Bnitish Journal of Ophthalmology 1995; 79: 277-281

Expression of c-Fos and c-Jun in the cornea, lens,and retina after ultraviolet irradiation of the rateye and effects of topical antisenseoligodeoxynucleotides

Frank Gillardon, Manfred Zimmermann, Eugen Uhlmann

AbstractAims-Immunohistochemical techniqueswere used to investigate c-Fos and c-Junproto-oncogene expression in the cornea,lens, and retina after ultraviolet irradia-tion of the rat eye.Methods-Eyes of anaesthetised rats wereexposed to 1 5 J/cm2 of ultraviolet radia-tion (280-380 nm). Animals were perfused1, 6, or 24 hours after irradiation andtissue sections were incubated withspecific antiserum to c-Fos and c-Jun,respectively.Results-Non-irradiated contralateraleyes displayed no c-Fos and c-Junimmunoreactivity. One and 6 hours afterultraviolet exposure numerous c-Fos andc-Jun immunopositive nuclei wereobserved mainly in the epithelial cell layersof the cornea and the lens epithelium.Scattered labelled nuclei were detectable inthe retinal ganglion cell layer and the innernuclear layer. Twenty four hours afterirradiation c-Fos and c-Jun proteinexpression returned to near control levels.Histological signs of ultraviolet damage(for example, chromatin condensation,nuclear fragmentation) were first recog-nisable in the corneal epithelium6 hours after irradiation and became moreapparent at later times.Conclusion-Thus, the rapid and sus-tained activation of c-Fos and c-Junexpression in the eye after single ultra-violet exposure may represent themolecular mechanism underlying ultra-violet induced photodamage and initia-tion of celi death. Furthermore, topicalapplication of a c-fos antisense oligode-oxynucleotide to the ultraviolet exposedrat eye inhibited the increase in c-Fosexpression in the cornea, suggestingtherapeutic activity of antisense drugs incorneal malignant and infectious diseases.(BrJ Ophthalmol 1995; 79: 277-281)

Ultraviolet (UV) radiation induced photo-damage of the eye has been demonstrated inboth humans and experimental animals.1 2Short wavelength UV C radiation (<280 nm)is cut off by the cornea. Excessive exposure toUV light results in inflaminatory damage of thecornea and even neoplastic alterations. Actionspectrum measurements for UV keratitis

revealed a maximum sensitivity at about 270nm, which corresponds to the absorption ofUV light by nucleic acids.3 In the cornealepithelium, UV radiation inhibits mitosis, pro-duces nuclear fragmentation, and desquama-tion.4 Neoplasms occur most frequently inthose portions of the eye exposed to UV radia-tion (conjunctiva, cornea, and eyelids).Although primary corneal tumours are exceed-ingly rare suggesting a relative resistance tomalignant transformation, an enhanced sus-ceptibility has been demonstrated in humanswho suffer from xeroderma pigmentosum.3 5

Longer wavelength UV radiation (UV-Brange: 280-320 nm) is strongly absorbed bythe lens. In cultured rabbit lens epithelial cells,cyototoxicity of UV radiation was mostefficient at 297 mm.6 Numerous epidemio-logical studies have shown that long termexposure to UV light correlates with anirreversible loss of human lens transparency,and cataract induction by UV-B radiation hasalso been demonstrated in albino mice.1 7 Invitro studies have identified amino acids (forexample, tryptophan, tyrosine) and DNA inlens epithelial cells as targets of UV-B radia-tion, and membrane alterations and DNAstrand breaks have been suggested as possiblemolecular mechanisms involved in acutecataract development.8 9UV-A wavelengths (320-440 nm) can

pass through all optic media and causephotochemical damage to the retina. At highintensities or in aphakic and pseudophakicpatients cellular damage occurs in thepigment epithelium, the photoreceptors, andoccasionally other retinal neurons.1 2 10

In mammalian skin, UV radiation promotessimilar cellular effects, ranging from growthinhibition and cell death to hyperproliferationand carcinogenesis. The underlying molecularmechanisms are as yet incompletely under-stood. Single UV exposure of mammalian cellsin culture leads to the induction of variousgenes, among them members of the Fos/Junfamily of transcription factors,'1-13 which areknown to be associated with cell growth anddifferentiation.14 Recently, the activation ofc-Fos protein expression has been demon-strated to precede both naturally occurring andUV induced programmed cell death in mam-malian skin.15a 15b However, little is knownabout fos/jun proto-oncogene expression in themammalian eye following UV exposure.Therefore, we investigated whether single UV

