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Vasanth Rao, Kelly Buddin, Rupalatha Maddala; Calponin-3 Regulated Lens Epithelial Contractile Activity Induces Fibrogenic Activity via Yap/Taz Transcriptional Activation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3488.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the functional significance of calponin-3, a contractile and actin binding protein expressed abundantly and preferentially in the lens epithelium
Expression of different calponin isoforms (CNN1, 2 & 3) and the distribution of acidic calponin (CNN3) in mouse lens were evaluated using cDNA microarray, RNA seq, immunoblot and immunofluorescence analyses. Phosphorylation of CNN3 (Ser 296 &Thr 288) in mouse and human lenses and its regulation by TGF-beta2 in mouse lens epithelial cell cultures was studied, while the effects of siRNA-mediated CNN3 deficiency on cell-cell junctions, actin stress fibers, connective tissue growth factor (CTGF), fibronectin, α-smooth muscle actin, Yap/Taz activation and myosin light chain (MLC) phosphorylation were evaluated in mouse lens epithelial explants and cell cultures, using immunoblotting and immunofluorescence analyses.
Unlike many other tissues which express all three calponin isoforms, the lens predominantly and abundantly expresses the CNN3 isoform which distributes preferentially to cell-cell junctions and colocalizes with actin stress fibers in the lens epithelium. CNN3 is phosphorylated on Ser 296 and Thr288 in both mouse and human lenses regulated by Rho kinase and MEKK1, respectively, and is stimulated by TGF-β2 in mouse lens epithelial cells. CNN3 deficiency in mouse lens epithelial cell cultures was found to induce formation of actin stress fibers, focal adhesions, cell plasticity, activation of mechanosensing transcriptional factors, Yap/Taz and disruption of cell-cell junctions, and increased the levels of CTGF, fibronectin, αSMA and MLC phosphorylation. CNN3 deficiency in mouse lens epithelial explants disrupted E-cadherin-based cell-cell junctions, induces formation of actin stress fibers and increases levels of αSMA.
This study uncovers the importance of CNN3, an inhibitor of myosin II ATPase activity in regulation of lens epithelial actin cytoskeletal organization, contractile activity and cell adhesive interactions. Significantly, augmented cell contractile force associated with CNN3 deficiency or via TGF-β2 –mediated phosphorylation of CNN3 in the lens, leads to epithelial to mesenchymal transition, Yap/Taz transcriptional activation and increases the levels of CTGF, fibronectin and αSMA, which are expected to induce the formation of posterior capsular cataract.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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