title: The c-Abl tyrosine kinase controls protein kinase Cδ-induced Fli-1 phosphorylation in human dermal fibroblasts.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Boston University School of Medicine, Arthritis Center-Rheumatology, Boston, Massachusetts 02118, USA.
authors.forename: Andreea M
authors.initials: AM
authors.lastname: Bujor
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Asano
authors.forename: Paul
authors.initials: P
authors.lastname: Haines
authors.forename: Robert
authors.initials: R
authors.lastname: Lafyatis
authors.forename: Maria
authors.initials: M
authors.lastname: Trojanowska
mesh_terms: D001549:Benzamides; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D020405:Dermis / Q000187:drug effects / Q000201:enzymology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D000068877:Imatinib Mesylate; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016315:Proto-Oncogene Proteins c-abl / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000201:enzymology* / Q000235:genetics; D015534:Trans-Activators; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D001549:Benzamides; D024042:Collagen Type I; C098179:FLII protein, human; D008840:Microfilament Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D000068877:Imatinib Mesylate; D016315:Proto-Oncogene Proteins c-abl; D051745:Protein Kinase C-delta