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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector All-Trans-Retinoic Acid Induces Interleukin-8 via the Nuclear Factor-jB and p38 Mitogen-Activated Protein Kinase Pathways in Normal Human Keratinocytes Xiuju Dai, Kenshi Yamasaki, Yuji Shirakata, Koji Sayama, and Koji Hashimoto Department of Dermatology, Ehime University School of Medicine, Ehime, Japan Retinoic acid derivatives have been used successfully for the treatment of various dermatoses, such as psoriasis; however, topical application of these compounds often elicits skin irritation. We hypothesized that this irritation was as a result of the local production of interleukin-8 (IL-8). To test this hypothesis, we investigated whether all- trans-retinoic acid (ATRA) induced IL-8 production in normal human keratinocytes. Stimulation with 10À7 M ATRA enhanced IL-8 mRNA expression and induced IL-8 production. We also studied the intracellular signaling mech- anisms of ATRA-induced IL-8 production in keratinocytes. ATRA increased the expression of RelA (p65), RelB, nuclear factor (NF)-jB2 (p52), and NF-jB1 (p50), and elevated the DNA-binding activity of p65 and phosphorylation of inhibitor jB(IjB) a. Introduction of a dominant-negative mutant of IjBa completely abolished ATRA-induced IL-8 production, which indicates that this process is NF-jB-dependent. We also studied the role of the p38 mitogen- activated protein kinase (MAPK) pathway in this phenomenon. ATRA phosphorylated the p38 MAPK, and SB202180 inhibited ATRA-induced IL-8 production, which indicates that the p38 MAPK is also involved in ATRA-induced IL-8 production. In summary, ATRA induces IL-8 production in both NF-jB- and p38 MAPK-dependent manners in normal human keratinocytes. Key words: skin irritation/chemokine/p65/retinoid/IkBa/SB202180 J Invest Dermatol 123:1078 –1085, 2004 Retinoids include natural and synthetic compounds with pathogenesis of inflammatory diseases, IL-8 has diverse specific biological activity similar to that of Vitamin A (retinol) biological properties, including chemotaxis of neutrophils (Orfanos et al, 1997). Retinol and its derivative acid are and T lymphocytes (Baggiolini et al, 1989), regulation of cell widely used to treat acne, psoriasis, and photo-aged skin. adhesion (Djeu et al, 1990), activation of neutrophils (Muk- Despite their many beneficial effects, however, topical ap- aida et al, 1992), and modulation of histamine release (Kuna plication of retinoids often causes local irritation that is et al, 1991). Therefore, it seems worthwhile to investigate manifested as redness, scaling, and dryness (Kang et al, the potential role of IL-8 in retinoic acid-induced skin in- 1995; Ale et al, 1997). The mechanism of retinoic acid-in- flammation. Various cell types, such as endothelial cells, duced inflammation is poorly understood. The important epithelial cells, synovial cells, T cells, fibroblasts, keratin- role of interleukin-8 (IL-8) in the progress of irritation has ocytes, chondrocytes, and some tumor cells, have been been demonstrated (Schroder, 1995). A recent report shown to produce IL-8 (Hoffmann et al, 2002). The classical showed that treatment with 2% retinol upregulated the lev- inducers of IL-8 are inflammatory stimuli, such as IL-1, el of IL-8 mRNA in the ear epidermis of BALB/c mice (Kim tumor necrosis factor (TNF)-a, bacterial lipopolysaccharides et al, 2003), and that anti-irritants significantly inhibited re- (LPS), 12-O-tetradecanoylphorbol-13-acetate, viruses, and tinoid-induced IL-8 in cultured keratinocytes (Kim et al, double-stranded RNA (Hoffmann et al, 2002). IL-8 produc- 2003), indicating that IL-8 plays a central role in retinoid- tion is regulated at both the transcriptional and post-tran- induced skin irritation. Although human keratinocytes pro- scriptional levels. The 30-end of the IL-8 transcript contains duce IL-8 in response to retinoids (Kim et al, 2003), the an AU-rich cis-element (ARE), which is responsible for the molecular mechanism involved is unclear. destabilization of a variety of cytokine mRNA (Shaw and IL-8 is a member of the C–X–C subfamily of chemokines Kamen, 1986). The sequence that spans nucleotides (nt) À1 (CXCL8; Zlotnik and Yoshie, 2000). As a key factor in the to À133 within the 50-flanking region of the IL-8 gene is essential and sufficient for transcriptional regulation of the gene. The core IL-8 promoter contains a nuclear factor-kB Abbreviations: ATRA, all-trans-retinoic acid; CT, cycle threshold; (NF-kB) element that is required for activation in all of the GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IkB, inhib- cell types studied, and it also contains activating protein itor kB; IL, interleukin; MAPK, mitogen-activated protein kinase; (AP)-1- and CCAAT/enhancer-binding protein (C/EBP)-bind- MOI, multiplicity of infection; NF-kB, nuclear factor kB; RAR, re- tinoic acid receptor; RARE, retinoic acid-responsive