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.
Cetin
Selma
S
Div. of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA.
Leaphart
Cynthia L
CL
Li
Jun
J
Ischenko
Irene
I
Hayman
Michael
M
Upperman
Jeffrey
J
Zamora
Ruben
R
Watkins
Simon
S
Ford
Henri R
HR
Wang
James
J
Hackam
David J
DJ
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D002464:Cell Migration Inhibition; D020345:Enterocolitis, Necrotizing / Q000473:pathology / Q000503:physiopathology; D020895:Enterocytes / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D009603:Nitroso Compounds / Q000494:pharmacology; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000502:physiology; D051381:Rats; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology
D047908:Intracellular Signaling Peptides and Proteins; D009603:Nitroso Compounds; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009569:Nitric Oxide; D051416:Focal Adhesion Protein-Tyrosine Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C516727:Ptpn11 protein, rat; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D020742:rhoA GTP-Binding Protein