Endoglin negatively regulates transforming growth factor beta1-induced profibrotic responses in intestinal fibroblasts.
BACKGROUND
Fibroblasts isolated from strictures in Crohn's disease (CD) exhibit reduced responsiveness to stimulation with transforming growth factor (TGF) beta1. TGF-beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. The membrane glycoprotein, endoglin, is a negative regulator of TGF-beta1.


METHODS
Intestinal fibroblasts were cultured from seromuscular biopsies of patients undergoing intestinal resection for CD strictures or from control patients. Endoglin expression was assessed using confocal microscopy, flow cytometry and western blot. The effect of small interfering (si) RNA-mediated knockdown and plasmid-mediated overexpression of endoglin on fibroblast responsiveness to TGF-beta1 was assessed by examining smad phosphorylation, smad binding element (SBE) promoter activity, connective tissue growth factor (CTGF) expression and ability to contract collagen.


RESULTS
Crohn's stricture fibroblasts expressed increased constitutive cell-surface and whole-cell endoglin relative to control cells. Endoglin co-localized with filamentous actin. Fibroblasts treated with siRNA directed against endoglin exhibited enhanced TGF-beta1-mediated smad-3 phosphorylation, and collagen contraction. Cells transfected with an endoglin plasmid did not respond to TGF-beta1 by exhibiting SBE promoter activity or producing CTGF.


CONCLUSIONS
Fibroblasts from strictures in CD express increased constitutive endoglin. Endoglin is a negative regulator of TGF-beta1 signalling in the intestinal fibroblast, modulating smad-3 phosphorylation, SBE promoter activity, CTGF production and collagen contraction.
Burke
J P
JP
Department of Surgery, St Vincent's University Hospital, Dublin, Ireland.
Watson
R W G
RW
Mulsow
J J
JJ
Docherty
N G
NG
Coffey
J C
JC
O'Connell
P R
PR
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015703:Antigens, CD / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D002478:Cells, Cultured; D003424:Crohn Disease / Q000378:metabolism; D000071063:Endoglin; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008875:Middle Aged; D011956:Receptors, Cell Surface / Q000378:metabolism / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface; D053773:Transforming Growth Factor beta1