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Budesonide Epimer R Or Dexamethasone Selectively Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3914–3919, March 1998 Medical Sciences Budesonide epimer R or dexamethasone selectively inhibit platelet-activating factor-induced or interleukin 1b-induced DNA binding activity of cis-acting transcription factors and cyclooxygenase-2 gene expression in human epidermal keratinocytes WALTER J. LUKIW*, RICARDO PALACIOS PELAEZ†,JORGE MARTINEZ†, AND NICOLAS G. BAZAN*‡ *Louisiana State University Medical Center, School of Medicine, Neuroscience Center of Excellence and Department of Ophthalmology, 2020 Gravier Street, Suite D, New Orleans, LA 70112-2272; and †Research and Development Department, IFIDESA-ARISTEGUI, Industrial Farmaceutica y de Especialidades SA, Maria de Molina 37, Madrid 28006 Spain Communicated by Ralph T. Holman, University of Minnesota, Austin, MN, January 2, 1998 (received for review July 23, 1997) ABSTRACT To further understand the molecular mecha- cytokines, matrix metalloproteinases, and cell adhesion molecules nism of glucocorticoid action on gene expression, DNA-binding known to be critical to both inflammation and the immune activities of the cis-acting transcription factors activator protein response (1, 2). Besides a direct “type 1” interaction with a 1 (AP1), AP2, Egr1 (zif268), NF-kB, the signal transducers and palindromic GC responsive element in GC-sensitive gene pro- activators of transcription proteins gamma interferon activation moters (3, 4), functions of the GC receptor also include a “type site (GAS), Sis-inducible element, and the TATA binding protein 2” interaction with chromatin-associated cis-acting transcription transcription factor II D (TFIID) were examined in human factors. For example, the GC receptor can physically interact with epidermal keratinocytes. The cytokine interleukin 1b (IL-1b) the Fos and Jun components of dimeric activator protein 1 (AP1) and platelet-activating factor (PAF), both potent mediators of (5, 6), the p65 subunit of NF-kB (7, 8), and the STAT5 family of inflammation, were used as triggers for gene expression. Budes- signal transducers and activators of transcription (9, 10) to onide epimer R (BUDeR) and dexamethasone (DEX) were modulate the trans-activation of gene promoters that contain no studied as potential antagonists. BUDeR or DEX before IL-1b- GC responsive elements. GCs also alter the nuclear availability of or PAF-mediated gene induction elicited strong inhibition of transcription factors as is the case when NF-kB activity is sup- AP1-, GAS-, and in particular NF-kB-DNA binding (P < 0.001, pressed by induction of the I-kB inhibitory protein, which se- ANOVA). Only small effects were noted on AP2, Egr1 (zif268), questers the regulatory element NF-kB as an inactive cytoplasmic and Sis-inducible element-DNA binding (P > 0.05). No signifi- complex (11, 12). cant effect was noted on the basal transcription factor TFIID GCs also moderate the inflammatory response by stimulating recognition of TATA-containing core promoter sequences (P > transcription from the interleukin 1 (IL-1) decoy receptor gene, 0.68). To test the hypothesis that changing cis-acting transcrip- which acts as a scavenger for free IL-1b (13, 14) while down- tion factor binding activity may be involved in inflammatory- regulating expression of the inducible cyclooxygenase-2 (COX-2) response related gene transcription, RNA message abundance gene when activated by inflammation or cytokines such as IL-1b for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was (15). The potent inflammation-related cis-activating transcription assessed in parallel by using reverse transcription–PCR. Al- factors AP1 (16, 17) and NF-kB (18, 19), as well as COX-2 (20) though the COX-1 gene was found to be expressed at constitu- and matrix metalloproteinase-1 and -9 gene expression (21), are tively low levels, the TATA-containing COX-2 gene, which con- also strongly induced by the inflammatory mediator platelet- tains AP1-like, GAS, and NF-kB DNA-binding sites in its im- activating factor (PAF; refs. 22–26). Although several synthetic mediate promoter, was found to be strongly induced by IL-1b or GCs have been intensively studied at the gene signaling level PAF (P < 0.001). BUDeR and DEX both suppressed COX-2 RNA (1–12) the molecular specificity and potency of the nonfluori- message generation; however, no correlation was associated with nated GC budesonide epimer R (BUDeR) remains incompletely TFIID–DNA binding. These results suggest that on stimulation characterized. BUDeR, which displays a 14-fold higher relative by mediators of inflammation, although the basal transcription affinity for the GC receptor when compared with dexamethasone machinery remains intact, modulation of cis-activating tran- (DEX), has a very high ratio