II PhysiologischesInstitut, UniversitAftHeidelberg, GermanyF GillardonM Zimmermann

Hoechst AG, GeneralPharma Research,Frankfirt, GermanyE Uhlmann

Correspondence to:Dr Frank Gillardon, IIPhysiologisches Institut,Universitat Heidelberg, INF326, 69120 Heidelberg,Germany.Accepted for publication27 September 1994

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Fig IA

Figa JB

Figure 1 Cross section of the albino rat eye 6UVirradiation in vivo. (A) c-Jun immunopoare concentrated in the basal layer of the cormepithelium. Magnification X320. (B) At lowmagnification nuclear c-Jun immunostainingin the cornea and in the central zone of the len(arrowhead). The separation of the corneal enfrom the stroma represents a fixation artefact.Magnification X240.

irradiation modulates c-Fos and c-Jexpression in the cornea, lens, and r

rat. Furthermore, we tested the in viof topically administered antisense cnucleotides (ODNs) in blocking cor

oncogene expression.

Materials and methodsMale albino Sprague-Dawley rats (

were purchased from the ZentraliVersuchstierzucht (Hannover,Animals were kept under a 12 hoihour dark cycle and given food anlibitum.

All animals were irradiated 4 houonset of the light period. The U'Tsource consisted of a high pressure nlamp (Osram HBO 100W/2), twquartz lenses, and a UV filter (SpHoyer UG 1 1). The eyes were irradintensity of 14 mW/cm2 in the focusdensor system. 16 Irradiance was meia UV radiometer (Laser InstrurSixty five per cent of the energy vw

within the UV-B wavelength rangenm), the remainder within the Us(320-380 nm). Animals were an

(Nembutal, 60 mglkg, intraperitorthe right eye (with lids clampedexposed once to 1-5 J/cm2, which isthe amount to produce thresholdThe left eye was shielded from the r

aluminium foil and used as a contro

After different survival times (1, 6, and 24hours, respectively) reanaesthetised rats (n=3per time point) were perfused transcardiallywith 4% paraformaldehyde in phosphatebuffered saline solution. Eyes were excised andstored in fixative overnight. Tissue was equi-librated in 30% sucrose and sectioned on afreezing microtome (20 ,um). Sections werethaw mounted onto gelatine coated slides andincubated for 48 hours at room temperature inrabbit antiserum to c-Fos (689/5) (1:10 000)or c-Jun (607/3) (1:1 000). Antibody bindingwas localised by the avidin-biotin-peroxidasemethod (Vector, Burlingame, VA, USA) usingdiaminobenzidene as chromogen. For histo-logical examination sections were stainedwith haematoxylin and eosin using standardprotocols.

Generation of antisera and their specificitya* has been described in detail elsewhere.17 18 In

short, all antibodies were raised againstbacteria expressed fusion proteins. As deter-

s mined by immunoprecipitation analysis of ini vitro translated proteins, the rabbit anti-c-Fos\ antibody showed a strong reaction with the

c-Fos protein, no cross reactivity with Fos Band a weak reaction with the Fos relatedantigen, Fra-1. The anti-c-Jun antiserum

S hours after. showed a very strong reaction with the c-Junsitive nuclezsitivea nucle

protein and no cross reactivity with both theer Jun B and Jun D proteins. Immunoreactivityis visible both was absent in sections after preabsorption of~s epitheliumedothelium the primary antisera with the respective

antigene, whereas incubation with relatedproteins did not affect immunoreactivity.

Oligodeoxynucleotides with 3',5'-terminal[un protein phosphorothioate internucleoside backbonesetina ofthe were synthesised and purified as describedivo efficacy elsewhere.15a A 20-mer c-fos antisense ODN)ligodeoxy- (5'-C*G*AGAACATCATGGTCGT*G*G-3')neal proto- was topically applied to the cornea (1 nmol in

10 ,ul sterile phosphate buffered saline) imme-diately after UV exposure. Control eyes wereexposed and treated with a random sequenceODN (5'-C*C*CTTA -TTTACTACTlTTC*G*

250-300 g) C-3') (asterisks indicate sites of phosphoro-institut fur thioate modification). ODN solution wasGermany). applied at 1 hour intervals, and 6 hour post-ur light-12 irradiation tissue was processed as describedd water ad above.

