The role of ROS levels on the activation of SMAD2 by TGFβ1 in corneal fibroblasts

Abstract

AbstractPurposesTGF‐1/TGFβR‐induced corneal myofibroblast genesis is mediated by the positive feedback loop (TGFβR ‐>> p‐SMAD2↑ ‐>> ROS↑‐>>TRPV1↑ ‐>> Ca↑‐>> p‐p38↑‐>> p‐SMAD2↑‐>ROS..‐> p‐SMAD2↑). Inhibition of pp38, ROS, TRPV1, Ca influx or N‐acetyl cysteine (NAC; 1 µM), a ROS scavenger, reduces SMAD2 phosphorylation (PLOS1, 2013: e77300). Our studies seek to identify signal transduction components of the loop.Methods4th generation cultures of fibroblasts derived from donor cadaver corneas (HCF) seeded in a FBS‐free fibroblast medium for 24h were incubated with inhibitors (15 min at 10‐50 x of inhibitor Kd) and the changes in p‐SMAD2 and p‐p38 upon addition of TGFβ1 (2 ng/ml for 5‐60 min) were determined by Western blot. Untreated HFCs showed nil p‐SMAD2 and a low p‐p38 level.ResultsAddition of TGFβ1 to uninhibited cells caused a 4.7‐fold p‐p38 increase (n=3) and the de novo accumulation of p‐SMAD2. Inhibition of maternal embryonic leucine zipper kinase (MELK) by 100 nM OTSSP168, phospholipase D by 1 µM FIPI, PI3Kα by 50 nM PI3K Inhibitor #2, or the superoxide conversion to H2O2 in the mitochondria by 500 µM Mito‐Tempo resulted in > 5‐fold increase in p‐p38 and pronounced or full inhibition of the de novo SMAD2 activation by TGFβ1. The increase in p‐p38 and inhibition of SMAD2 activation was also elicited by incubation with the cellular oxidant p‐Nitrophenyldisulfide (NPDS; 10 nM). Next, we treated the cells with NAC, 0‐500 nM NPDS and TGFβ. As the [NPDS] was increased, the inhibitory effect of NAC on both [p‐p38] and [p‐SMAD2] was decreased and canceled at [NPDS] = 15 nM. Further increases in [NPDS] thought, elevated [p‐p38] but inhibited SMAD2 activation by TGFβ1.ConclusionFibroblast activation by TGFβ1/TGFβR requires a very precise ROS level. The inhibitory effect of some signal transduction pathways may occur indirectly through their effect of the redox cellular status. Inflammations or infections that increases ROS may delay the wound healing response.