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• W1972906142 abstract "Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damageand activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 °C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominante over nuclear effects. UV-induced IL-6 release is mediated via NFκB since the NFκB inhibitor MG132 or transfection of cells with a super-repressor form of the NFκB inhibitor IκB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage. Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damageand activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 °C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominante over nuclear effects. UV-induced IL-6 release is mediated via NFκB since the NFκB inhibitor MG132 or transfection of cells with a super-repressor form of the NFκB inhibitor IκB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage. nuclear factor κB interleukin tumor necrosis factor TNF receptor TNF-R-associated factor-2 enzyme-linked immunosorbent assay fetal calf serum phosphate-buffered saline polymerase chain reaction Ultraviolet radiation (UV) and, in particular, UVB with a wave length range between 290 and 320 nm represents one, if not the most, important environmental factor of inducible health hazards for mankind, which include the induction of skin cancer (1.De Gruijl F.R. Sterenborg H.J. Forbes P.D. Davies R.E. Cole C. Kelfkens G. van Weelden H. Slaper H. van der Leun J.C. Cancer Res. 1993; 53: 53-60PubMed Google Scholar), suppression of the immune system (2.Beissert S. Schwarz T. J. Invest. Dermatol. Symp. Proc. 1999; 4: 61-64Abstract Full Text PDF PubMed Scopus (120) Google Scholar), and chronic skin damage including premature skin aging (3.Fisher G.J. Datta S.C. Talwar H.S. Wang Z.Q. Varani J. Kang S. Voorhees J.J. Nature. 1996; 379: 335-339Crossref PubMed Scopus (1209) Google Scholar). Similar to chemical agents, UV has the ability to alter mammalian gene expression (4.Herrlich P. Ponta H. Rahmsdorf H.J. Rev. Physiol. Biochem. Pharmacol. 1992; 119: 187-223Crossref PubMed Scopus (183) Google Scholar, 5.Herrlich P. Rahmsdorf H.J. Curr. Opin. Cell Biol. 1994; 6: 425-431Crossref PubMed Scopus (51) Google Scholar, 6.Schenk H. Klein M. Erdbrügger W. Dröge W. Schulze-Osthoff K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1672-1676Crossref PubMed Scopus (644) Google Scholar). Elucidation of the mechanisms by which UV affects gene expression is crucial for understanding how UV exerts its biological effects and how it develops its pathogenic properties. In this context, one of the most frequently but also controversially discussed issues is whether the cellular UV response is initiated at the cell membrane or in the nucleus (reviewed in Refs. 7.Schwarz T. J. Photochem. Photobiol. B. 1998; 44: 91-96Crossref PubMed Scopus (77) Google Scholarand 8.Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. Photochem. Photobiol. 1997; 37: 1-17Crossref Scopus (233) Google Scholar). The biological effects of UV are multiple and include the release of soluble mediators, the induction of apoptosis, and alterations of surface molecule expression, just to name a few. To exert these biological effects, UV must first be absorbed by a chromophore within the cell, which then transduces energy into a biochemical signal. A number of chromophores have been identified, e.g.porphyrins, aromatic amino acids, urocanic acid, and DNA. Among these, DNA has been regarded as the most important one, since the wavelength dependences of various UV effects match that of DNA absorption (9.Petit-Frére C. Clingen P.H. Grewe M. Krutmann J. Roza L. Arlett C.F. Green M.H.L. J. Invest. Dermatol. 1998; 111: 354-359Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In addition, removal of UV-induced DNA damage, e.g. by enhancing DNA repair, reduces or even inhibits some of the biological UV effects (10.Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7516-7520Crossref PubMed Scopus (468) Google Scholar, 11.Wolf P. Cox P. Yarosh D.B. Kripke M.L. J. Invest. Dermatol. 1995; 104: 287-292Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 12.Kibitel J. Hejmadi V. Alas L. O'Connor A. Sutherland B.M. Yarosh D. Photochem. Photobiol. 1998; 67: 541-546Crossref PubMed Scopus (68) Google Scholar, 13.Nishigori C. Yarosh D.B. Ullrich S.E. Vink A.A. Bucana C.B. Roza L Kripke M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10354-10359Crossref PubMed Scopus (222) Google Scholar). Finally, lower UV doses induce some of the biological effects at the same magnitude in DNA repair-deficient cells as in cells with normal DNA repair (14.Krutmann J. Bohnert E. Jung E.G. J. Invest. Dermatol. 