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Scott Bowman, Anna Korol, Madhuja Gupta, Judith West-Mays; Smad3 and MRTF regulation of TGFβ−induced EMT in mouse lens explants. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2957.
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Epithelial-to-mesenchymal transition (EMT) is a pathological process leading to the formation of posterior capsular opacification (PCO). The cytokine TGFβ is known to induce EMT in the lens and previous studies from our lab have shown that in rat lens explants myocardin-related transcription factor A (MRTF-A) acts downstream of TGFβ. Smad3 is another known mediator of TGFβ−induced EMT in the lens. Using the ex vivo lens explant model, the following study examines the relationship between Smad3 and MRTF-A in the mouse lens.
Anterior lens capsules, with epithelia attached, were isolated from wild-type and Smad3 KO mice and maintained in culture. Confluent LEC explants were then stimulated with 500 pg/ml of recombinant TGFβ for timepoints ranging from 15 to 72 hours (hrs), and then fixed and immunostained for alpha-smooth muscle actin (αSMA) and MRTF-A.
Following treatment with TGFβ for 15 hrs (and all subsequent time points) lens epithelia from wild-type mouse explants transformed from a tightly packed cuboidal morphology into an elongated ‘spindle-shaped’ morphology, expressing αSMA. In comparison, Smad3 KO epithelial explants were found to transform and express αSMA at a much later time (72hrs), following treatment with TGFβ. MRTF-A immunolocalization revealed that it was predominantly nuclear in both wild-type and Smad3 KO mice and this did not change upon treatment with TGFβ. This is in contrast to findings for the rat in which MRTF-A localization was found to change from primarily cytoplasmic in untreated explants to nuclear in explants following TGFβ treatment.
The current findings indicate that TGFβ-induced EMT in Smad3 KO mouse explants is delayed and reduced compared to those of wild-type littermates, suggesting a time-dependent role for Smad3 in EMT in the lens. In other systems, MRTF-A has been shown to interact with Smad3 to control EMT. We did not observe any change in MRTF-A subcellular localization following TGFβ treatment, indicating that it may not be involved in TGFβ-induced EMT in the mouse lens. Further studies will be carried out to examine this further and any potential interaction with Smad3.
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