SMAD2/3 activation and myofibroblast conversion of corneal fibroblasts is fully dependent on the activation of MELK by the noncanonical TGFβ1 pathway

Abstract

AbstractPurpose: To identify the role of noncanonical responses to TGFβ on the early canonical response and the subsequent fibroblasts into myofibroblasts conversion (FMC).Methods: Human corneal fibroblasts were seeded in a serum‐free medium for 48 h. TGFβ1 (TGFβ‐2 n/ml) was added and cultures were lysed 1–15 min later. Kinase inhibitors were added 10 min beforehand. Identical amounts of soluble protein were then used for Western blot, to measure the level of expression of p.SMAD2 (S465/467), p.SMAD3(S423/425), p.TAK1 (S412), p.JNK1/2 (JNK;T183/185), p.p38 (T180/182), p.MELK(T167/171), α‐SMA, and GAPDH. MELK esiRNA was electroporated 48 h prior to the addition of TGFβ. FMC was assessed by α‐SMA immunohistology and morphology.Results: In the corneal fibroblasts, the TGFβ‐induced SMAD2/3 phosphorylation (canonical response), displayed a 6–8 min latency period that was followed by rapid, p.SMAD2/3 accumulation to peak response within 10 min. Overt de novo expression of α‐SMA and morphological FMC was observed after 48 h. Inhibition of MELK, a leucine zipper kinase, by OTSS167 or MELK8A, or its downregulation by esiRNA, blocked both responses. In contrast to the delayed canonical response, phosphorylation of TAK1 (noncanonical response), reached its peak within 1 min, as did JNK and p38, TAK1 downstream targets in the mitogen‐activated kinase cascade. The temporal difference was exploited to examine the relationship between the two responses. Inhibition of TAK1 by TAKinib or 5Z‐7‐oxozeaenol blocked the phosphorylation of JNK, p38, MELK, and SMAD2/3 and the FMC. In turn, inhibition of JNK by SP600125 blocked the phosphorylation of MELK and SMAD2/3; p38 inhibition had no effect. MELK inhibition did not inhibit the phosphorylation of either TAK1 or JNK. Finally, phosphorylation of JNK and MELK via TNFα or LPS, did not induce p.SMAD2/3 or blocked the effect of TGFβ on SMAD. These results are consistent with the event sequence, TGFβ → p.TAK → p.JNK1/2 → p.MELK + TGFβ → p.SMAD2/3.Conclusions: SMAD2/3 activation is fully dependent on TAK1 activity, challenging the notion of separated canonical ‐ noncanonical responses. MELK, a previously unknown component of the response to TGFβ, plays a central role in SMAD phosphorylation. The molecular interaction between the TGFβ receptor, MELK, and SMAD2/3 remains to be elucidated.