P2Y2 receptor transcription is increased by NF-kappa B and stimulates cyclooxygenase-2 expression and PGE2 released by intestinal epithelial cells

Abstract

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y(2) mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn's disease and ulcerative colitis. However, the transcriptional events regulating P2Y(2) receptor (P2Y(2)R) expression are not known. We have identified a putative transcription start site in the P2Y(2)R gene and demonstrated acetylation of Lys(14) on histone H3 and Lys(8) on histone H4, thus suggesting that the chromatin associated with the P2Y(2) promoter is accessible to transcription factors. We also showed that the transcription factor NF-kappaB p65 regulates P2Y(2)R transcription under both proinflammatory and basal conditions. A NF-kappaB-responsive element was identified at -181 to -172 bp in the promoter region of P2Y(2). Hence, activation of P2Y(2)R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE(2) secretion by intestinal epithelial cells. These findings demonstrate that P2Y(2)R expression is regulated during intestinal inflammation through an NF-kappaB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

FIGURE 1

P2Y₂R mRNA expression in IECs is enhanced by proinflammatory stimuli. P2Y₂R mRNA expression was determined by quantitative RT-PCR in confluent IEC-6 and 3-day-postconfluent Caco-2 cells. A and B, Increases in mRNA expression were significant after a 3- and 6-h incubation with 0.5% (w/v) DSS and 24 h of incubation with 12.5 μg/ml LPS in Caco-2 cells. C and D, Increases in mRNA expression were significant after 12 h of incubation with 12.5 μg/ml LPS and 18 h of treatment with 10 ng/ml IL-6 in IEC-6 cells. Data are expressed as P2Y₂R mRNA expression induced by stimuli relative to the untreated control. Results were normalized to the expression of TATA-binding protein mRNA. Values are the means ± SEM of results from three separate experiments done in duplicate. Statistical significance was determined by unpaired t test; *, p < 0.05 and **, p < 0.01, as compared with control.

FIGURE 2

NF-κB p65 trans activates the P2Y₂R promoter region under basal conditions and this trans activation is enhanced following DSS-induced stress in Caco-2 cells. A, P2Y₂R promoter constructs. B, Subconfluent Caco-2 cells were transiently cotransfected with the P2Y₂R promoter-luciferase construct (prP2Y₂-luc) and the NF-κB p65-expressing vector or the empty pGL4.10 vector (control). Cells were incubated with or without 0.5% (w/v) DSS for 6 h, and luciferase activity was assayed after 48 h. C, Subconfluent Caco-2 cells were cotransfected with the P2Y₂R promoter-deletion luciferase constructs prP2Y₂ Δ-350bp, prP2Y₂Δ-784bp, or prP2Y₂Δ-1165bp and the NF-κB p65-expressing or control vector. D, Subconfluent Caco-2 cells were cotransfected with the P2Y₂R promoter-deletion luciferase constructs prP2Y₂Δ-350/PmlI or prP2Y₂Δ-350/StuI and the NF-κB p65-expressing or control vector. E, prP2Y₂ΔNBM1, prP2Y₂ΔNBM2, prP2Y₂ΔNBM3, P2Y₂R full-length promoter constructs or prP2Y₂Δ-350bp promoter construct were transiently cotransfected with the NF-κB p65-expressing or control vector. Luciferase activity is expressed as the fold increase relative to the activity of the empty vector cotransfected with the NF-κB p65-expressing vector. Data are the means ± SEM of results from at least four separate experiments done in triplicate. Statistical analysis was performed by an ANOVA test; *, p < 0.05 and **, p< 0.01, as compared with respective controls and indicated on the figures. E, §§, p < 0.01, as compared with empty vector control (EV).

FIGURE 3

NF-κB p65 binds to the P2Y₂R promoter sequence. A, Nuclear extracts from Caco-2 cells were incubated with the putative [γ-³²P]ATP-labeled NF-κB p65 DNA-binding site probes NBM1, NBM2, or NBM3 and anti-NF-κB p65 Abs for electrophoretic mobility and supershift assays. DNA-protein complexes were separated from the free probe on a native polyacrylamide gel. Arrow, NF-κB p65 DNAbinding and supershifted complexes. B, Nuclear extracts from Caco-2 cells were incubated with the putative [γ-³²P]ATP-labeled NF-κB p65 DNA-binding site probe NBM2 and 10× or 100× unlabeled NBM2 for electrophoretic mobility and competition assays. DNA-protein complexes were separated from the free probe on a native polyacrylamide gel. Results are representative of three independent experiments. NF-κB p65 DNA-binding complex is indicated by the arrow. C, Chromatin was immunoprecipitated with or without rabbit IgG Ab or anti- NF-κB p65 Ab. The re-ChIP assay was performed with anti-p300 Ab following the first immunoprecipitation with anti-p65 Ab. Samples were verified by quantitative RT-PCR analysis with oligonucleotides amplifying the −221 bp to −155 bp region of the P2Y₂R promoter and expressed as fold increase over normal rabbit IgG normalized to input. Results are representative of three independent experiments.

FIGURE 4

ATP and UTP stimulate COX-2 expression and PGE₂ released by Caco-2. Addition of 100 μM ATP (A) or UTP (B) rapidly stimulated the mRNA expression of COX-2 in Caco-2 cells. C, Caco-2 cells were stimulated with 100 μM ATP or UTP for 0 (control; ctrl), 3, 6, 18, and 24 h. COX-2 protein expression was detected by Western analysis. D, PGE₂ released in the cell culture medium was determined after a 24-h stimulation of Caco-2 cells with 100 μM ATP or UTP. For A, B, and D, data are the means ± SEM of results from three separate experiments done in duplicate. Statistical analysis was performed by an unpaired t test; *, p < 0.05; **, p < 0.01; and ***, p < 0.001, in comparison with unstimulated cells (ctrl). C, Blot is typical of three separate sets of experiments.