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A Role for NF-Κb–Dependent Gene Transactivation in Sunburn

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A Role for NF-Κb–Dependent Gene Transactivation in Sunburn A role for NF-κB–dependent gene transactivation in sunburn Kazuhiro Abeyama, … , Paul R. Bergstresser, Akira Takashima J Clin Invest. 2000;105(12):1751-1759. https://doi.org/10.1172/JCI9745. Article Exposure of skin to ultraviolet (UV) radiation is known to induce NF-κB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-κB–dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-κB cis element (NF-κB decoy ODNs). UV-induced secretion of IL-1, IL- 6, TNF-α, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-κB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-κB target genes, rather than from nonspecific changes associated with tissue damage. Find the latest version: https://jci.me/9745/pdf A role for NF-κB–dependent gene transactivation in sunburn Kazuhiro Abeyama,1 William Eng,1 James V. Jester,2 Arie A. Vink,3 Dale Edelbaum,1 Clay J. Cockerell,1 Paul R. Bergstresser,1 and Akira Takashima1 1Department of Dermatology, and 2Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA 3TNO Nutrition and Food Research Institute, Zeist, The Netherlands Address correspondence to: Akira Takashima, Department of Dermatology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-0969, USA. Phone: (214) 648-3419; Fax: (214) 648-3472; E-mail: [email protected]. Received for publication March 1, 2000, and accepted in revised form May 8, 2000. Exposure of skin to ultraviolet (UV) radiation is known to induce NF-κB activation, but the func- tional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be pre- vented by blocking UV-induced, NF-κB–dependent gene transactivation with oligodeoxynu- cleotides (ODNs) containing the NF-κB cis element (NF-κB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-α, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-κB decoy ODNs also inhib- ited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proin- flammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Impor- tantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irri- tant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-κB target genes, rather than from nonspecific changes associated with tissue damage. J. Clin. Invest. 105:1751–1759 (2000). Introduction responses, including genes that encode cytokines, Ultraviolet (UV) radiation causes sunburn reactions, adhesion molecules, and regulators of cell growth and immunosuppression, accelerated skin aging, and skin death (24, 25). Thus, we sought to determine the cancer, and it is considered to be an important envi- causative relationship between UV-dependent NF-κB ronmental hazard for humans (1). At cellular levels, UV activation and the development of sunburn reactions. radiation has been shown to trigger cytokine produc- For this aim, we used the recently developed technol- tion (2), regulate surface expression of adhesion mole- ogy of decoy ODNs. The original concept of using syn- cules (3), affect cellular mitosis (4), and induce apop- thetic double-stranded ODN as “decoy” cis elements to totic cell death (5, 6). With respect to molecular block the binding of nuclear factors to promoter mechanisms, UV radiation is known to alter cellular regions of target genes was introduced in 1990 by Sul- function via DNA damage (7–10), generation of reac- lenger et al. and Bielinska et al. (26, 27). In 1997, Mor- tive oxygen intermediates (ROIs) (11, 12), ligand-inde- ishita et al. reported the first in vivo application of this pendent signaling through cell surface receptors, such technology; they prevented myocardial infarction after as EGF, TNF, and IL-1 receptors (13–15), phosphory- reperfusion in rats by direct infusion of synthetic dou- lation of receptor-associated tyrosin kinases and pro- ble-stranded 20-bp ODNs containing the NF-κB cis ele- tein kinase C (15–18), and activation of selected tran- ment into cannulated coronary arteries (28). Subse- scription factors, including NF-κB, AP-1, and AP-2 (13, quently, Ono et al. prevented cerebral angiopathy after 19–21). These mechanisms are by no means mutually subarachnoid hemorrhage in rabbits by delivering NF- exclusive; rather, they are likely to operate in an inter- κB decoy ODNs into the subarachnoid space (29), and dependent manner. For example, NF-κB activation can Kawamura et al. showed that direct injection of NF-κB be induced experimentally by damaging