Transgelin as a regulator of myofibroblast differentiation in fibrosis
Date
2023
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Cellular differentiation into highly contractile myofibroblasts is a major underlying cause of morbidity in fibrotic diseases. The cells responsible for fibrosis progression are myofibroblasts. Filamentous (F-)actin reorganization into stress fibers and incorporation of alpha smooth muscle actin (αSMA) into stress fibers play a critical role in myofibroblast differentiation. Understanding F-actin reorganization regulation could aid in the development of therapeutic interventions against fibrosis. Epithelial to mesenchymal transition (EMT) is a form of fibrosis in which epithelial cells transdifferentiate into myofibroblasts. Transgelin (TAGLN), an actin associated molecule, is highly upregulated early EMT. It is speculated that TAGLN is required to cross-link F-actin for stress fibers formation. The early upregulation prior to αSMA may suggest a potential role for TAGLN in regulating the incorporation of αSMA and thus regulating myofibroblast differentiation. This led us to test the hypotheses that TAGLN is required for the differentiation of epithelial cells to myofibroblasts by promoting (1) stress fibers formation and (2) the incorporation of αsma into stress fibers. ☐ We developed an ex vivo whole organ model system to study lens EMT by culturing whole isolated lenses in medium 199. Using this model, we determined molecular expression changes that mimicked in vivo lens EMT including an upregulation of TAGLN protein (at 1day) that preceded αSMA (4day). We determined that Tagln mRNA levels were up as early as 6 hours of lens culture. Concomitant with early changes in Tagln mRNA, is F-actin reorganization which occurs as early as 4 hours. Thus, we investigated if TAGLN is required for stress fiber formation. We determined that lenses from TAGLN(-/-) mice suppresses, but does not completely prevent, of stress fiber formation. ☐ Next, we used an in vitro model of fibrosis to further determine the association of TAGLN with stress fibers and αSMA. We exposed cultured immortalized mouse lens epithelial cells(imLECs) to TGFꞵ2 resulted in co-upregulation of TAGLN and αSMA mRNA and protein levels. Using fluorescence confocal microscopy, we found that all cells organized F-actin into stress fibers, and out of these cells 80% do not express TAGLN. However, we determined that all the cells that incorporated αSMA express TAGLN. Exposure of imLECs to TGFꞵ2 further increases the proportion of cells that have TAGLN associated with stress fibers and that have incorporated αSMA. Knockdown of was able to inhibit αSMA incorporation. ☐ Taken together, our data suggests that stress fiber formation is not necessarily dependent on TAGLN, but that αSMA incorporation may be dependent on TAGLN. Understanding TAGLN’s role in myofibroblast differentiation will provide insight if targeting TAGLN could be a means of preventing the transition of cells into myofibroblasts during fibrosis.
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Keywords
Alpha smooth muscle actin, Cataracts, Epithelial to mesenchymal transition, Fibrosis, Myofibroblasts, Transgelin