Abstract
Purpose:
To elucidate effects of p38 inhibitor on TGF-β2 signal in trabecular meshwork (TM) cells, and to compare the effects with ROCK inhibitor.
Methods:
Cultured human TM cells, with or without pretreatment with the 10 μM p38 inhibitor SB203580, were stimulated with 2.5 ng/ml TGF-β2. The mRNA levels were evaluated by real-time reverse transcription-polymerase chain reaction. The protein expression level and its phosphorylation ratio were detected by Western blot analysis. Transcriptional activities were estimated by luciferase reporter assays. Nuclear translocation of Smad2/3 was examined by immunofluorescence microscopy. For ROCK inhibition, 10 μM Y-27632 was used. Rho activation was assessed using a Rho-GTPase pull-down assay.
Results:
Treatment with TGF-β2 increased promoter activity, mRNA synthesis, and protein expression of COL1A2, and these effects were significantly inhibited by SB203580. SB203580 also partly inhibited phosphorylation and nuclear translocation of Smad2/3 (P <0.05). Effects of Y-27632 on the expression of COL1A2 and activation of Smad2/3 were limited. On the other hand, TGF-β2 also increased RhoA activity, polymerized actin, and phosphorylation of myosin light chain 2 (MLC2). Those effects on actin cytoskeleton were diminished by Y-27632, while SB27632 did not show significant effects on actin regulation.
Conclusions:
Our results indicated that p38 contributed TGF-β2 induced collagen production via Smad2/3 activation, while ROCK is involved in TGF-β2 induced actin polymerization independently of Smad2/3 activation in TM cells.
Keywords: 735 trabecular meshwork •
519 extracellular matrix •
543 growth factors/growth factor receptors