Budesonide epimer R or dexamethasone selectively inhibit platelet-activating factor-induced or interleukin 1β-induced DNA binding activity ofcis-acting transcription factors and cyclooxygenase-2 gene expression in human epidermal keratinocytes

Abstract

To further understand the molecular mechanism of glucocorticoid action on gene expression, DNA-binding activities of the cis-acting transcription factors activator protein 1 (AP1), AP2, Egr1 (zif268), NF-B, the signal transducers and activators of transcription proteins gamma interferon activation site (GAS), Sis-inducible element, and the TATA binding protein transcription factor II D (TFIID) were examined in human epidermal keratinocytes. The cytokine interleukin 1␤ (IL-1␤) and platelet-activating factor (PAF), both potent mediators of inflammation, were used as triggers for gene expression. Budesonide epimer R (BUDeR) and dexamethasone (DEX) were studied as potential antagonists. BUDeR or DEX before IL-1␤-or PAF-mediated gene induction elicited strong inhibition of AP1-, GAS-, and in particular NF-B-DNA binding (P < 0.001, ANOVA). Only small effects were noted on AP2, Egr1 (zif268), and Sis-inducible element-DNA binding (P > 0.05). No significant effect was noted on the basal transcription factor TFIID recognition of TATA-containing core promoter sequences (P > 0.68). To test the hypothesis that changing cis-acting transcription factor binding activity may be involved in inflammatoryresponse related gene transcription, RNA message abundance for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was assessed in parallel by using reverse transcription-PCR. Although the COX-1 gene was found to be expressed at constitutively low levels, the TATA-containing COX-2 gene, which contains AP1-like, GAS, and NF-B DNA-binding sites in its immediate promoter, was found to be strongly induced by IL-1␤ or PAF (P < 0.001). BUDeR and DEX both suppressed COX-2 RNA message generation; however, no correlation was associated with TFIID-DNA binding. These results suggest that on stimulation by mediators of inflammation, although the basal transcription machinery remains intact, modulation of cis-activating transcription factor AP1, GAS, and NF-B-DNA binding by the glucocorticoids BUDeR and DEX play important regulatory roles in the extent of specific promoter activation and hence the expression of key genes involved in the inflammatory response.Glucocorticosteroids (GCs) have long been used therapeutically as immunosuppressive and anti-inflammatory agents; however, the mechanism of their activity is only recently becoming understood at the genetic level. GCs interact with an intracellular GC receptor, which subsequently translocates to the nucleus as a ligand-activated transcriptional modulator. In turn, the GC receptor regulates the expression of genes such as those encoding cytokines, matrix metalloproteinases, and cell adhesion molecules known to be critical to both inflammation and the immune response (1, 2). Besides a direct "type 1" interaction with a palindromic GC responsive element in GC-sensitive gene promoters (3, 4), functions of the GC receptor also include a "type 2" interaction with chromatin-associated cis-acting transcription factors. For example, the GC receptor can physically interact with the Fos and J...

Cyclooxygenase-2 and Presenilin-1 Gene Expression Induced by Interleukin-1β and Amyloid β42 Peptide Is Potentiated by Hypoxia in Primary Human Neural Cells

“…1, A and B). Both the observed and predicted RNA message stability analyses indicated that COX-2 and PS1 RNA messages have relatively short half-lives, on the order of 3 and 4 -5 h, respectively; PS2 RNA message half-life was Ͼ10 h, and, in agreement with previous reports, brain ␤-actin RNA half-life was Ͼ12 h (4,24).…”

“…4. As previous studies have shown, human AP1, HIF-1␣, NF-Bp50/p65, and STAT1␣ gel shifts could each be super-shifted using their TF N-terminal-specific antibodies (24,32). Electrophoretic mobility shift assay analysis showed that, during normoxia in HN cells, AP1-, HIF-1␣-, NF-Bp50/p65-, and STAT1␣-DNA binding increased 1.1-, 1.6-, 1.5-, and 1.1-fold, respectively, over controls 1 h after IL-1␤, and by 1.1-, 1.2-, 1.1-, and 1.2-fold 3 h after A␤42, as compared with control cells treated with A␤42s.…”

“…Normal human neural (HN) progenitor cells (CC-2599), NPMM (neural progenitor maintenance medium), human epidermal and fibroblast growth factors, gentamicin/amphotericin B (G/A1000), and neural survival factor-1 were obtained from Clonetics (Walkersville, MD). AP1, HIF-1␣, NF-Bp50/p65, and STAT1␣ gel-shift consensus and mutant oligonucleotides (9,21,24) were synthesized at the Louisiana State University Health Sciences Center (LSUHSC) core facility or were purchased from Promega (Madison, WI). AP1, HIF-1␣, NF-Bp50/p65, and STAT1␣ supershift and COX-2 and PS1 antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA).…”

“…Electrophoretic Mobility Shift Assay and Immunochemistry of Human AP1, HIF-1␣, NF-Bp50/p65, and STAT1␣-Nuclear protein extracts (NPXTs) were prepared from one to three 3.5-cm-diameter wells of HN cells or 100 mg (wet weight) brain tissue and quantitated as described previously (9,21,24). NPXTs (5 g) derived from HN cells were incubated with [␥-32 P]ATP (ϳ3000 Ci/mmol) end-labeled AP1, HIF-1␣, NF-Bp50/p65, or STAT1␣ consensus and mutant oligonucleotides in 5-l volumes, reacted for 30 min on ice, analyzed on 5% acrylamide/90 mM Tris borate, pH 8.4, 1 mM EDTA (TBE) gels, and dried onto 2-mm Whatman filter paper at 80°C for 2 h and phosphorimaging was performed.…”

“…In the human association neocortex in AD, DNA-binding activities for these TFs, as well as IL-1␤, are sharply elevated (9,24,26). Therefore, IL-1␤-triggered AP1-, NF-Bp50/p65-, and STAT1␣-sensitive genes have strong potential to propagate inflammatory signaling in the brain (20 -27).…”