1994; 102: 428-432Abstract Full Text PDF PubMed Google Scholar). Considering these observations, it is understandable that for quite a long time DNA was regarded as the only molecular target for UV, and why the dogma existed that any biological effect must be a direct consequence of DNA damage. On the other hand, a variety of groups have provided convincing evidence that UV may exert biological effects without the need of a nuclear signal. Utilizing enucleated cells, Devary et al.(15.Devary Y. Rosette C. DiDonato J.A. Karin M. Science. 1993; 261: 1442-1445Crossref PubMed Scopus (578) Google Scholar) demonstrated that activation of the transcription factor nuclear factor κB (NFκB)1 does not require a nuclear signal. This was confirmed by the observation that UV exposure of cytosolic extracts containing NFκB in its inactive form supplemented with cellular membranes causes activation of NFκB (16.Simon M.M. Aragane Y. Schwarz A. Luger T.A. Schwarz T. J. Invest. Dermatol. 1994; 120: 422-427Crossref Scopus (152) Google Scholar). In addition, growth factor receptors appear to be involved in the UV response since UV activates the epidermal growth factor receptor by directly initiating tyrosine phosphorylation (17.Sachsenmaier C. Radler-Pohl A. Zinck R. Nordheim A. Herrlich P. Rahmsdorf H.J. Cell. 1994; 78: 963-972Abstract Full Text PDF PubMed Scopus (407) Google Scholar). Rosette and Karin (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar) reported for the first time that UV and osmotic shock, respectively, can activate cell surface receptors by inducing their oligomerization. Triggering of receptors in such a way by UV takes place without the binding of any ligand and independently of DNA damage (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar, 19.Sheikh M.S. Antinore M.J. Huang Y. Fornace Jr., A.J. Oncogene. 1998; 17: 2555-2563Crossref PubMed Scopus (114) Google Scholar). One of the most important biological effects of UV is the induction of apoptotic cell death (20.Young A.R. Photodermatology. 1987; 4: 127-134PubMed Google Scholar). Convincing data exist that nuclear events especially UV-induced DNA damage determine whether a cell undergoes apoptosis or not (21.Ziegler A. Jonason J.S. Leffel D.W. Simon J.A. Sharma A.W. Kimmelman J. Remington L. Jacks T. Brash D.E. Nature. 1994; 372: 773-776Crossref PubMed Scopus (1352) Google Scholar). On the other hand, it was clearly demonstrated that activation of death receptors such as CD95 (Fas/APO-1) on the cell surface by UV induces the apoptotic machinery without being connected with DNA damage (22.Aragane Y. Kulms D. Metze D. Kothny G. Pöppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar, 23.Rehemtulla A. Hamilton C.A. Chinnaiyan A.M. Dixit V.M. J. Biol. Chem. 1997; 272: 25783-25786Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). In addition, Sheikh et al. (19.Sheikh M.S. Antinore M.J. Huang Y. Fornace Jr., A.J. Oncogene. 1998; 17: 2555-2563Crossref PubMed Scopus (114) Google Scholar) proposed that UV-stimulated ligand independent activation of the tumor necrosis factor receptor plays a major role in mediating the apoptotic effects of UV. Considering these data, which on first glance appear conflicting, we recently tried to determine the relative contribution of nuclear and membrane effects in UV-induced apoptosis (24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar). Removal of UV-induced DNA damage by enhancing DNA repair using the repair enzyme photolyase significantly reduced the apoptosis rate in HeLa cells. On the other hand, exposure of HeLa cells to UV at 4 °C, which prevents death receptor clustering (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar, 22.Aragane Y. Kulms D. Metze D. Kothny G. Pöppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar), also reduced the apoptosis rate, although to a lesser extent. It is important to mention that neither of these strategies alone was able to completely prevent UV-mediated apoptosis. However, when both strategies were combined,i.e. when cells were exposed to UV at 4 °C and DNA damage removed by photolyase, UV-induced apoptosis was completely inhibited (24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar). Hence, these data indicated that, although nuclear effects are predominant in comparison