The c-Abl tyrosine kinase controls protein kinase Cδ-induced Fli-1 phosphorylation in human dermal fibroblasts

Arthritis Rheum. 2011 Jun;63(6):1729-37. doi: 10.1002/art.30284.

Abstract

Objective: We have previously demonstrated that in response to transforming growth factor β (TGFβ), Fli-1 activity is repressed through a series of sequential posttranslational modifications, consisting of protein kinase Cδ (PKCδ)-induced Thr312 phosphorylation, acetylation by p300/CREB binding protein-associated factor, and detachment from the collagen promoter. The purpose of this study was to further investigate the upstream events that lead to Fli-1 phosphorylation in response to TGFβ.

Methods: Dermal fibroblasts were isolated from systemic sclerosis (SSc) patients and healthy control subjects matched for age, sex, and ethnicity. Western blotting was used to analyze protein levels and real-time quantitative reverse transcription-polymerase chain reaction analysis was used to measure messenger RNA expression. Cells were transduced with constitutively active PKCδ adenovirus or were transiently transfected with a Bcr-Abl-overexpressing plasmid. Subcellular localization of PKCδ was examined by immunocytochemistry.

Results: Western blot analysis of cell lysates demonstrated that the levels of phospho-Fli-1 (Thr312) were up-regulated in SSc fibroblasts, correlating with increased levels of type I collagen and c-Abl protein. Experiments using a constitutively activated form of c-Abl, small interfering RNA against c-Abl and the specific tyrosine kinase inhibitor imatinib, demonstrated the requirement of c-Abl for the TGFβ-induced phosphorylation of Fli-1. Additionally, we showed that c-Abl kinase activity was required for nuclear localization of PKCδ.

Conclusion: Our results demonstrate that in SSc fibroblasts, c-Abl is an upstream regulator of the profibrotic PKCδ/phospho-Fli-1 pathway, via induction of PKCδ nuclear localization. Additionally, the finding that Fli-1 is phosphorylated at higher levels in SSc fibroblasts supports the notion that the c-Abl/PKCδ/phospho-Fli-1 pathway is constitutively activated in these cells. Thus, blocking the TGFβ/c-Abl/PKCδ/phospho-Fli-1 pathway could be an attractive alternative approach to therapy for scleroderma.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzamides
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Collagen Type I / biosynthesis
  • Dermis / drug effects
  • Dermis / enzymology*
  • Female
  • Fibroblasts / drug effects
  • Fibroblasts / enzymology*
  • Humans
  • Imatinib Mesylate
  • Male
  • Microfilament Proteins / metabolism*
  • Phosphorylation
  • Piperazines / pharmacology
  • Protein Kinase C-delta / genetics
  • Protein Kinase C-delta / metabolism*
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-abl / biosynthesis
  • Proto-Oncogene Proteins c-abl / genetics
  • Proto-Oncogene Proteins c-abl / metabolism*
  • Pyrimidines / pharmacology
  • RNA, Small Interfering / pharmacology
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Scleroderma, Systemic / enzymology*
  • Scleroderma, Systemic / genetics
  • Trans-Activators
  • Transduction, Genetic
  • Transforming Growth Factor beta / pharmacology

Substances

  • Benzamides
  • Collagen Type I
  • FLII protein, human
  • Microfilament Proteins
  • Piperazines
  • Protein Kinase Inhibitors
  • Pyrimidines
  • RNA, Small Interfering
  • Receptors, Cytoplasmic and Nuclear
  • Trans-Activators
  • Transforming Growth Factor beta
  • Imatinib Mesylate
  • Proto-Oncogene Proteins c-abl
  • Protein Kinase C-delta