DNA chemi- decoy ODNs into implanted tumors in mice inhibited cally (6), exposure to hydrogen peroxide (22), cluster- cachexia, without affecting the tumor growth (30). ing of surface receptors (13), or phosphorylation of Here we report the first application to our knowledge tyrosin kinases or protein kinase C (17, 23, 24). NF-κB of the decoy ODN technology to the skin (as a target activation, in turn, leads to the expression of many organ) and to prevent inflammatory responses to an genes involved in immunological and inflammatory environmental hazard (as a target disease). The Journal of Clinical Investigation | June 2000 | Volume 105 | Number 12 1751 Methods macokinetics, XS106 cells were incubated with 10 µM Animals and cell lines. Female BALB/c mice (4–6 weeks FITC-conjugated NF-κB decoy ODN or FITC-conju- old) were housed in the pathogen-free facility of the gated scrambled ODN at either 4°C or 37°C and then Animal Resource Center at the University of Texas analyzed for the uptake of FITC-conjugated ODN Southwestern Medical Center, and all the animal probes by FACS or under a Leica CLSM laser scanning experiments were approved by the Institutional Review confocal microscope (Leica, Deerfield, Illinois, USA) as Board and conducted according to guidelines of the described previously (37). National Institutes of Health (NIH). The Pam 212 ker- Assays to assess the impact of NF-κB decoy ODNs. Before atinocyte line (established from BALB/c mouse skin) and after UV irradiation, ear skin thickness was meas- (31) and the NS47 fibroblast line (from BALB/c mouse ured by a third experimenter “blinded” to sample iden- skin) (32) were maintained and expanded in complete tity, using a caliper-type engineer’s micrometer (34). RPMI 1640 containing 10% FCS (32). The XS106 Some ear skin samples were fixed in formalin for his- Langerhans cell line (from A/J mouse skin) (33) was tological evaluation. Thickness of ear skin sections, cultured in complete RPMI 1640 in the presence of 1 number of keratinocyte layers in the epidermis, num- ng/mL mouse recombinant GM-CSF and 10% ber of infiltrating leukocytes, and frequency of sun- (vol/vol) NS47 fibroblast culture supernatant as burn cells were measured by light microscopy after described previously (34). hematoxylin and eosin (H&E) staining. To determine UV radiation. The dorsal surfaces of unrestrained the impact of NF-κB decoy ODN on UV-induced DNA BALB/c mice were exposed from above to a bank of damage, ear skin samples were harvested 5 minutes four unfiltered FS20 sunlamps (Westinghouse, Pitts- after UV radiation, and ear skin sections were exam- burgh, Pennsylvania, USA) that emit a spectrum with ined for cyclobutane pyrimidine dimers (CPDs) by high irradiance in the UVB region and a peak at 313 using mAb H3 (38) and FITC-conjugated goat anti- nm (35). Cell lines cultured in 100-mm tissue culture mouse IgG (Jackson ImmunoResearch Laboratories plates were washed twice with PBS and then exposed to Inc., West Grove, Pennsylvania, USA) as described pre- either FS20 sunlamps or a solar simulator with 1 kW viously (39). Surface densities of Langerhans cells in Xenon arc lamp (Model 81190; Oriel Instruments, ear skin were examined 24 hours after UV radiation as Stratford, Connecticut, USA). An IL 700 research described elsewhere (40). Briefly, the epidermis was radiometer equipped with a SEE 240 photodetector separated as an intact sheet from the remaining der- (International Light Inc., Newburyport, Massachusetts, mis after a 20-minute incubation in 0.5 M ammonium USA) was used to measure the irradiance in the UVB thiocyanate at 37°C, fixed in acetone, and then stained region (sunburn spectrum) for both light sources. with FITC-conjugated anti-IA mAb 2G9 (PharMingen, NF-κB decoy ODN treatment. Phosphorothioated, San Diego, California, USA). Numbers of IA-positive double-stranded 20-bp NF-κB decoy ODNs epidermal cells (i.e., Langerhans cells) were counted (5′-CCTTGAAGGGATTTCCCTCC-3′) and scrambled under a fluorescence microscope. Some ear specimens ODNs (5′-TTGCCGTACCTGACTTAGCC-3′) were synthe- were extracted in 10 mM HEPES/10 mM KCl buffer sized according to Morishita et al. (28). NF-κB decoy (pH 7.9) with 0.08% NP-40, 0.1 mM EDTA, 1 mM ODNs or scrambled ODNs (0.5 mM solution in PBS) EGTA, 0.5 mM DTT, and 0.5 mM PMSF, and the were injected intraperitoneally (200 µL/injection per resulting soluble fractions were then examined for mouse) 24 hours and 1 hour before irradiation.
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