to membrane events, both are necessary to obtain the complete apoptotic response. Inspired by these observations, we were interested to determine whether the participation of both nuclear and membrane components is specific for UV-induced apoptosis or is also essential for other UV responses. Here, we studied the induction of the release of the inflammatory cytokine interleukin 6 (IL-6) in HeLa cells by UV. Using this system, we demonstrate that removal of DNA damage by enhancing DNA repair does not cause reduction of IL-6 release, implying that UV-induced DNA damage is not an important intermediate in this type of UV response. On the other hand, prevention of triggering cell surface receptors by maintaining HeLa cells at 4 °C during UV exposure resulted in complete inhibition of UV-mediated IL-6 secretion. Using dominant negative mutants, we provide evidence that NFκB is involved in this signaling process and that the tumor necrosis factor type 1 receptor (TNF-R1) seems to be a major target at the cell membrane in this UV response. Together, these data indicate that UV can also exert its biological effects by exclusively acting on the cell membrane without the necessity of a nuclear signal. In addition, our findings suggest that the multiple biological effects of UV on mammalian cells do not only differ in their final outcome but are also dependent on how they are generated. The human epithelial carcinoma cell line HeLa (American Tissue Culture Collection) was cultured in RPMI 1640 with 10% FCS. Human recombinant tumor necrosis factor α (TNFα) was obtained from Endogen (Woburn, CA). IL-6 release from cells was measured by subjecting supernatants (10 μl each) to an IL-6 ELISA kit (Diaclone, Besancon, France). Measurements were performed according to the manufacturer's guidelines. The proteasome inhibitor MG132 was purchased from Calbiochem (San Diego, CA). The plasmid allowing overexpression of a mutated IκB variant was kindly provided by K. Schulze-Osthoff (University of Münster, Münster, Germany) (25.Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1995; 14 (2838): 2876Crossref PubMed Scopus (934) Google Scholar), the plasmid overexpressing a dominant negative mutant of TRAF-2 was kindly provided by David Goeddel (Tularik Inc., San Francisco, CA) (26.Hsu H. Shu H.B. Pan M. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar). UV irradiation was performed as described previously with slight modifications (24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar). Briefly, subconfluent cells were washed with PBS and exposed to UV light through colorless medium without FCS. For UV irradiation, we used a bank of six TL12 fluorescent bulbs (Philips, Eindhoven, The Netherlands), which emit most of their energy within the UVB range (290–320 nm) with an emission peak at 313 nm. Throughout this study, a dose of 400 J/m2 was used. Control cells were subjected to the identical procedure without being exposed to UV. UV irradiation at low temperature was carried out by keeping cells at 4 °C for 10 min before UV exposure and during exposure, which lasted 40 s. Cells were kept at 4 °C for another 20 min before incubation at 37 °C for 16 or 24 h. Osmotic shock was induced by incubating cells with 1 msorbitol (Sigma, Munich) in FCS-free medium for 30 min either at 37 °C or at 4 °C (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar). Thereafter cells were washed with PBS, supplemented with normal RPMI medium, and incubated for 16 h or 24 h at 37 °C. Stimulation of cells with TNFα at low temperature was carried out by adding TNFα (100 ng/ml) to cells that had been kept at 4 °C for 10 min. Cells were kept at 4 °C for another 20 min and then cultured at 37 °C for 16 or 24 h. Photolyase was encapsulated into liposomes (Photosomes®, AGI Dermatics, Freeport, NY) at a concentration of 1.2 mg/ml (27.Yarosh D. Klein J. Trends Photochem. Photobiol. 