Rho kinase blockade prevents inflammation via nuclear factor κB inhibition: evidence in Crohn’s disease and experimental colitis

doi:10.1016/S0016-5085(03)00283-X

Gastroenterology

Volume 124, Issue 5, May 2003, Pages 1180-1187

https://doi.org/10.1016/S0016-5085(03)00283-X Get rights and content

Abstract

Background & Aims:

Rho proteins are involved in the regulation of several cellular functions. Data from in vitro studies suggest that RhoA could be involved in the inflammatory response. We investigated the role of RhoA and its downstream effector Rho kinase in intestinal inflammation.

Methods:

Activation of RhoA was assessed by pull-down assays. A specific inhibitor of Rho kinase, Y-27632, was used to examine the role of Rho kinase in inflammatory response in vivo and in vitro by molecular biology and by immunological and biochemical approaches.

Results:

Increased activation of RhoA was found in inflamed intestinal mucosa of patients with Crohn’s disease and of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Oral administration of Y-27632 in rats significantly reduced the colonic inflammation. In vitro, activation of RhoA alone was sufficient to induce tumor necrosis factor production. Y-27632 inhibited production of tumor necrosis factor-α and interleukin-1β by lamina propria and peripheral blood mononuclear cells. Rho kinase inhibition prevented nuclear factor κB activation and I-κB phosphorylation and degradation. We showed that Rho kinase associates with and activates I-κB kinase α and that Y-27632 prevents I-κB kinase activation.

Conclusions:

Our study provides the first evidence that Rho kinase activates I-κB kinase and, thus, nuclear factor κB, suggesting a key role of Rho kinase in inflammatory responses and intestinal inflammation. Specific inhibition of Rho kinase may be a promising approach for the treatment of patients with Crohn’s disease.

Section snippets

Patients

Eighteen patients with CD (8 men and 10 women, aged 18–72 years) and 6 healthy controls (3 men and 3 women, aged 22–67 years) were prospectively included. At the time of the study, 6 patients were receiving steroids, and 6 were receiving azathioprine alone or with steroids. Six CD patients and all controls were not receiving any medication. All patients underwent endoscopy for medical reasons. Colonic biopsy specimens were obtained from inflamed and/or noninflamed mucosa. All patients gave

Activation of the RhoA/Rho kinase pathway in intestinal inflammation

Activation of small GTPase RhoA in intestinal mucosa was assessed by a pull-down assay.23 The level of active GTP-bound RhoA was higher in biopsy specimens taken from inflamed mucosa of patients with CD as compared with noninflamed regions and with the mucosa of control patients (Figure 1A). Total RhoA amounts were equivalent between samples as assessed by Western blot of protein extracts. We also observed a similar increase in RhoA activation in inflamed mucosa of patients with ulcerative

Discussion

This study provides the first in vivo evidence for a role of a RhoA-dependent signaling pathway in inflammatory diseases and defines a new therapeutic target for the treatment of CD. Results reported here show that (1) RhoA and its effector ROCK are activated in CD and TNBS-induced colitis and that (2) blockade of ROCK inhibits proinflammatory cytokine production via inhibition of IKK and NF-κB activation and markedly reduces intestinal inflammation in rats with TNBS-induced colitis.

In vitro

Acknowledgements

The authors thank Dr. P. Boquet for the gift of cytotoxic necrotizing factor 1. The authors are grateful to Dr. C. H. Régnier for her helpful discussion and review of the manuscript.

References (30)

C. Fiocchi
Inflammatory bowel diseaseetiology and pathogenesis
Gastroenterology
(1998)
A.J. Ridley
Rho proteins, PI 3-kinases, and monocyte/macrophage motility
FEBS Lett
(2001)
J.L. Wallace et al.
Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease
Gastroenterology
(1989)
T. Espevik et al.
A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes
J Immunol Methods
(1986)
C.H. Regnier et al.
Identification and characterization of an IkappaB kinase
Cell
(1997)
V. Sauzeau et al.
Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca²⁺ sensitization of contraction in vascular smooth muscle
J Biol Chem
(2000)
S. Montaner et al.
Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases
J Biol Chem
(1998)
J.A. Frost et al.
Stimulation of NFkappa B activity by multiple signaling pathways requires PAK1
J Biol Chem
(2000)
S. Hippenstiel et al.
Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells
Blood
(2000)
M.S. Cammarano et al.
Dbl and the Rho GTPases activate NFκB by IκB kinase (IKK)-dependent and IKK-independent pathways
J Biol Chem
(2001)

Y.R. Mahida

The key role of macrophages in the immunopathogenesis of inflammatory bowel disease

Inflamm Bowel Dis

(2000)

M. Karin et al.

Phosphorylation meets ubiquitinationthe control of NF-κB activity

Annu Rev Immunol

(2000)

S. Schreiber et al.

Activation of nuclear factor kappa B inflammatory bowel disease

Gut

(1998)

M.F. Neurath et al.

Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice

Nat Med

(1996)

A.S. Baldwin

The transcription factor NFκB and human disease

J Clin Invest

(2001)

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^☆: Supported by grants from the Institut National de la Santé et de la Recherche Médicale. Y-27632 was a gift from the Institut International de Recherche Servier.

¹: J.-P. Segain and D. Raingeard de la Blétière contributed equally to this work.

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Rapid communicationRho kinase blockade prevents inflammation via nuclear factor κB inhibition: evidence in Crohn’s disease and experimental colitis☆

Abstract

Background & Aims:

Methods:

Results:

Conclusions:

Section snippets

Patients

Activation of the RhoA/Rho kinase pathway in intestinal inflammation

Discussion

Acknowledgements

Gastroenterology

FEBS Lett

Gastroenterology

J Immunol Methods

Cell

J Biol Chem

J Biol Chem

J Biol Chem

Blood

J Biol Chem

The key role of macrophages in the immunopathogenesis of inflammatory bowel disease

Inflamm Bowel Dis

Phosphorylation meets ubiquitinationthe control of NF-κB activity

Annu Rev Immunol

Activation of nuclear factor kappa B inflammatory bowel disease

Gut

Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice

Nat Med

The transcription factor NFκB and human disease

J Clin Invest

Rapid communication
Rho kinase blockade prevents inflammation via nuclear factor κB inhibition: evidence in Crohn’s disease and experimental colitis☆