between topical and systemic activity scription factor AP1, GAS, and NF-kB-DNA binding by the (27), making it active directly at the site of inflammation such as glucocorticoids BUDeR and DEX play important regulatory in asthma and rhinitis where inhalation therapy is the drug roles in the extent of specific promoter activation and hence the delivery system of choice. expression of key genes involved in the inflammatory response. To further understand the genetic mechanism of GC anti- inflammatory activity, here we have compared the effects of BUDeR with those of DEX on IL-1b- or PAF-stimulated gene Glucocorticosteroids (GCs) have long been used therapeutically expression in human epidermal keratinocyte (HEK) cells in as immunosuppressive and anti-inflammatory agents; however, primary culture. DNA binding activities for the cis-acting tran- the mechanism of their activity is only recently becoming under- stood at the genetic level. GCs interact with an intracellular GC receptor, which subsequently translocates to the nucleus as a Abbreviations: AP1, activator protein 1; BUDeR, budesonide epimer R; COX, cyclooxygenase; DEX, dexamethasone; EMSA, electro- ligand-activated transcriptional modulator. In turn, the GC re- phoretic mobility shift assay; GAS, gamma interferon activation site; ceptor regulates the expression of genes such as those encoding GC, glucocorticoid; HEK, human epidermal keratinocyte; IL-1b, interleukin 1b; KGM, keratinocyte growth medium; NPXT, nuclear The publication costs of this article were defrayed in part by page charge protein extract; PAF, platelet-activating factor; PMSF, phenylmeth- ylsulfonyl fluoride; RT-PCR, reverse transcription–PCR; SIE, Sis- payment. This article must therefore be hereby marked ‘‘advertisement’’ in inducible element; STAT, signal transducers and activators of tran- accordance with 18 U.S.C. §1734 solely to indicate this fact. scription; TFIID, transcription factor II D. © 1998 by The National Academy of Sciences 0027-8424y98y953914-6$2.00y0 ‡To whom reprint requests should be addressed at: 2020 Gravier PNAS is available online at http:yywww.pnas.org. Street, Suite D, New Orleans, LA 70112. e-mail: [email protected]. 3914 Downloaded by guest on September 26, 2021 Medical Sciences: Lukiw et al. Proc. Natl. Acad. Sci. USA 95 (1998) 3915 scription factors AP1 (16, 17), AP2 (28), Egr1 (also known as taining 15 mgyml bovine pituitary extract, 0.2 ngyml human zif268, krox24, tis-8, d2, and NGF1A; refs. 29 and 30); NF-kB (18, epidermal growth factor, 0.5 mgyml insulin, 0.15 mM gentamy- 31, 32); the STAT elements SIE (Sis inducible element) and GAS cinyamphotericin-B, and 0.5 mgyml hydrocortisone until 80% (gamma interferon activation site; refs. 9, 10, and 33); and the confluent. For maximal GC effect, cells were serum-deprived for TATA binding protein TFIID (transcription factor II D; refs. 34 24 hr to 0.5% of normal serum levels before the addition of test and 35) were quantitated in HEK nuclear protein extracts compounds. Without serum deprivation, only small effects on (NPXTs) by using electrophoretic mobility shift assay (EMSA). transcription factor binding were observed with a pattern similar To correlate transcription factor availability with gene expression, to those observed with serum deprivation. For each experimental RNA message abundance for two genes critically involved in the condition, normal HEK cells were cultured in triplicate T75 flasks inflammatory response, the constitutively expressed COX-1 and for preparation of NPXTs and total RNA message extraction. the highly inducible COX-2 gene (36–40), were measured in Harvesting of Cells and Preparation of NPXTs. NPXTs were parallel by using reverse transcription–PCR (RT-PCR). The isolated at 4°C in the presence of proteolytic enzyme inhibitors TATA-containing COX-2 gene, a key regulator of prostaglandin phenylmethylsulfonyl fluoride (PMSF, Sigma P-7626; 1 mM), biosynthesis, and which contains DNA binding sites for AP1-like, aprotinin (Sigma A-6279; 0.05 mgyml), leupeptin (Sigma L-2884; AP2, NF-kB, GAS, SIE, Egr1 (zif268), and TFIID located 0.05 mgyml), and pepstatin A (Sigma P-4265, 0.025 mgyml). After between 21,000 bp and 1165 bp of its promoter (36, 39) was incubation with test compounds, ligand containing KGM was found to be strongly induced in HEK cells after IL-1b or PAF decanted and replaced with Dulbecco’s PBS (GIBCOyBRL stimulation. Our results also suggest that the GCs BUDeR and 14040–018) containing 1 mM PMSF and 0.05 mgyml of aprotinin, DEX each down-regulate COX-2 gene expression because of the and this suspension was pelleted and gently resuspended in 500 reduced ability of the cis-acting transcription factors AP1, GAS, ml of nuclei preparation buffer (NPB) consisting of 20 mM Hepes and NF-kB to interact with their target
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