Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement
The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.
Activation of the TGFbeta/c-Abl/PKCdelta/Fli-1 Pathway Is a Major Contributor to SSc Fibrosis
Bujor, Andreea M., Asano, Yoshihide, Hant, Faye N., Trojanowska, Maria
Scleroderma (SSc) is a connective tissue disease characterized by prominent skin fibrosis. Previous studies have linked PKCd and Fli1 to SSc pathogenesis and the phosphorylation of Fli1(Thr312) by PKCd downstream of TGFb has been shown to inhibit the repressor function of Fli1 on the collagen promoter. Despite this, the upstream signaling events underlying TGFb induced PKCd activation remain unknown. Overexpression of c-abl was reported in SSc. Furthermore, in other cells c-abl induced PKCd tyrosine phosphorylation and nuclear translocation. The aim of this study was to further define the molecular mechanisms that mediate the TGFb induced Fli1 phosphorylation in dermal fibroblasts.
Protein expression of collagen type I, c-abl and P-Fli1 (Thr312) was measured in adult SSc fibroblasts from skin biopsies and matched normal controls by western blotting (n=4). Normal dermal fibroblasts were either transduced with an adenovirus overexpressing constitutively active PKCd (CA-PKCd) or transiently transfected with a plasmid containing bcr-abl (a constitutively activated form of c-abl) in the presence or absence of TGFb and Imatinib. Western blot analysis was used to measure the effects on the protein levels of collagen type I and P-Fli1 (Thr312). Quantitative real-time PCR was performed to measure the mRNA levels of COL1A1 and COL1A2.
Data analysis showed significantly increased expression of P-Fli1 (Thr312) in cultured SSc fibroblasts compared to normal controls in all of the pairs tested. The enhanced Fli1 phosphorylation in SSc fibroblasts correlated with an increase in the protein levels of collagen type I and c-abl in these cells. Cells transduced with the CA-PKCd had a two fold increase in collagen levels. Blockade of c-abl using Imatinib was followed by a 70% down-regulation of the mRNA and protein levels of collagen type I which was consistently rescued by over-expression of CA-PKCd. The levels of P-Fli1 (Thr312) where significantly increased after 2h of TGFb addition and pretreatment with Imatinib prevented the TGFb effect on Fli1 phosphorylation. In agreement with these observations, constitutive activation of c-abl by bcr-abl over-expression was sufficient to induce the phosphorylation of Fli1 on Thr312 to levels similar to those obtained after TGFb treatment.
This study shows for the first time that Fli1 is constitutively phosphorylated on Thr312 in SSc fibroblasts in culture. Additionally we show that c-abl is required for the TGFb induced Fli1 phosphorylation in dermal fibroblasts. Our results suggest that the constitutive activation of TGFb/c-abl/PKCd/Fli1 pathway in SSc fibroblasts could contribute to fibrosis by inhibiting Fli1 induced repression of collagen promoter. Blockade of the TGFb/c-abl/PKCd/Fli1 pathway by Imatinib further clarifies the antifibrotic mechanisms of this molecule and suggests that SSc patients with constitutive activation of this pathway could benefit from Imatinib treatment.
To cite this abstract, please use the following information:
Bujor, Andreea M., Asano, Yoshihide, Hant, Faye N., Trojanowska, Maria; Activation of the TGFbeta/c-Abl/PKCdelta/Fli-1 Pathway Is a Major Contributor to SSc Fibrosis [abstract]. Arthritis Rheum 2009;60 Suppl 10 :1072