title: Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes in human trabecular meshwork cells.
abstract: OBJECTIVE
  The profibrotic cytokine TGFβ is associated with glaucoma and plays an important role in the regulation of extracellular matrix metabolism in the trabecular meshwork (TM). The purpose of this study was to determine whether expression of ECM cross-linking LOX genes is regulated by TGFβ in TM cells.
  
  
  METHODS
  Expression of the five LOX genes (LOX, LOXL1, LOXL2, LOXL3, and LOXL4) was examined in cultured human TM cells by using RT-PCR, quantitative RT-PCR, and Western immunoblot analysis. TM cells were treated with recombinant TGFβ1, -2, and -3, to determine the effects on LOX and LOXL1 to -4 expression. The TM cells were pretreated with TGFBR inhibitors (LY364947, SB431542), canonical Smad signaling pathway (SIS3 or Smad2, -3, and -4 siRNAs) inhibitors, or inhibitors of the non-Smad signaling pathways (SP600125, SR11302), to identify the signaling pathway(s) involved in TGFβ induction of LOX and LOXL gene and protein expression. A novel LOX activity assay was used to determine the effects of the LOX inhibitor BAPN on tropoelastin cross-linking.
  
  
  RESULTS
  All five LOX genes (LOX, LOXL1 to -4) were expressed in cultured human TM cells and were induced by all three isoforms of TGFβ. This TGFβ induction of LOX and LOXL expression was blocked by TGFβ inhibitors as well as by inhibitors of the canonical Smad2, -3, and -4 signaling and non-Smad JNK/AP-1 signaling pathways (P < 0.05).
  
  
  CONCLUSIONS
  Both Smad and non-Smad signaling pathways are involved in TGFβ-mediated LOX induction, suggesting complex regulation of these important extracellular matrix cross-linking enzymes. Increased LOX activity may be at least partially responsible for TGFβ-mediated IOP elevation and increased aqueous humor outflow resistance.
authors.affiliation: Department of Cell Biology and Anatomy, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA.
authors.forename: Anirudh
authors.initials: A
authors.lastname: Sethi
authors.forename: Weiming
authors.initials: W
authors.lastname: Mao
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wordinger
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004790:Enzyme Induction / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009994:Osmolar Concentration; D020033:Protein Isoforms / Q000494:pharmacology; D008249:Protein-Lysine 6-Oxidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000378:metabolism; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D003432:Cross-Linking Reagents; D016326:Extracellular Matrix Proteins; D007527:Isoenzymes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D051785:Smad Proteins; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D000594:Amino Acid Oxidoreductases; C445313:LOXL1 protein, human; D008249:Protein-Lysine 6-Oxidase; D048031:JNK Mitogen-Activated Protein Kinases