1994; 3: 175-181Google Scholar). Liposomes consisted of the lipids egg phosphatidylcholine, egg phosphatidyl trans-ethanolamine, oleic acid, and the membrane stabilizer cholesterol hemisuccinate. Empty liposomes were used as negative controls, referred to as liposomes. For photoreactivation, HeLa cells were irradiated as described above and either Photosomes® or liposomes (40 μl/ml each) were added. Cells were incubated at 37 °C for 1 h in the dark, followed by illumination with photoreactivating light. As a light source for photoreactivating light, UVA fluorescent bulbs (TL09, Philips) filtered through a 6-mm glass plate with peak emission at 365 nm were used. Cells were exposed for 20 min, which corresponds to a photoreactivating light fluence of 12 kJ/m2. After photoreactivation, cells were supplemented with normal RPMI medium containing 10% FCS and incubated for 24 h at 37 °C. 16 h after stimulation cells were detached from dishes, and apoptosis analyzed by a cell death detection ELISA (Cell Death Detection ELISAPLUS, Roche Molecular Biochemicals). The enrichment of mono- and oligonucleosomes released into the cytoplasm of cell lysates is detected by biotinylated anti-histone- and peroxidase-coupled anti-DNA antibodies and is calculated as follows: absorbance of sample cells/absorbance of control cells. Unless otherwise stated, this factor was used as a parameter of apoptosis and is given as the mean ± S.D. of three independently performed experiments. At 4 h after stimulation, total RNA was extracted from cells according to the protocol described by Chomczynski and Sacchi (28.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Total RNA (1 μg) was reverse transcribed with SuperScript RNase H reverse transcriptase (Life Technologies, Inc.). The amount of template needed was titrated by β-actin PCR in a 20-μl reaction utilizing the RedTaq polymerase system from Sigma and evaluated densitometrically. A hIL-6-amplimer set fromCLONTECH (Palo Alto, CA) was used as primers for IL-6 PCR. Western blot analysis was performed as recently described (29.Kothny-Wilkes G. Kulms D. Pöppelmann B. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1998; 273: 29247-29253Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly, cells were lysed in lysis buffer (50 mm Hepes pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 100 mm NaF, 10 mmpyrophosphate, 0.01% NaN3, and Complete™ protease inhibitor mixture) for 15 min on ice. After centrifugation, supernatants were collected, and the protein content measured by Bio-Rad protein assay kit. The protein samples were subjected to 12% SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and incubated with antibodies directed against IκB (Upstate Biotechnology, Inc., Lake Placid, NY). Equal loading was determined by reprobing membranes with an antibody directed against α-tubulin (Calbiochem, San Diego, CA). Signals were detected with an ECL™ kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). HeLa cells (6 × 106) were washed once with PBS and resuspended in 600 μl of FCS-free RPMI medium, 2% Me2SO. Cells were electroporated with 20 μg of each plasmid DNA (pCMV-IκB-DN or pRK-F-TRAF-2-DN) according to the method described by Melkonyan et al. (30.Melkonyan H. Sorg C. Klempt M. Nucleic Acids Res. 1996; 24: 4356-4357Crossref PubMed Scopus (98) Google Scholar). Transfection efficacy of cells cotransfected with a plasmid encoding β-galactosidase (pcDNA6-VS-His-lacZ; Invitrogen, San Diego, CA) was determined 36 h later by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (100 μg/ml) in 5 mm potassium ferricyanide, 5 mmpotassium ferrocyanide, and 1 mm MgCl2 in PBS. Transfection efficacies ranged from 30% to 50%. Both nuclear and membrane events have been shown previously to contribute independently to UV-induced apoptosis (24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar). Since both components are essential to obtain the complete apoptotic response, inhibition of aggregation of death receptors expressed on the cell surface, such as CD95, by keeping cells at 4 °C during UV exposure only partially inhibits UV-induced apoptosis (Fig.1). Osmotic shock was recently demonstrated to induce receptor aggregation similar to UV (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar). However, in contrast to UV, osmotic shock does not induce DNA damage and can thus be used as a stimulus that acts on the cell membrane exclusively. Rosette and Karin (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar) predicted that any receptor whose activation mechanism involves multimerization should be activable by UV or osmotic shock. Hence, osmotic shock should also activate the CD95 receptor and thus induce apoptosis. As predicted, exposure of HeLa cells to osmotic shock caused apoptotic cell death (Fig. 1). When HeLa cells were kept at 4 °C during osmotic shock, which prevents receptor clustering (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar), apoptosis was completely inhibited. The complete prevention of apoptosis by inhibiting receptor aggregation thus confirms that osmotic shock, in contrast to UV, acts exclusively at the cell membrane when inducing apoptosis. TNFα is known to induce apoptosis via activation of the TNF receptor 1 (TNF-R1) (31.Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). To determine the effect of low temperature on TNFα-induced apoptosis, HeLa cells were exposed to TNFα, maintained at 4 °C during the first 30 min of exposure, and then kept at 37 °C. 16 h later apoptosis was measured. Under these conditions, low temperature had no effect on TNFα-induced apoptosis, since the death rate was the same irrespective of whether the cells were kept at 4 °C for the first 30 min or at 37 °C throughout the entire incubation period (Fig. 1). To further determine whether the involvement of both nuclear and membrane events is unique for UV-mediated apoptosis or also relevant for other biological effects caused by UV, we studied UV-mediated release of IL-6 by HeLa cells. HeLa cells do not constitutively secrete IL-6. However, UV irradiation resulted in a significant secretion of IL-6 (Fig.2). Exposure of HeLa cells to TNFα, a well known inducer of IL-6, also caused enhanced IL-6 levels in the supernatants. Exposing HeLa cells to osmotic shock also induced IL-6 production, which may best be explained by activation of TNF-R1 (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar). Accordingly, osmosis-induced IL-6 release was drastically reduced, when cells were kept at 4 °C during exposure to osmotic shock (Fig. 2). Likewise, when cells were stimulated with TNFα at 4 °C for 30 min, TNFα removed after that period by medium change and cells cultured for another 24 h at 37 °C, no induction of IL-6 was observed (data not shown). In contrast, keeping TNFα-stimulated cells at 4 °C for 30 min did not have any inhibitory impact on TNFα-induced IL-6 release provided that TNFα was not washed off but left in the medium for the rest of the incubation period of 24 h at 37 °C (Fig. 2). This confirms that keeping cells at low temperature for such a limited period by itself does not cause reduced IL-6 production by inhibition of molecular processes within the cell, e.g.transcription, but just interferes with membrane receptor activation (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar, 22.Aragane Y. Kulms D. Metze D. Kothny G. Pöppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar). Surprisingly, when HeLa cells were kept at 4 °C during UV exposure, UV-induced IL-6 release was strongly reduced close to base-line levels. Since inhibition of UV-induced IL-6 release was almost as pronounced as the inhibition of IL-6 release induced by osmotic shock, a purely membrane-located event, this implies that UV-induced IL-6 release appears to be primarily mediated via membrane and not nuclear events. To confirm the above presented observations, we tested the effect of accelerated removal of UV-mediated DNA damage by enhancing DNA repair on UV-mediated IL-6 release. This approach was used previously to demonstrate the importance of DNA damage in mediating UV effects (10.Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7516-7520Crossref PubMed Scopus (468) Google Scholar, 11.Wolf P. Cox P. Yarosh D.B. Kripke M.L. J. Invest. Dermatol. 1995; 104: 287-292Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 12.Kibitel J. Hejmadi V. Alas L. O'Connor A. Sutherland B.M. Yarosh D. Photochem. Photobiol. 1998; 67: 541-546Crossref PubMed Scopus (68) Google Scholar, 13.Nishigori C. Yarosh D.B. Ullrich S.E. Vink A.A. Bucana C.B. Roza L Kripke M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10354-10359Crossref PubMed Scopus (222) Google Scholar, 24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar). To induce repair of UV-mediated DNA damage, we utilized the photoreactivating enzyme photolyase. Photolyase binds to UV-induced cyclobutane pyrimidine dimers in DNA and catalyzes its splitting by electron transfer from absorbing wavelengths above 320 nm (photoreactivating light) (32.Eker A.P. Kooiman P. Hessels J.K.C. Yasui A. J. Biol. Chem. 1990; 265: 8009-8015Abstract Full Text PDF PubMed Google Scholar). To enable uptake of the enzyme into the cells, photolyase was encapsulated into liposomes (Photosomes®) (27.Yarosh D. Klein J. Trends Photochem. Photobiol. 