irs after theV radiation Resultsnercury arc One and 6 hours after UV exposure numerousto concave c-Fos and c-Jun positive nuclei were visible)indler and mainly in the basal cell layer of the cornealliated at an epithelium, whereas less immunoreactive cellsof the con- were observed in the corneal stroma andasured with endothelium (Fig 1). At the same timepointsnentation). the lens epithelial cell layer exhibited c-Fos andras emitted c-Jun positive nuclei in the central zone(280-320 (Fig 1B) and to a significant higher level in the

V-A region germinative zone (Fig 2A). Cornea, lens, andLaesthetised retina of non-irradiated control eyes displayedaeally) and no detectable nuclear c-Fos and c-Junopen) was immunostaining (Figs 2B and 3B). Scatteredjust below c-Fos and c-Jun immunostained nuclei werekeratitis.3 observed in the retinal ganglion cell layer and

adiation by in the inner nuclear layer after irradiation;1. however, no staining of photoreceptor nuclei

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Expression ofc-Fos and c-Jun in the cornea, lens, and retina after ultraviolet irradiation of the rat eye

Fig 2A

Fig 2B

Figure 2 (A) c-Jun immunoreactive nuclei in thegerminative zone of the lens epithelial cell layer 6 hoursafter UV exposure. Magnification X320. (B) Noimmunostaining is seen in the cornea and in the lensepithelium (arrowhead) of the non-irradiated contralateraleye. Magnification X240.

could be detected (Fig 3A). Similar temporaland spatial expression patterns were obtainedfor both proto-oncogenes. Twenty four hoursafter UV irradiation c-Fos and c-Jun immuno-reactivity returned to control levels.The earliest histological alterations (chro-

matin condensation, pyknotic nuclei) wereobserved in the corneal epithelium 6 hoursafter exposure (Fig 4). Twenty four hours afterirradiation nuclear fragmentation, vacuolisa-tion and desquamation became visible in allepithelial cell layers of the cornea. Pyknoticnuclei were less frequent in the UV irradiatedlens epithelium and not detectable in thecorneal stroma and endothelium and the retina(not shown).

Topical administration of a terminal phos-phorothioated c-Fos antisense ODN inhibitedthe UV induced increase in c-Fos immuno-reactive cells in the cornea but not in theunderlying lens epithelium, whereas a randomsequence control ODN did not show signifi-cant inhibitory activity (Fig 5).

DiscussionIn the present study, a rapid and sustainedactivation of both c-Fos and c-Jun expressionhas been demonstrated in different cell popula-tions of the rat eye following single UVexposure in vivo. The time course of inductionof Fos/Jun transcription factors in the UVirradiated cornea, lens, and retina is verysimilar to that observed both in the rat epider-mis and in epidermal cells in culture,'1 12 15aand seems to be a general response of

Fig 3B

Figure 3 Retinal cross sections (counterstained with eosin)6 hours after UV exposure in vivo. (A) c-Fosimmunostaining is visible in a ganglion cell of the retinalganglion cell layer (arrow) and in a presumptive amacrinecell of the inner nuclear layer (arrowhead).(B) Non-irradiated contralateral retina displays noimmunoreactivity. Magnification X600.

mammalian cells when exposed to environ-mental stress.13 The molecular mechanismseliciting the activation of c-Fos and c-Jun inthe UV exposed eye remain unknown. Inkeratinocytes in culture, a role for DNAdamage and oxidative stress in mediating theUV response has been described,13 and DNAdamage and hydrogen peroxide formation havealready been detected in cultured lenses afterUV irradiation.8 9 Furthermore, platelet acti-vating factor, an inflammatory mediatorrapidly appearing in the cornea after alkaliinjury, induces c-Fos and c-Jun expression inepithelial cells of cultured rabbit corneaswithin 1 hour,'9 and mechanical wounding ofrabbit corneal epithelium in vivo activatesc-Fos expression within 15 minutes.20 In thesestudies, however, proto-oncogene expressionwas induced only transiently in non-injuredepithelial cells of the cornea, whereas in thepresent study c-Fos and c-Jun activation inputative photodamaged cells lasted at least6 hours after irradiation.

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Figure 4 Haematoxylin and eosin stained sections of non-irradiated control cornea (left), UV irradiated cornea 6 hoursafter exposure (middle), demonstrating chromatin condensation (arrow), and cornea 24 hours after irradiation (right),showing extensive nuclearfragmentation and vacuolisation. Magnification X5100.

Fig 5Aa

1.

