Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism.(Intestine)

Gut 2004;53:514-522. doi: 10.1136/gut.2003.027540

Background: L-Arginine is a nutritional supplement that may be useful for promoting intestinal repair. Arginine is metabolised by the oxidative deiminase pathway to form nitric oxide (NO) and by the arginase pathway to yield ornithine and polyamines.

Aims: To determine if arginine stimulates restitution via activation of NO synthesis and/or polyamine synthesis.

Methods: We determined the effects of arginine on cultured intestinal cell migration, NO production, polyamine levels, and activation of focal adhesion kinase, a key mediator of cell migration.

Results: Arginine increased the rate of cell migration in a dose dependent biphasic manner, and was additive with bovine serum concentrate (BSC). Arginine and an NO donor activated focal adhesion kinase (a tyrosine kinase which localises to cell matrix contacts and mediates [beta]1 integrin signalling) after wounding. Arginine stimulated cell migration was dependent on focal adhesion kinase (FAK) signalling, as demonstrated using adenovirus mediated transfection with a kinase negative mutant of FAK. Arginine stimulated migration was dependent on NO production and was blocked by NO synthase inhibitors. Arginine dependent migration required synthesis of polyamines but elevating extracellular arginine concentration above 0.4 mM did not enhance cellular polyamine levels.

Conclusions: These results showed that L-arginine stimulates cell migration through NO and FAK dependent pathways and that combination therapy with arginine and BSC may enhance intestinal restitution via separate and convergent pathways.

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After acute mucosal injury, rapid restoration of epithelial continuity depends on migration of uninjured epithelial cells to cover denuded sections of basement membrane. (1) Enterocyte migration is a mitosis independent process that is completed within 6-12 hours. Cell adhesion and migration are mediated by key cytoskeletal and signalling proteins that are organised in lamellipodial extensions. (2) At these focal adhesions, numerous proteins colocalise, including p120 focal adhesion kinase (FAK), (3) talin, alpha-actinin, vinculin, paxillin, p130Cas, and p60c-src. Several of these proteins are phosphorylated and activated by FAK. (4-8)

Arginine (ARG) has been reported to lessen intestinal damage in animal models of necrotising enterocolitis. (9-11) ARG is metabolised by two major pathways in enterocytes: conversion by arginase to ornithine, the precursor of polyamines, and conversion by nitric oxide synthase (NOS) to nitric oxide (NO) and citrulline. Polyamines are polycations that are required for normal formation of Iamellipodia and stress fibres during cell migration. NO is a lipophilic free radical that stimulates mucus secretion and water absorption, produces smooth muscle relaxation, and regulates bowel permeability. (12 13) In the current studies, we determined the effect of ARG on enterocyte migration through FAK phosphorylation after wounding of cultured cells (14) as a first step in intestinal repair. Bovine serum concentrate (BSC) was used as a positive control. BSC has been shown by our group to facilitate intestinal villous regrowth and improve bowel permeability in experimental cryptosporidial diarrhoea, (15) and serum activates ornithine decarboxylase (ODC) in intestinal cells. (16) BSC is inexpensive and approved by the United States Department of Agriculture for use as a dietary supplement (Immunolin or NutraGammax; Proliant Inc., Ames, Iowa, USA) in health food stores. BSC contains active IgG, IgM, and IgA, transforming growth factor [beta] (TGF-[beta]), and insulin-like growth factor 1 (IGF-1).

METHODS

Chemicals

Acrylamide and bisacrylamide were from National Diagnostics (Atlanta, Georgia, USA). Protease and phosphatase inhibitors (aprotinin, leupeptin, bestatin, 4-nitrophenyl phosphate, pepstatin, dithiothreitol, and NP-40) were from Boehringer Mannheim (Indianapolis, Indiana, USA). Herbimycin was from Gibco BRL (Gaithersburg, Maryland, USA). Tyrphostins (AG213 and 216) were from Professor Alex Levitski (Hebrew University, Jerusalem, Israel). BSC was obtained from Proliant Inc. (Ames, Iowa, USA). BSC powder contains approximately 80% protein, of which 60% is albumin and 25% is immunoglobulin G (IgG). The manufacturer has measured significant levels of IGF-I (6000 ng/g protein) and TGF-[beta]1 (90 ng/g) in BSC. All other chemicals, including 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (Deta-NONOate), were obtained from Sigma (St Louis, Missouri, USA).

Antibodies

Mouse monoclonal antibody IgG1 to FAK (clone 4.47) was obtained from Upstate Biotechnology (Lake Placid, New Jersey, USA). Mouse monoclonal antiphosphotyrosine (PY-20) and rabbit polyclonal anti-nitric oxide sythase II (anti-NOS II) antibodies were obtained from Transduction Laboratories (Lexington, Kentucky, USA).

Cells

We selected IPEC-J2 cells derived from newborn piglet jejunum because of their differentiated characteristics, (17) and Cdx2 transformed IEC-6 cells because of a more differentiated phenotype, including a fourfold increased rate of cell migration. (18 19) IPEC-J2 cells were obtained from H Berschneider (North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina, USA). IPEC-J2 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/F12 medium with 5% serum, split weekly, and were studied at passages 28-56. The Cdx2 transformed rat crypt cell line IEC-6 (18 19) was obtained from Dr J-Y Wang (University of Maryland, Baltimore, USA). Cdx2 transformed IEC-6 cells were cultured in DMEM with isopropyl-[beta]-D-thioguanine (4 mM), which served as the inducer for Cdx2 directed by the LacSwitch system (Stratagene, La Jolla, California, USA), for four weeks prior to the experiments and were studied at…