element; RXR, ing sites. The latter two sites are dispensable for transcrip- retinoid X receptor; TNF, tumor necrosis factor tional activation in some cell types, but contribute to Copyright r 2004 by The Society for Investigative Dermatology, Inc. 1078 123:6 DECEMBER 2004 ATRA INDUCES IL-8 VIA THE NF-kB AND p38 MAPK PATHWAYS IN KERATINOCYTES 1079 activation in other cell types. Thus, unlike the NF-kB site, the AP-1 and C/EBP sites are not essential for IL-8 gene induction, but are required for maximal gene expression (Hoffmann et al, 2002). Cytokines and chemokines play important roles in the pathogenesis of various dermatoses. Based of the results of biochemical analyses and in vitro studies, it is clear that IL-8 contributes significantly to the pathological changes seen in psoriasis (Sticherling et al, 1991a, b, 1999; Takematsu and Tagami, 1993). The involvement of IL-8 in other dermatoses, such as bullous pemphigoid (Baroni et al, 2002), erythema migrans (Grygorczuk et al, 2002), atopic dermatitis, and al- lergic contact dermatitis (Albanesi et al, 2001), has also been demonstrated. The erythematous reaction in retinoid- induced skin irritation is clinically similar to a mild irritant dermatitis (Fisher et al, 1991; Ale et al, 1997; Grygorczuk et al, 2002). In particular, activated keratinocytes are an im- portant source of chemotactic factors that direct the re- cruitment of specific leukocyte populations and regulate the quality, magnitude, and duration of the inflammatory re- sponse. Normal cultured human keratinocytes have been shown to produce IL-8 after appropriate stimulation (Larsen et al, 1989; Boorsma et al, 1994; Stoof et al, 2001). Retinoic acid modulates immunological and inflammato- ry responses, most probably by regulating cytokine pro- duction, although it also affects epidermal cell growth and differentiation (Zitnik et al, 1994; Sawatsri et al, 2000). As retinoic acid upregulates IL-8 expression in fibroblasts (Zhang et al, 1992), neuroblastoma cells (Yang et al, 1993), and a human ovarian carcinoma cell line (Harant et al, 1995), we hypothesized that retinoic acid might also induce IL-8 Figure 1 production in human keratinocytes. To test this hypothesis, All-trans-retinoic acid (ATRA) simulates the production of inter- we investigated whether all-trans-retinoic acid (ATRA) was leukin (IL)-8 by human keratinocytes. (A) Subconfluent keratinocyte À6 able to induce IL-8 production in cultured normal human cultures were treated with IL-1a (1 ng per mL), ATRA (10 M), or ve- hicle (dimethyl sulfoxide). After 36 h of incubation, the culture supe- keratinocytes, and we assessed the involvement of the NF- rnatants were collected and subjected to ELISA for the detection of kB and p38 mitogen-activated protein kinase (MAPK) path- IL-8. (B) Keratinocytes were stimulated with ATRA (10À9–10À5 M). IL-8 ways in this process. mRNA expression was monitored by RT-PCR after a 24-h incubation. (C) Keratinocytes were stimulated with ATRA for 36 h, and the level of secreted IL-8 was measured by ELISA. Results (Fig 1B). The release of IL-8 into the culture medium was ATRA increases the production of IL-8 in keratin- upregulated in a concentration-dependent manner, and was ocytes IL-1a is the prominent inducer of IL-8 (Kristensen consistent with the increase in IL-8 mRNA (Fig 1C). et al, 1991; Hoffmann et al, 2002). Moreover, retinoids re- duce IL-1 induction of IL-8 production in human monocytes (Gross et al, 1993). Therefore, we initially investigated the ATRA increases the expression of the NF-jB family effect of ATRA on IL-1a-induced IL-8 production. Keratin- members in human keratinocytes NF-kB binding is es- ocytes were treated with IL-1a (1 ng per mL) or IL-1a plus sential for the activation of IL-8 gene transcription (Kunsch ATRA (10À6 M), and the IL-8 levels in the culture supernat- et al, 1994). Several members of the NF-kB family, such as ants were measured after 36 h of incubation. Marked in- p50 (NF-kB1), p65 (RelA), c-Rel, and p52 (NF-kB2), have duction of IL-8 was observed in IL-1a-stimulated cells, and been shown to bind to the NF-kB motif of the IL-8 promoter ATRA increased IL-8 production in a synergistic manner (Kunsch and Rosen, 1993; Stein et al, 1993; Harant et al, (Fig 1A). 1996a). To characterize the molecular mechanisms under- To assess the effect of ATRA on IL-8 production in ker- lying ATRA-induced IL-8 production in keratinocytes, the atinocytes, we stimulated the keratinocytes with different expression of NF-kB family members in ATRA-treated ker- concentrations of ATRA. IL-8 mRNA expression was atinocytes was examined using the RPA. The expression scarcely detectable by the ribonuclease protection assay levels of p65, RelB, p52, and p50 mRNA started to increase (RPA) in samples that contained the vehicle (dimethyl sulf- 1 h after the addition of ATRA, and persisted for more than oxide (DMSO)) alone (Fig 1B).

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