Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.
OBJECTIVE
In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD).


METHODS
Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration.


RESULTS
TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut.


CONCLUSIONS
Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.
Di Sabatino
A
A
First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura delle Malattie Infiammatorie Croniche Intestinali, University of Pavia, Pavia, Italy.
Jackson
C L
CL
Pickard
K M
KM
Buckley
M
M
Rovedatti
L
L
Leakey
N A B
NA
Picariello
L
L
Cazzola
P
P
Monteleone
G
G
Tonelli
F
F
Corazza
G R
GR
MacDonald
T T
TT
Pender
S L
SL
D000328:Adult; D000368:Aged; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods; D016022:Case-Control Studies; D002478:Cells, Cultured; D016922:Cellular Senescence; D003106:Colon / Q000473:pathology; D003424:Crohn Disease / Q000378:metabolism* / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism; D005355:Fibrosis; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D053508:Matrix Metalloproteinase 12 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000032:analysis / Q000096:biosynthesis*; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000502:physiology*; D051899:Smad2 Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D055815:Young Adult
C497782:SMAD2 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D053508:Matrix Metalloproteinase 12