title: Regulation of SPARC by transforming growth factor β2 in human trabecular meshwork.
abstract: OBJECTIVE
  An increased aqueous level of TGF-β2 has been found in many primary open-angle glaucoma patients. Secreted Protein, Acidic, and Rich in Cysteine (SPARC)-null mice have a lower intraocular pressure. The mechanistic relationship between SPARC and TGF-β2 in trabecular meshwork (TM) is unknown. We hypothesized that TGF-β2 upregulates SPARC expression in TM.
  
  
  METHODS
  Cultured TM cells were incubated with selective inhibitors for p38 MAP kinase (p38), Smad3, p42, JNK, RhoA, PI3K, or TGF-β2 receptor for 2 hours, and then TGF-β2 was added for 24 hours in serum-free media. Quantitative polymerase chain reaction (qPCR) and immunoblot analysis were performed. Immunofluorescent microscopy was used to determine nuclear translocation of signaling proteins. Ad5.hSPARC and Lentiviral shRNA for p38 and Smad3 were constructed, and infected human TM cells.
  
  
  RESULTS
  SPARC was upregulated by TGF-β2 in the human TM cells (3.8 ± 1.7-fold, n = 6, P = 0.01 for protein and 7.1 ± 3.7-fold, n = 6, P = 0.01 for mRNA), while upregulation of SPARC had no effect on TGF-β2. TGF-β2-induced SPARC expression was suppressed by inhibitors against p38 (-40.3 ± 20.9%, n = 10, P = 0.0001), Smad3 (-56.2 ± 18.9%, n = 10, P = 0.0001), JNK (-49.1 ± 24.6%, n = 10, P = 0.0001), and TGF-β2 receptor (-83.6 ± 14.4%, n = 6, P = 0.003). Phosphorylation and translocation of Smad3, p38, and MAPKAPK2 were detected at 30 minutes and 1 hour, respectively, following TGF-β2 treatment. Phosphorylation of JNK and c-jun was detected before TGF-β2 treatment. SPARC was suppressed 31 ± 13% (n = 5, P < 0.0001) by shRNA-p38 and 41 ± 3% (n = 5, P < 0.0001) by shRNA-Smad3.
  
  
  CONCLUSIONS
  TGF-β2 upregulates SPARC expression in human TM through Smad-dependent (Smad2/3) or -independent (p38) signaling pathways. SPARC may be a downstream regulatory node of TGF-β2-mediated IOP elevation.
authors.affiliation: Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Min Hyung
authors.initials: MH
authors.lastname: Kang
authors.forename: Dong-Jin
authors.initials: DJ
authors.lastname: Oh
authors.forename: Ja-heon
authors.initials: JH
authors.lastname: Kang
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Rhee
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D002648:Child; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D020935:MAP Kinase Signaling System / Q000502:physiology; D008875:Middle Aged; D015676:Osteonectin; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D004791:Enzyme Inhibitors; D015676:Osteonectin; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C577241:SPARC protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D053781:Transforming Growth Factor beta2; D025521:Tumor Suppressor Proteins