Volume 134, Issue 4, Pages 1070-1082 (April 2008) Gastrin-Mediated Interleukin-8 and Cyclooxygenase-2 Gene Expression: Differential Transcriptional and Posttranscriptional Mechanisms  Dharmalingam Subramaniam, Satish Ramalingam, Randal May, Brian K. Dieckgraefe, Douglas E. Berg, Charalabos Pothoulakis, Courtney W. Houchen, Timothy C. Wang, Shrikant Anant  Gastroenterology  Volume 134, Issue 4, Pages 1070-1082 (April 2008) DOI: 10.1053/j.gastro.2008.01.040 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Gastrin treatment induces COX-2 and IL-8 gene expression. (A and B) Real-time PCR. AGS-E cells were treated with 10 nmol/L of gastrin for 0–4 hours. Gastrin induces the (A) COX-2 and (B) IL-8 mRNA expression within 30 minutes. *P < .05, **P < .001. (C) Western blot analysis for COX-2 protein. The protein was increased significantly at 2 hours. (D) Enzyme-linked immunosorbent assay analysis for IL-8. The protein levels increased at 2 hours. **P < .001. (E and F) Gastrin-dependent IL-8 expression is mediated in part by COX-2–derived PGE2. Cells were treated with the specific COX-2 inhibitor, NS398 (NS, 50 μmol/L). Real-time (E) RT-PCR and (F) enzyme-linked immunosorbent assay analyses show that PGE2 (1 μmol/L) partially rescues NS398-inhibited gastrin-mediated IL-8. Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Gastrin-mediated activation of Akt is downstream of p38 MAPK. (A) Gastrin induces Akt, Erk, and p38 phosphorylation. Cell lysates from gastrin-treated cells were analyzed for total and phosphorylated Akt, Erk, and p38. Gastrin treatment phosphorylates Akt at 1 minute, followed by a robust induction at 10 minutes. However, gastrin induces Erk and p38 phosphorylation within 1 minute. (B) Inhibiting Akt activation does not affect Erk or p38 phosphorylation. The cells were pretreated with PI3K inhibitor LY294002 (ly, 15 μmol/L) for 2 hours and subsequently treated with gastrin for 15 minutes. LY294002 treatment inhibited Akt phosphorylation, but not Erk or p38 phosphorylation. (C) Inhibiting Erk activation does not affect Akt or p38 phosphorylation. Erk inhibitor PD98059 (pd, 10 μmol/L) treatment inhibited Erk phosphorylation, but not Akt or p38 phosphorylation. (D) Inhibiting p38 activation also suppresses Akt phosphorylation. Treatment with p38 inhibitor SB203580 (sb, 10 μmol/L), inhibited both p38 and Akt but not Erk phosphorylation. Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Gastrin regulates COX-2 and IL-8 through Akt and p38 MAPK pathways. Cells were pretreated with LY294002 (ly, 15 μmol/L), SB203580 (sb, 10 μmol/L), or PD98059 (pd, 10 μmol/L) for 2 hours before pretreatment with 10 nmol/L gastrin for 2 hours. (A and B) COX-2 mRNA and protein expression were inhibited by both Akt and p38 inhibitors (P < .05). (C) Cells were transfected with a plasmid encoding a human COX-2 promoter–driven luciferase before treatment with the inhibitors. Akt and p38 but not Erk inhibitor significantly inhibited gastrin-mediated COX-2 promoter activity (P < .05). (D and E) IL-8 mRNA and protein expression were inhibited by Akt and p38 inhibitors (P < .001). (F) AGS-E cells were transfected with an IL-8 promoter–driven luciferase reporter plasmid and subsequently treated as described in C. Both Akt and p38 inhibitors, but not Erk inhibitor, inhibited IL-8 promoter activity (P < .01). Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Gastrin regulates NF-κB and AP-1 activity. (A) Nuclear extracts were subjected to EMSA analysis using an NF-κB oligonucleotide probe. Gastrin increased nuclear NF-κB levels in 30 minutes. (B) Supershift was observed with antibodies against NFkB1 and RelA. (C) EMSA analysis for AP-1 activation. Gastrin treatment resulted in AP-1 activation within 30 minutes. (D) EMSA analyses for NF-κB and AP-1 after treatment of the cells with proteosomal inhibitor MG132. NF-κB activity was suppressed whereas AP-1 activity was increased by MG132. (E) Western blot analyses show that MG132 inhibits nuclear localization of NF-κB subunits NFkB1 and RelA. (F) Western blot analyses show that MG132 further increases expression of AP-1 subunits c-Fos and c-Jun. Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Gastrin-mediated COX-2 expression is independent of NF-κB, whereas that of IL-8 is dependent on NF-κB. MG132 treatment does not affect gastrin-mediated expression of either (A) COX-2 mRNA or (B) protein. However, MG132 inhibited gastrin-mediated expression of both (C) IL-8 mRNA and (D) protein in the cells. To confirm that NF-κB only regulates IL-8 expression, and not that of COX-2, siRNA-mediated down-regulation of p65 RelA subunit (siRelA) was performed before gastrin treatment. (E) Knockdown of p65 RelA was confirmed by Western blot analysis of nuclear extracts. (F) RelA knockdown did not affect COX-2 protein levels, (G) but inhibited gastrin-mediated IL-8 protein expression. Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Gastrin enhances COX-2 and IL-8 mRNA stability in a p38 MAPK-dependent manner. Cells were treated with 10 nmol/L gastrin in either in the presence or absence of PI3K/Akt (A and D, LY:LY249002), Erk (B and E, PD:PD98059), or p38 (C and F, SB:SB203580) inhibitors, and the stability of (A–C) COX-2 and (D–F) IL-8 mRNA were determined after addition of actinomycin D. Gastrin increases the half-life of COX-2 and IL-8 mRNA from 31 minutes to 8 hours and approximately 4 hours, respectively. (A–C and B–F). The addition of the PI3K/Akt or Erk inhibitors did not affect the COX-2 (A and B) and IL-8 (D and E) mRNA. On the other hand, p38 inhibitor abrogated the stability of both transcripts (C and F). Half-life measurements for (G) COX-2 and (H) IL-8 were determined. Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Gastrin regulates HuR expression and cytoplasmic localization through p38 MAPK. (A) Lysates from gastrin-treated cells were subjected to Western blot analysis. HuR protein was increased significantly for 2 hours. (B) Cells were treated with increasing doses of gastrin (0–100 nmol/L) for 2 hours. The protein was induced to the maximum level at a gastrin dose of 10 nmol/L. (C) Increased binding of HuR binding to COX-2 mRNA after gastrin treatment. Whole-cell extract (t) were immunoprecipitated with anti-HuR antibody, and RNA bound to the immunoprecipitates (p) and supernatant (s) were isolated and subjected to RT-PCR for COX-2 mRNA. Data show increased COX-2 mRNA in the pellet of gastrin-treated cells. (D) Gastrin-mediated HuR levels are reduced in cells pretreated with p38 inhibitor SB203580 (sb, 10 μmol/L) for 2 hours. (E) Immunocytochemistry for HuR after gastrin treatment in the presence or absence of p38 inhibitor. Cells transiently expressing FLAG-tagged HuR before treatment with p38 inhibitor and gastrin. The protein is nuclear in control untreated cells (a). Gastrin treatment results in relocalization of HuR from the nucleus to cytoplasm (b). Treatment with the p38 inhibitor did not affect the localization of the protein (c). Pretreatment with the p38 inhibitor followed by gastrin treatment resulted in a predominant nuclear localization of the HuR protein (d). Gastroenterology 2008 134, 1070-1082DOI: (10.1053/j.gastro.2008.01.040) Copyright © 2008 AGA Institute Terms and Conditions