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induced MMP-1 expression, and does not decrease UVB orUVA1 radiation-induced gene expression (Fig. 3B).

Electron transport chain activity as a determinant of the IRAresponse

Most of the ROS generated by mitochondria are derivedfrom the mitochondrial electron transport chain (ETC) as a by-product of oxidative phosphorylation [28]. Infrared A radiationcan be absorbed by components of the ETC [10], indicating thethe ETC is a possible intramitochondrial source for IRAradiation-induced ROS production. In order to test thishypothesis, we next pretreated human dermal fibroblasts 24 hprior to irradiation with nonlethal concentrations of chemicalinhibitors of the mitochondrial respiratory chain [29]. As seen inFig. 4, rotenone, antimycin A, and thenouyltrifluoroacetonepretreatment almost completely inhibited IRA radiation-induced MMP-1 expression. This inhibition was specific,because pretreatment with the same inhibitors did not affectUVA1 or UVB radiation-induced MMP-1 expression (Fig. 4).

These results are in line with the assumption that the ETCplays a role in IRA radiation-induced gene expression, whileother mitochondria-independent mechanisms might be impor-tant for UVA- (R. Wiesner, 2006, personal communication) orUVB- [30] induced effects. In order to further test thispossibility we next assessed IRA radiation-induced MMP-1expression in fibroblasts with decreased or increased ETCactivity. We hypothesized that if ETC activity were importantfor IRA radiation-induced gene expression, lowering ETCactivity should weaken IRA responsiveness of cells, whereasenhancing it should have the opposite effect.

A model for cells with low ETC activity are ρ0 cells, whichcompletely lack mtDNA [31]. Unfortunately such cells are notavailable in a primary fibroblast background. In the presentstudy we have therefore used immortalized (hTert) human skinfibroblasts which were gradually depleted of mtDNA throughtreatment with low concentrations of ethidium bromide [32]. A2-week treatment with 50 ng/mL ethidium bromide induced aρ0 phenocopy as it decreased mtDNA content and oxygenconsumption below the respective detection limits (supportinginformation, Figs. S-2A and S-2B). Exposure of these cells todifferent doses of IRA, UVA1, or UVB radiation revealed thatfibroblasts with low ETC activity were not less susceptible toUVA1 or UVB radiation, whereas their capacity to respond toIRA radiation by increasing transcriptional expression of theMMP-1 gene was significantly reduced (Fig. 5A).

In order to generate a model for skin fibroblasts withincreased ETC content we have transiently transfected primaryhuman skin fibroblasts with a plasmid encoding for PGC-1[16,33]. As previously reported for cardiac myocytes [34] andhuman diploid fibroblasts [16], overexpression of PGC-1(supporting information, Fig. S-3A), but not respective controltransfection, led to increased mitochondrial biogenesis, indi-cated by increased amounts of mtDNA (supporting information,Fig. S-3B) and COX-1 protein levels (supporting information,Fig. S-3C). In such cells, IRA irradiation caused an increasedtranscriptional expression of MMP-1, which was approximately

twofold stronger in PGC-1 overexpressing fibroblasts, ascompared with control transfected cells (Fig. 5B). BasalMMP-1 expression in PGC-1 transfected cells did notsignificantly differ from that observed in control transfectedcells (supporting information, Table S-1). Taken together, thesedata imply that the amount of mitochondria, especially ETCcomponents, is a factor that determines the susceptibility of cellstoward IRA radiation-induced gene expression.

Discussion

Infrared A radiation induces retrograde mitochondrialsignaling

The present study provides clear evidence that the IRAradiation response in human skin fibroblasts critically involvesROS that are derived from mitochondria. This conclusion isbased on three major observations: (i) IRA radiation causesintracellular ROS formation and a concomitant change in theredox state of irradiated cells, and this oxidative stress responseis relevant for IRA radiation-induced signaling because anoverall increase in antioxidant defense prior to irradiationrenders cells resistant toward IRA radiation-induced geneexpression; (ii) specifically, IRA radiation induces superoxideanion formation at the mitochondria of cells, which mediate

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IRA radiation-induced signaling, because mitochondriallytargeted coenzyme Q10 was highly effective in preventingIRA radiation-induced gene expression, and (iii) three differentand independent strategies to manipulate the activity of themitochondrial ETC, i.e., its chemical inhibition and the use ofρ0 cells with low ETC activity and of PGC-1 overexpressingcells with high ETC activity suggest that the capacity of a givenfibroblast to respond to IRA radiation critically depends on theactivity of its mitochondrial ETC.

Specificity of the IRA response

The observations noted above are specific for IRA radiation.Other signaling responses that are induced in mammalian cellsby exogenous factors are thought to be mediated by ROS aswell. Prominent examples are stress responses elicited by UVBand, in particular, UVA radiation, which similar to IRAradiation can activate MAPKs and increase MMP-1 expression[4,6,7,9,29,35,36]. The present study indicates that mitochon-dria-derived ROS (mtROS) are not involved in UVA1 or inUVB radiation-induced MMP-1 expression, because mito-chondrially targeted coenzyme Q10 or manipulation of ETCactivity did not affect these responses. It is thus likely thatintracellular sources different from the mitochondrial ETC areinvolved in UVB or UVA1 radiation-induced signaling. Indeed,recent evidence indicates that plasma membrane electrontransport systems play a role in UVA radiation-induced celldeath (R. Wiesner, 2006, personal communication). The presentstudy for the first time demonstrates that exposure ofmammalian cells to an exogenous stimulus, which is part ofnatural sunlight, causes ROS formation by mitochondria.

