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Miyuki Mochita Inoue, Toshihiro Inoue, Tomokazu Fujimoto, Nanako Awai-Kasaoka, Hidenobu Tanihara; Effects of p38 inhibitor on TGF-β2 signal in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5691.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate effects of p38 inhibitor on TGF-β2 signal in trabecular meshwork (TM) cells, and to compare the effects with ROCK inhibitor.
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.
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.
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.
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