title: Acid sphingomyelinase deficiency contributes to resistance of scleroderma fibroblasts to Fas-mediated apoptosis.
abstract: BACKGROUND
  Scleroderma (SSc) is characterized by excess production and deposition of extracellular matrix (ECM) proteins. Activated fibroblasts play a key role in fibrosis in SSc and are resistant to Fas-mediated apoptosis. Acid sphingomyelinase (ASMase), a major sphingolipid enzyme, plays an important role in the Fas-mediated apoptosis.
  
  
  OBJECTIVE
  We investigated whether dysregulation of ASMase contributes to Fas-mediated apoptosis resistance in SSc fibroblasts.
  
  
  METHODS
  Fibroblasts were isolated from SSc patients and healthy controls. Western blot was performed to analyze protein levels and quantitative real time RT-PCR was used to determine mRNA expression. Cells were transiently transfected with siRNA oligos against ASMase or transduced with adenoviruses overexpressing ASMase. Apoptosis was induced using anti-Fas antibody (1 μg/mL) and analyzed using caspase-3 antibody or Cell Death Detection ELISA.
  
  
  RESULTS
  SSc fibroblasts showed increased resistance to Fas-mediated apoptosis. ASMase expression was decreased in SSc fibroblasts and Transforming Growth Factor beta (TGFβ), the major fibrogenic cytokine involved in the pathogenesis of SSc, downregulated ASMase in normal fibroblasts. Forced expression of ASMase in SSc fibroblasts restored sensitivity of these cells to Fas-mediated apoptosis while blockade of ASMase was sufficient to induce partial resistance to Fas-induced apoptosis in normal fibroblasts. In addition, ASMase blockade decreased activity of protein phosphatase 2A (PP2A) through phosphorylation on Tyr(307) and resulted in activation of extracellular regulated kinase 1/2 (Erk1/2) and protein kinase B (Akt/PKB).
  
  
  CONCLUSIONS
  In conclusion, this study suggests that ASMase deficiency promotes apoptosis resistance and contributes to activation of profibrotic signaling in SSc fibroblasts.
authors: Arthritis Center, Division of Rheumatology, Boston University Medical Campus, Boston, MA 02118, USA.
authors: Glady Hazitha
authors: GH
authors: Samuel
authors: Stefania
authors: S
authors: Lenna
authors: Andreea M
authors: AM
authors: Bujor
authors: Robert
authors: R
authors: Lafyatis
authors: Maria
authors: M
authors: Trojanowska
mesh_terms: D017209:Apoptosis; D016207:Cytokines / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005355:Fibrosis; D006801:Humans; D058628:Myofibroblasts / Q000378:metabolism; D052536:Niemann-Pick Disease, Type A / Q000150:complications* / Q000503:physiopathology; D009841:Oligonucleotides / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014945:Wound Healing; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D016207:Cytokines; D009841:Oligonucleotides; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D019014:fas Receptor; D048049:Extracellular Signal-Regulated MAP Kinases; D054648:Protein Phosphatase 2; C543066:SMPD1 protein, human; D013108:Sphingomyelin Phosphodiesterase