Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes in human trabecular meshwork cells

• Published in: Investigative ophthalmology & visual science Vol. 52; no. 8; pp. 5240 - 5250
• Main Authors: Sethi, Anirudh, Mao, Weiming, Wordinger, Robert J, Clark, Abbot F
• Format: Journal Article
• Language: English
• Published: United States Association for Research in Vision and Ophthalmology, Inc 15.07.2011
• Subjects: Amino Acid Oxidoreductases - metabolism; Cells, Cultured; Cross-Linking Reagents - metabolism; Enzyme Induction - physiology; Extracellular Matrix Proteins - metabolism; Humans; Isoenzymes - metabolism; JNK Mitogen-Activated Protein Kinases - metabolism; Osmolar Concentration; Protein Isoforms - pharmacology; Protein-Lysine 6-Oxidase - metabolism; Recombinant Proteins - pharmacology; Signal Transduction - physiology; Smad Proteins - metabolism; Time Factors; Trabecular Meshwork - cytology; Trabecular Meshwork - metabolism; Transcription Factor AP-1 - metabolism; Transforming Growth Factor beta - pharmacology
• ISSN: 1552-5783; 0146-0404; 1552-5783
• DOI: 10.1167/iovs.11-7287

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. 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. 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). 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.