Research

Nitric Oxide Inhibits Enterocyte Migration Through Activation of RhoA-GTPase in a SHP-2-dependent Manner

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved.
 
The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC.
 
Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.
 
 
NO increases stress fiber formation in enterocytes in a RhoA-dependent manner. IEC-6 cells were transiently transfected with cDNA encoding hemagglutinin-tagged constructs encoding either wild-type (WT-) RhoA, dominant negative (DN-) RhoA, DN-RhoB, or DN-RhoC. Cells were then either left untreated (A and B) or were treated with detaNONOate (250 _M) for 14 h (C–J), then were immunostained with antibodies against hemagglutinin and rhodamine phalloidin and were examined by confocal microscopy such that the distribution of F-actin appears in red (phalloidin; A, C, E, G, and I) and the hemagglutinin stain identifying transfected cells appears in green (anti-HA; B, D, F, H, and J). Arrows identify the formation of stress fibers in NO-treated cells transfected with WT-RhoA, DN-RhoB, and DN-RhoC and indicate the lack of stress fibers in untreated cells and those transfected with DN-RhoA. There is no effect of inhibition of RhoB or RhoC on NO-induced stress fiber formation. Bar _ 10 _m. Representative images are from 3 individual experiments with over 50 transfected cells per experiment.

Authors
Cetin S, Leaphart CL, Li J, Ischenko I, Hayman M, Upperman J, Zamora R, Watkins S, Ford HR, Wang J, Hackam DJ
Am J Physiol Gastrointest Liver Physiol
2007 May
 
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Last Update
January 22, 2011
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Last Update
January 22, 2011
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