Transforming Growth Factor-β Induces Extracellular Matrix Protein Cross-Linking Lysyl Oxidase (LOX) Genes in Human Trabecular Meshwork Cells

PURPOSE. The profibrotic cytokine TGF beta 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 TGF beta 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 TGF beta 1, -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 TGF beta 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 TGF beta. This TGF beta induction of LOX and LOXL expression was blocked by TGF beta 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 TGF beta-mediated LOX induction, suggesting complex regulation of these important extracellular matrix cross-linking enzymes. Increased LOX activity may be at least partially responsible for TGF beta-mediated IOP elevation and increased aqueous humor outflow resistance. (Invest Ophthalmol Vis Sci. 2011; 52:5240-5250) DOI: 10.1167/iovs.11-7287