Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate.
OBJECTIVE
Activation of Smad1 signaling has recently been implicated in the development of fibrosis. The goal of the present study was to gain further insights into activation of the Smad1 pathway in fibrosis in systemic sclerosis (SSc) and to determine whether this pathway is targeted by the antifibrotic drug imatinib mesylate.


METHODS
Levels of phosphorylated Smad1 and total Smad1 were examined in SSc and control skin biopsy samples by immunohistochemistry and in cultured fibroblasts by Western blotting. Activity of the CCN2 promoter was examined by a luciferase reporter gene assay. Interactions of Smad1 with the CCN2 promoter were examined by in vitro and in vivo DNA binding assays. Expression of the nonreceptor tyrosine kinase c-Abl and Smad1 was blocked using respective small interfering RNA.


RESULTS
Total and phosphorylated Smad1 levels were significantly elevated in SSc skin biopsy samples and in cultured SSc fibroblasts and correlated with elevated CCN2 protein and CCN2 promoter activity. DNA binding assays demonstrated that Smad1 was a direct activator of the CCN2 gene. Small interfering RNA-mediated depletion of Smad1 in SSc fibroblasts normalized the production of CCN2 and collagen. Imatinib mesylate blocked activation of the Smad1 pathway in transforming growth factor beta-stimulated control fibroblasts and reversed activation of this pathway in SSc fibroblasts. Likewise, blockade of c-Abl abrogated activation of the Smad1 pathway in SSc fibroblasts.


CONCLUSIONS
Our findings demonstrate that activation of Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. Demonstration that the Smad1/CCN2 pathway is blocked by imatinib mesylate further clarifies the mechanism of the antifibrotic effects of this compound. This study suggests that SSc patients with activated Smad1 signaling may benefit from imatinib mesylate treatment.
Pannu
Jaspreet
J
Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, SC 29425-6370, USA.
Asano
Yoshihide
Y
Nakerakanti
Sashidhar
S
Smith
Edwin
E
Jablonska
Stefania
S
Blaszczyk
Maria
M
ten Dijke
Peter
P
Trojanowska
Maria
M
D000328:Adult; D000368:Aged; D001549:Benzamides; D001706:Biopsy; D016022:Case-Control Studies; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D006801:Humans; D000068877:Imatinib Mesylate; D017874:Immediate-Early Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology* / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D011743:Pyrimidines / Q000494:pharmacology* / Q000627:therapeutic use; D012595:Scleroderma, Systemic / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D012867:Skin / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051898:Smad1 Protein / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism
D001549:Benzamides; C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C497399:SMAD1 protein, human; D051898:Smad1 Protein; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D000068877:Imatinib Mesylate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3