Chromophores for IRA and potential mechanism of action

In order to elicit intramitochondrial ROS production, IRAradiation must be absorbed by a chromophore. There iscompelling evidence that mitochondria absorb in the IRArange. Accordingly, IRA irradiation of isolated mitochondriaenhanced ATP synthesis [37] and IRA irradiation of wholefibroblasts altered the uptake of rhodamine 123, which wasinterpreted as a perturbation of mitochondrial energy productionand membrane potential [10,38]. In addition, IRA radiationfrom a laser source induced cell proliferation [39,40] andanalysis of the action spectrum of this proliferation increase inHeLa cells indicated cytochrome c oxidase as the relevantchromophore [10,41]. Cytochrome c oxidase is the terminalenzyme of the ETC in eukaryotic cells, which mediates thetransfer of electrons from cytochrome c to molecular oxygen. Itis a large multicomponent and structurally complex membraneprotein of 200 kDa, which contains two heme moieties, tworedox-active copper sites, one zinc, and one magnesium ion aspotential chromophores for visible light and possibly also IRAradiation. The following four photochemical and photobiologi-cal consequences that might possibly result from the absorbanceof IRA radiation by cytochrome c oxidase have been discussed:(i) changes in the redox properties of the respiratory chaincomponents following photoexcitation of their electronic states,

(ii) generation of singlet oxygen, (iii) localized transient heatingof absorbing chromophores, and (iv) increased superoxideanion production with subsequent increase of its dismutationproduct, i.e., H2O2 [10]. In support of the latter possibility is thepresent observation that IRA radiation increases the fluores-cence of the superoxide anion-specific dye MitoSox. The factthat ETC inhibitors, which act upstream of complex IV,abrogate IRA radiation-induced signaling might be explainedby IRA radiation disturbing the function of cytochrome coxidase and thereby the electron flow which in consequencecauses additional leakage of superoxide anions at complexes 1and 3 [42]. Further experiments, which are, however, beyondthe scope of the present study, which we concentrated on generegulation, are required to determine whether IRA radiationalters the function, i.e., kinetics and/or amplitude of the workingcycle intermediates of cytochrome c oxidase in our system.

Molecular pathways involved in the IRA response

Mitochondrial retrograde signaling has been defined as apathway of communication from mitochondria to the nucleusthat influences many cellular functions under both normal andpathophysiological conditions [11,43,44]. Implicit in thisdefinition is that mitochondrial signaling is opposite in directionto that of the more familiar anterograde regulation characterizedby the transfer of information from the nucleus to mitochondria.The data presented in this study are consistent with the view thatIRA radiation-induced MMP-1 expression represents a mito-chondrial retrograde signaling response. In previous studies wehave shown that IRA radiation-induced MMP-1 expressiondepends on the activation of ERK1/2 [5]. The signaling stepsbetween IRA radiation-induced intramitochondial superoxideanion production and activation of ERK1/2 located in thecytoplasm of cells are currently not known. In general,mitochondrial signaling cascades in mammalian cells havebeen shown to involve elevated cytosolic free Ca2+ andactivation of Ca2+/calmodulin-responsive calcineurin [43,45].In addition, in mammalian cells retrograde signaling is alsolinked to TOR signaling, but the precise signals and connectionsthat interlink these pathways are unclear [46,47].

Physiological implications

The major natural source of daily IRA radiation exposure isthe sun. In addition, artificial IRA radiation devices areincreasingly used for therapeutical and cosmetic purposes.Human skin is exposed to significant amounts of solar IRAradiation with an average dose of 75 J/cm2 /h (summertime,Munich, Germany) and the IRA radiation doses used in thepresent study are of physiological relevance. We show that anexogenous factor, which is part of natural sunlight, can elicit aretrograde signaling response in human skin.

Conclusions

Biological consequences of IRA radiation exposure arethought to include the generation of skin cancer and, in

134 P. Schroeder et al. / Free Radical Biology & Medicine 43 (2007) 128–135

particular, premature (photo)aging of human skin. In thisregard, IRA radiation-induced MMP-1 expression is ofimportance because IRA-irradiated skin fibroblasts have anincreased capacity to proteolytically degrade dermal collagenfibers and collagen degradation is a key feature of photoagedhuman skin [17]. The present study indicates that retrogrademitochondrial signaling processes in human dermal fibroblastscontribute to actinic damage of human skin. The observationthat this exogenously triggered signaling cascade is mediated byROS derived from the mtETC suggests that administration ofproper antioxidants to human skin may protect against IRAradiation-induced skin damage.

Acknowledgments

This work was supported by Deutsche Forschungsge-meinschaft, SFB 728, TP B4, the Bundesministerium fuerUmwelt, Naturschutz und Reaktorsicherheit, Berlin Germanyand by the Research Committee of the Medical Faculty of theHeinrich-Heine-University, Duesseldorf, Germany.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.freeradbiomed.2007.04.002.

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