Pre-activated p38 inhibits phosphorylation of SMAD2 by TGFβ1-activated TGFβR in corneal fibroblasts

Abstract

Purpose : TGFβ1/TGFβR-induced corneal myofibroblast genesis is mediated by the positive feedback loop (TGFβR ->> p-SMAD2↑ ->> ROS↑->>TRPV1↑ ->> Ca²⁺↑->> p-p38↑->> p-SMAD2↑->>..), where inhibition of p-p38, ROS, TRPV1 or Ca²⁺ influx markedly reduces SMAD2 phosphorylation (PLOS1, 2013: e77300). Accordingly, the original purpose of these studies was to identify signal transduction components of this self-reinforcing loop.

Methods : Human cornea fibroblasts (HCFs, 4^th or 5^th generation) seeded in a FBS-free fibroblast medium for 24h were incubated with inhibitors (Inhb.) using 10-50 x of the reported Ki. After 15 min, 1 ng/ml TFGβ1 was added and the changes in p-SMAD2 and p-p38 30 min later were determined by Western blot. Electroporation of siRNAs was used to attain > 70 % reduction of [protein] prior to TGFβ1 addition.

Results : The unperturbed HFCs showed nil p-SMAD2 and a low p-p38 level. Addition of TGFβ1 caused the de novo accumulation of p-SMAD2 (n =3) and a 4.7-fold p-p38 increase (n=3). Inhibition of maternal embryonic leucine zipper kinase, or MELK (100 nM OTSSP168), phospholipase D (1 µM FIPI), PI3K (200 nM Wortmannin), PI3Kα and β (50 nM PI3K Inhb. #2 or 50 nM ), or mitochondrial ROS (500 µM MitoTempo) did not induce p-SMAD2 but caused a 9 to12 fold p-p38 increase (n = 2, see Figure), Unexpectedly, the addition of TGFβ after this p38 activation failed to activate SMAD2 or modify the elevated p-p38. Dose-response studies showed that the degree of inhb.-induced increase in p-p38 was directly related to the inhibition of TGFβ generated SMAD2 activation. The effect of downregulation of MELK by its siRNAs confirmed that MELK was the OTSSP168 target. The inhibition of PKC (GF109203X), Ca2+/calmodulin-dependent kinase II (KN62), phospholipase C (U73122), 3- p-inositide-dependent protein kinase (GSK 2334470), did not modify the unperturbed or post-TGFβ1 pSMAD2 and p-p38 HCF values.

Conclusions : Presently unknown mechanisms result in p38 activation when MELK, PLD, PK3I and ROS are inhibited in HFCs. Directly or as an epi-phenomenon, this p38 activation is associated with inhibition of the canonical TGFβ response. This effect maybe provide novel venues to modulate the fibrosis.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Effect of kinase and mitochondrial ROS inhibitors on p-p38 and p-SMAD2 in human corneal fibroblasts