Abstract
Purpose :
To identify the role of noncanonical responses to TGFβ on the early canonical response and the subsequent fibroblasts into myofibroblasts conversion (FMC).
Methods :
Fibroblasts derived from NDRI-procured corneas were cultured for 48 h in a serum-free medium, TGFβ1 (TGFβ-2n/ml) was added, and cultures were lysed 1 to 30 min later. Kinase inhibitors were added 10 min beforehand. MELK siRNA was electroporated 48 h prior to the addition of TGFβ. Western blot was used to measure p.TAK1 (S412), p.JNK1/2 (JNK;T183/185), p.ASK1 p.p38 (T180/182), p.MELK(T167/171), p.SMAD2 (S465/467), p.SMAD3(S423/425), α-SMA, and GAPDH. FMC was assessed by α-SMA immunohistology and morphology after a 48 or 72 h incubation.
Results :
The TGFβ1-induced SMAD2/3 phosphorylation or canonical response displayed a 5-6 min latency period. p.SMAD2/3 rapidly accumulated to a peak response over the next 15 min. Phosphorylation of TAK1, JNK, and p38 part of the noncanonical responses to TGFβ1, reached its peak within 1 min and remained nearly unchanged for the next 20 min. A kinase inhibitor screen revealed that p.SMAD2/3 was blocked by inhibitors of MELK (OTSP167 and MELK8A) and its siRNA. Maximal p.MELK was attained within 2 min after TGFβ1 addition. The canonical-noncanonical temporal difference was exploited to examine the relationship between the two responses. Inhibiting TAK1 (TAKinib or 5Z-7-oxozeaenol) blocked the phosphorylation of JNK, p38, MELK, and SMAD2/3. Inhibition of JNK (SP600125 or JNK VI) blocked the phosphorylation of MELK and SMAD2/3 but not of TAK1. MELK inhibition did not inhibit the phosphorylation of either TAK1 or JNK. Inhibition of p38 by 4 agents did not interfere with the p.SMAD2/3 increase. Phosphorylation of JNK and MELK via TNFα, did not induce p.SMAD2/3 or blocked the effect of TGFβ1 on the SMADs. Inhibitors of TAK1, JNK, or MELK or MELK siRNA blocked the FMC.
Conclusions :
The canonical TGFβ pathway is fully dependent on the activation sequence, TGFβ → p.TAK → p.JNK1/2 → p.MELK + TGFβ → p.SMAD2/3, challenging the notion of independent canonical and noncanonical responses. MELK, plays a central role in SMAD2/3 phosphorylation and the FMC. The molecular interaction between the TGFβ receptor, MELK, and SMAD2/3 remains to be elucidated. This finding opens the door to new ways to control corneal fibrosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.