Epidermal COX-2 Induction Following Ultraviolet Irradiation: Suggested Mechanism for the Role of COX-2 Inhibition in Photoprotection  Catherine S. Tripp, Eric A.G. Blomme, Kevin S. Chinn, Medora M. Hardy, Peter LaCelle, Alice P. Pentland  Journal of Investigative Dermatology  Volume 121, Issue 4, Pages 853-861 (October 2003) DOI: 10.1046/j.1523-1747.2003.12495.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Emission spectrum of UVA 340 sunlamps compared to the solar spectrum. Journal of Investigative Dermatology 2003 121, 853-861DOI: (10.1046/j.1523-1747.2003.12495.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Characterization of UVA/B-induced epidermal injury in SKH-1 mice. (A) Photomicrographs of normal dorsal mouse skin (N) and of skin 24, 72, 96, or 168 h after UVA/B irradiation. Note the epidermal hyperplasia (black arrow), hyperkeratosis (white arrow), and the perivascular inflammation (arrowheads) present 72 h after UVA/B irradiation. At 168 h post UVA/B irradiation, the epidermis has returned to near normal. Hematoxylin and eosin stain. Scale bar: 50 μm. (B) UVA/B-induced edema expressed as increased skin thickness measured with a caliper. Data are expressed as mean±SEM; *p<0.05 versus normal. (C) Keratinocyte proliferation measured by immunohistochemical detection of BrdU. Data are expressed as mean±SEM; *p<0.05 versus normal; n=3–5. Filaggrin score was obtained by blinded observers as noted in Methods. Data are expressed as mean±SEM; **p<0.01 versus normal; n=5. (D) UV-induced apoptosis in the interfollicular epidermis was visualized using TUNEL staining with fluorescein-labeled dUTP. The cells undergoing apoptosis (white arrows) were visualized by fluorescent microscopy in the normal epidermis (N) and 24 or 72 h after UVA/B irradiation. Scale bar: 50 μm. Journal of Investigative Dermatology 2003 121, 853-861DOI: (10.1046/j.1523-1747.2003.12495.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 COX-2 protein expression is induced rapidly after UV irradiation of the epidermis. COX-1 or COX-2 expression was analyzed by western analysis following UVA/B irradiation. (A) SKH-1 mice were exposed to varying doses of UVA/B light as indicated (expressed as mJ per cm of UVB). Skin biopsies from mice were studied 24 h later by western analysis for COX protein as described in Methods. (B) Mice were exposed to UVA/B light (180 mJ per cm of UVB light), and at the indicated times skin biopsies were analyzed for COX-1 and COX-2 protein by western analysis (std, recombinant murine COX-1 or murine COX-2 protein). (C) Immunohistochemical detection of COX-2 in mouse epidermis following UVA/B irradiation (180 mJ per cm of UVB light). COX-2 could not be detected in the interfollicular epidermis of normal (N) skin but was markedly induced in basal keratinocytes at 24–72 h following UVA/B irradiation (arrows). Scale bar: 50 μm, hematoxylin counterstain. Journal of Investigative Dermatology 2003 121, 853-861DOI: (10.1046/j.1523-1747.2003.12495.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In vivo selectivity of COX inhibitors was defined in the carrageenan air pouch adapted to the SKH-1 mouse. An air pouch was formed and carrageenan was injected on day 6 to induce inflammation; saline was injected in control animals. One hour prior to the induction of inflammation with carrageenan, mice were treated orally with 2 mg per kg indomethacin or 50 mg per kg dexamethasone. (A) Five hours after inflammation induction, the air pouch was analyzed for COX-2 and COX-1 protein expression from pooled samples (n=6 mice) by immunoprecipitation followed by western blot analysis. The saline-injected air pouch is compared with the carrageenan-injected air pouch in the absence and presence of drugs indomethacin (Indo) and dexamethasone (Dex). (B) The effect of varying doses of SC-791 on PGE2 production from the carrageenan-induced murine air pouch and TxA2 production from clotted whole blood. Levels of TxA2, measured as TxB2, and PGE2 were determined by enzyme immunoassay. Veh, vehicle. PGE2 and TxA2 were also measured from the saline-injected air pouch (sal) in vehicle-treated mice and from the carrageenan-injected air pouch (carra) in mice treated with indomethacin or dexamethasone. Journal of Investigative Dermatology 2003 121, 853-861DOI: (10.1046/j.1523-1747.2003.12495.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 COX-2 inhibition reduces UV-induced keratinocyte proliferation and decreases filaggrin expression stimulated by UVA/B irradiation. Mice were dosed with vehicle (Veh), SC-791, or indomethacin followed by UVA/B irradiation (UVB, 180 mJ per cm). (A) Keratinocyte proliferation measured by BrdU incorporation into DNA. Data are expressed as the mean±SEM; *p<0.05 versus vehicle at each time point. (B) Histology demonstrates UVA/B-induced hyperplasia and filaggrin expression at 96 h in the absence and presence of the drugs indomethacin (Indo) and SC-791 (SC). Filaggrin score was determined for mice treated with vehicle, SC-791, or indomethacin followed by UV irradiation. Data from vehicle-treated animals shown in Figure 2a) is repeated for convenience. The values represent the mean of three to five mice±SEM; *p>0.05 versus vehicle-treated mice at the same time. Journal of Investigative Dermatology 2003 121, 853-861DOI: (10.1046/j.1523-1747.2003.12495.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 COX-2 inhibition results in increased keratinocyte apoptosis following UV irradiation of mouse skin. SKH-1 mice were dosed with vehicle, SC-791, or indomethacin, and then irradiated with UVA/B (180 mJ per cm of UVB) light as described in Methods. UV-induced apoptosis in the interfollicular epidermis was visualized using the TUNEL method and fluorescein-labeled dUTP. Apoptosis was quantified at various times after irradiation by counting cells in (A) the basal layer or (B) the granulosum layer of the interfollicular epidermis. Data represent the mean of three to five mice±SEM; *p>0.01. Journal of Investigative Dermatology 2003 121, 853-861DOI: (10.1046/j.1523-1747.2003.12495.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions