Volume 124, Issue 5, Pages 1180-1187 (May 2003) Rho kinase blockade prevents inflammation via nuclear factor κB inhibition: evidence in Crohn’s disease and experimental colitis  Jean-Pierre Segain, Diane Raingeard de la Blétière, Vincent Sauzeau, Arnaud Bourreille, Gréory Hilaret, Chrystelle Cario-Toumaniantz, Pierre Pacaud, Jean-Paul Galmiche, Gervaise Loirand  Gastroenterology  Volume 124, Issue 5, Pages 1180-1187 (May 2003) DOI: 10.1016/S0016-5085(03)00283-X

Figure 1 Activation of the RhoA/ROCK pathway in intestinal inflammation. (A–D) RhoA activity (A and C ) and TNF concentration (B and D) in intestinal mucosa from (A and B) CD (inflamed and noninflamed; n = 12) and control patients (n = 6) and from (C and D) rats with TNBS-induced colitis and control rats, respectively (n = 5 each). (A and C ) Pull-down assay (upper panel) and densitometrical quantification (mean ± SEM, lower panel) of active GTP-bound RhoA. Total RhoA amounts were equivalent between samples as assessed by Western blot of colon protein extracts. (B and D) Biopsies were cultured for 24 hours, and TNF concentration (mean ± SEM) in the culture supernatant was determined by bioassay. (E and F ) Effect of the ROCK inhibitor Y-27632 on TNF production by LPMC isolated from inflamed mucosa (E ) of CD patients (n = 6) and (F ) of rats with TNBS colitis. Boxes represent the median and interquartile range of TNF concentration in 24-hour culture supernatants. ∗P < 0.05; ∗∗P < 0.01. Gastroenterology 2003 124, 1180-1187DOI: (10.1016/S0016-5085(03)00283-X)

Figure 2 TNBS-induced colitis is improved by oral treatment with the ROCK inhibitor Y-27632. (A) Weight changes (mean ± SEM; n = 10 per group) in rats with TNBS-induced colitis treated with vehicle (empty squares) or Y-27632 (filled squares). Y-27632 significantly improved intestinal inflammation evaluated in rats (mean ± SEM; n = 10 per group) at day 7 of treatment by macroscopic score (B), colon wall thickness (C), and histological score (D). Colon sections (magnification, 200×) from rats with TNBS-induced colitis after treatment with vehicle (E) or Y-27632 (F) were stained with H&E. Y-27632 treatment significantly reduced the inflammatory infiltrate in the lamina propria and the submucosa. ∗P < 0.05; ∗∗P < 0.01. Gastroenterology 2003 124, 1180-1187DOI: (10.1016/S0016-5085(03)00283-X)

Figure 3 Role of the RhoA/ROCK pathway in proinflammatory cytokine expression. (A and B) PBMC were stimulated with the RhoA-activating CNF-1 toxin for 6 hours, and supernatants were analyzed by bioassay. (A) CNF-1 dose-dependently stimulated TNF production. (B) Kinetic of TNF production by PBMC stimulated with CNF-1 (1 nmol/L). The inhibitory effect of Y-27632 on CNF-induced TNF production is shown at 6 hours (triangle). (C) PBMC were cultured without or with LPS for 6 hours and lysed for the evaluation of the level of active GTP-RhoA by pull-down assay (upper bands). Total RhoA in unstimulated or LPS-stimulated cell lysates was equivalent as assessed by Western blot (lower bands). LPS stimulation increased the level of active GTP-RhoA. (D) PBMC were stimulated with LPS in the absence or presence of Y-27632 for 6 hours. TNF-α, IL-1β, and IL-10 production was evaluated by bioassay (for TNF) and enzyme-linked immunosorbent assay. Data are mean ± SEM of at least 4 independent experiments. ∗P < 0.05. Gastroenterology 2003 124, 1180-1187DOI: (10.1016/S0016-5085(03)00283-X)

Figure 4 ROCK associates with IKK in vivo and induces IKK activation (A ). Y-27632 prevents NF-κB activation and I-κBα degradation in PBMC stimulated with LPS or TNF-α. Immunofluorescence staining (upper panel) of the activated form of the NF-κB p65 subunit in control PBMC and in PBMC stimulated with LPS or TNF-α in the absence or presence of Y-27632 (10 μmol/L) for 30 minutes. I-κBα levels, determined in each condition by Western blot analysis (lower panel), are shown below the corresponding photomicrograph. (B) ROCK inhibition decreased NF-κB transcriptional activity. THP-1 cells were transfected with an NF-κB-dependent luciferase gene reporter plasmid, pretreated 10 minutes with or without Y-27632 (10 μmol/L), and stimulated with IL-1β for 4 hours. Cells were then lysed for measurement of luciferase activity (RLU, relative light unit; mean ± SEM; n = 4). (C, D, and E) THP-1 cells were pretreated for 10 minutes without or with Y-27632 (10 μmol/L) and stimulated with IL-1β (10 ng/mL) for 15 minutes. Cell lysates were then prepared for Western blot, immunoprecipitation, and I-κBα kinase assay as described in Materials and Methods. (C) ROCK inhibition prevents I-κBα phosphorylation and degradation induced by IL-1β. I-κBα and phosphorylated I-κBα levels were determined by Western blot analysis (D) ROCK associates with and activates IKK. The IKKα immune complex was assayed for I-κBα kinase activity (upper panel). The IKKα immune complex was analyzed by Western blot (wb) by using IKKα (middle panel) and ROCK (lower panel) Ab. Neither IKK activity nor the presence of ROCK was detected with control immunoglobulin G immunoprecipitates (not shown). (E) Active ROCK is present in the IKKα immune complex. Y-27632-sensitive kinase activity in the IL-1β-activated IKKα immune complex has been detected by in vitro S6 kinase substrate phosphorylation assay, in the absence and presence of Y-27632 (10 μmol/L). (F) In vitro phosphorylation experiment on glutathione S-transferase-I-κBα showing that recombinant ROCK, in the absence or presence of Y-27632, did not phosphorylate I-κBα (upper panel), whereas it induced S6 kinase substrate peptide phosphorylation that was inhibited by Y-27632 (lower panel). S6 kinase substrate peptide phosphorylation in the absence of Y-27632 was designated as 100%. Results are representative of at least 3 independent experiments. Gastroenterology 2003 124, 1180-1187DOI: (10.1016/S0016-5085(03)00283-X)