Fig SB

Figure 5 Immunohistochemical analysis of c-Fos expression in UVirradiatafter topical administration ofoligodeoxynucleotides (ODNs). (A) Six hour!exposure c-Fos immunoreactivity is absent in the cornea treated with a c-fos o

ODN, whereas UV induced c-Fos expression in the underlying lens epitheliuaffected (arrowhead). (B) c-Fos positive nuclei are still present in the cornearandom sequence control ODN after irradiation. Magnification X1230.

The physiological significance of the UVinduced activation of proto-oncogene expres-

* sion in the eye remains speculative. The rapidinduction of fos/jun expression after singleUV exposure is not related to corneal cell pro-liferation, since mitotic activity in UV irradi-ated rat corneal epithelium is stronglyinhibited as early as 1 hour for up to 24 hoursafter high dose UV exposure.4 Furthermore,

*@ our histochemical analysis revealed a UVinduced increase in fos/jun immunoreactivitythroughout all corneal epithelial cell layers andno restriction to proliferating basal cells. Therat lens displayed positive nuclei both in theUV exposed non-mitotic central zone of the

j ff epithelium, and in the mitotic germinativezone, which is barely protected from UV radia-tion by the non-pigmented iris of the albinorat. Nevertheless, a higher degree of nuclearstaining was observed in mitotic areas, whichmay be explained by the higher susceptibilityof proliferating cells to UV induced celldeath.2' UV light is mostly absorbed by thecornea and the lens,1 2 and transmittance ofthe isolated rat lens decreases to <2% at310-330 nm.22a In cell culture, the action

I spectrum for UV induced c-Fos expressionpeaks at 275 nm and falls off steeply at longerwavelengths.22b Consistently, few nuclei wereimmunostained in the retinal ganglion celllayer and the inner nuclear layer, where c-Fosexpression is usually not detectable during thelight period.22c 23 Additionally, the retina didnot show significant histopathological altera-tions, suggesting the photochemical damagemay occur after quite a long latency or afterlonger exposure times.2 10UV radiation is a potent agent for the induc-

tion of programmed cell death/apoptosis inhuman skin, and different cell lines undergoapoptosis en masse within 2-3 hours afterUW-B irradiation.24 The induction of c-Fos

ted rat eyes and c-Jun expression in response to stimulisafter UV evoking apoptosis has been demonstrated inantisense various systems,25 and c-Fos expression has

mtreated with a recently been localised in apoptotic fibro-blasts.w5b There is evidence that apoptosis is

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the molecular mechanism of cell death in dif-ferent types of retinal degeneration,26 and fol-lowing UV exposure histological signs ofapoptosis (for example, chromatin condensa-tion, nuclear fragmentation) are visible in therat cornea, the lens, and retina of albino mice,and in the aphakic monkey retina.4 10 27Consistent with our histological data, studiesin rodents have revealed major cellular damagein the corneal epithelium following UV irradia-tion with a latent period of several hours andhave suggested that stromal and endothelialcells may be protected by a higher unscheduledDNA repair activity.4 28 Thus, the rapid induc-tion of c-Fos and c-Jun expression precedesmorphological signs of UV phototoxicity andmay reflect the initiation of programmed celldeath at least in some affected cells of the UVirradiated eye.

Finally, topical administration of a terminalphosphorothioated c-fos antisense ODN to theUV exposed cornea blocked the activation ofc-Fos expression. These findings demonstratethat ionic antisense ODNs when instilled aseyedrops are able to penetrate into the corneaand to sequence specifically modulate cornealgene expression. This may be of clinicalrelevance.The authors thank R Bravo and T Herdegen for providing theFostJun antibodies and M Zipp for skilful technical assistance.

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3 Schein OD. Phototoxicity and the cornea. J Nad Med Assoc1992; 84: 579-83.

4 Buschke W, Friedenwald JS, Moses SG. Effects of ultra-violet irradiation on corneal epithelium: mitosis, nuclearfragmentation, post-traumatic cell movements, loss oftissue cohesion. J CeU Comp Physiol 1945; 26: 147-64.

5 Kraemer KH, Lee MM, Scotto J. Zeroderma pigmentosum.Arch Dermatol 1987; 123: 241-50.

6 Andley UP, Lewis RM, Reddan JR, Kochevar IE. Actionspectrum for cytotoxicity in the UVA- and UVB-wave-length region in cultured lens epithelial cells. InvestOphthalmol Vis Sci 1994; 35: 367-73.

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