1994; 3: 175-181Google Scholar). HeLa cells were irradiated with 400 J/m2 UV. Immediately thereafter, Photosomes® or empty liposomes were added and cells kept in the dark for 1 h, followed by exposure to photoreactivating light. As demonstrated previously (24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar), the combination of Photosomes® and photoreactivating light significantly reduces UV-induced DNA damage in HeLa cells. However, enhancement of DNA repair by Photosomes® had no effect on UV-induced IL-6 release, implying that in this case DNA damage is not an important mediator (Fig. 3). Likewise, addition of empty liposomes did not affect UV-stimulated IL-6 secretion. As already demonstrated in Fig. 2, the most effective way to inhibit UV-induced IL-6 release was keeping cells at 4 °C during UV exposure. The combination of inhibiting aggregation of cell surface receptors by keeping cells at low temperature and enhancement of DNA repair by adding Photosomes® did not result in further inhibition of UV-mediated IL-6 release (Fig. 3), although UV-induced pyrimidine dimers were reduced by 50% to 70%, as demonstrated by Southwestern dot blot analysis using an antibody directed against pyrimidine dimers (24.Kulms D. Pöppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7974-7979Crossref PubMed Scopus (179) Google Scholar). Taken together, these data indicate that DNA damage might not be of importance for mediating IL-6 release following UV exposure, further suggesting that membrane events may predominate over nuclear events concerning UV-induced IL-6 release in HeLa cells. Next, we addressed whether blocking UV-mediated receptor oligomerization by keeping cells at low temperature inhibits induction of IL-6 mRNA transcription. Therefore, semiquantitative RT-PCR utilizing primers amplifying parts of the IL-6 gene was performed. HeLa cells were UV-irradiated at 37 °C or at 4 °C, as described before, or alternatively exposed to osmotic shock at either 37 °C or 4 °C. In addition, cells were stimulated with TNFα at 37 °C or 4 °C. After incubating cells at 37 °C for another 4 h, RNA was extracted and semiquantitative PCR performed on reverse transcribed templates. Inhibition of UV- or osmosis-induced receptor clustering by low temperature significantly reduced IL-6 mRNA expression (Fig.4). In contrast, TNFα induced equal levels of IL-6 transcripts, irrespective of whether cells were kept at 37 °C or 4 °C during the initial period of stimulation. These data indicate that membrane effects, most likely receptor aggregation, induced either by UV or osmotic shock cause induction of IL-6 mRNA transcription. In addition to the experiments described above, these results exclude the unlikely possibility that low temperature inhibits UV-induced IL-6 release by interfering with protein translation or secretion. TNFα stimulates HeLa cells to produce enhanced amounts of IL-6 via activation of TNF-R1. TNF-R1 belongs to the group of receptors that are only biologically active when trimerized. Since UV directly induces receptor oligomerization independently of the respective ligands (18.Rosette C. Karin M. Science. 1996; 274: 1194-1197Crossref PubMed Scopus (945) Google Scholar, 19.Sheikh M.S. Antinore M.J. Huang Y. Fornace Jr., A.J. Oncogene. 1998; 17: 2555-2563Crossref PubMed Scopus (114) Google Scholar, 22.Aragane Y. Kulms D. Metze D. Kothny G. Pöppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar, 23.Rehemtulla A. Hamilton C.A. Chinnaiyan A.M. Dixit V.M. J. Biol. Chem. 1997; 272: 25783-25786Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), UV-induced IL-6 release may be due to direct activation of TNF-R1 by UV light. One consequence of triggering TNF-R1 is activation of the transcription factor NFκB. In addition, the IL-6 promoter contains several NFκB binding sites (34.Zhang Y. Broser M. Rom W.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2225-2229Crossref PubMed Scopus (165) Google Scholar). Hence, we postulated that, if activation of TNF-R1 is the initial signaling step in UV- or osmosis-induced IL-6 release, NFκB should be involved in the signaling cascade. Activation of NFκB is associated with degradation of the inhibitory protein IκB by the proteasome pathway. Upon a" @default.
• W1972906142 created "2016-06-24" @default.
• W1972906142 creator A5019087013 @default.
• W1972906142 creator A5053596948 @default.
• W1972906142 creator A5073671212 @default.
• W1972906142 date "2000-05-01" @default.
• W1972906142 modified "2023-10-15" @default.
• W1972906142 title "Ultraviolet Radiation-induced Interleukin 6 Release in HeLa Cells Is Mediated via Membrane Events in a DNA Damage-independent Way" @default.
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