Purpose.: 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.

Purpose.: The bone morphogenic protein (BMP) antagonist gremlin is elevated in glaucomatous trabecular meshwork (TM) cells and tissues and elevates intraocular pressure (IOP). Gremlin also blocks BMP4 inhibition of transforming growth factor (TGF)-β2 induction of TM extracellular matrix (ECM) proteins. The purpose of this study was to determine whether Gremlin regulates ECM proteins in cultured human TM cells. Methods.: Human TM cells were treated with recombinant gremlin to determine the effects on ECM gene and protein expression. Expression of the ECM genes FN, COL1, PAI1, and ELN was examined in cultured human TM cells by quantitative RT-PCR and Western immunoblot analysis. TM cells were pretreated with TGFBR inhibitors (LY364947, SB431542 or TGFBR1/TGFB2 siRNAs), inhibitors of the Smad signaling pathway (SIS3 or Smad2/3/4 siRNAs), or CTGF siRNA to identify the signaling pathway(s) involved in gremlin induction of ECM gene and protein expression. Results.: All ECM genes analyzed (FN, COL1, PAI1, and ELN) were induced by gremlin. This gremlin induction of ECM genes and protein expression was blocked by inhibitors of TGFBR and the canonical Smad2/3/4 and CTGF signaling pathways. Conclusions.: Gremlin employs canonical TGFβ2/Smad signaling to induce ECM genes and proteins in cultured human TM cells. Gremlin also induces both TGFβ2 and CTGF, which can act downstream to mediate some of these ECM changes in TM cells.