Tpm3.1 association with actin stress fibers is required for lens epithelial to mesenchymal transition

Abstract

PurposeEpithelial to mesenchymal transition (EMT) is a cause of anterior and posterior subcapsular cataracts. Central to EMT is the formation of actin stress fibers. Targeting specific, stress fiber associated tropomyosin in epithelial cells may be a means to prevent stress fiber formation and repress lens EMT.\n\nMethodsWe identified Tpm isoforms in mouse immortalized lens epithelial cells and isolated whole lenses by semi-quantitative PCR followed Sanger sequencing. We focused on the role of one particular tropomyosin isoform, Tpm3.1, in EMT. To stimulate EMT, we cultured cells or native lenses in TGF{beta}2. To test the function of Tpm3.1, we exposed cells or whole lenses to a Tpm3.1-specific chemical inhibitor, TR100, as well as investigated lenses from Tpm3.1 knockout mice. We examined stress fiber formation by confocal microscopy and assessed EMT progression by sma mRNA (qPCR) and protein (WES immunoassay) analysis.\n\nResultsLens epithelial cells express eight tropomyosin isoforms. Cell culture studies showed that TGF{beta}2 treatment results in an upregulation of Tpm3.1, which associates with actin in stress fibers. TR100 prevents stress fiber formation and reduces sma in TGF{beta}2 treated cells. We confirmed the role of Tpm3.1 in lens epithelial cells in the native lens ex vivo. Culture of whole lenses in the presence of TGF{beta}2 results in stress fiber formation at the basal regions of epithelial cells. Knockout of Tpm3.1 or treatment of lenses with TR100 prevents basal stress fiber formation and reduces epithelial sma levels.\n\nConclusionTargeting specific stress fiber associated tropomyosin isoform, Tpm3.1, is a means to represses lens EMT.