Smurf1 modulates Smad signaling pathway in fibrotic cataract formation [cell line]
Purpose: TGF-ß/BMP signaling pathway has a significant role in fibrotic cataract. Smurf1, a ubiquitin protein ligase, regulates the TGF-ß/BMP signaling pathway through the ubiquitin-proteasome system (UPS). This study aims to investigate the role of Smurf1 in the progression of fibrotic cataract and its underlying mechanism.Method: We employed a mouse injury-induced anterior subcapsular cataract (ASC) model and administered Smurf1 inhibitor A01 through anterior chamber injection for in vivo investigation. RNA sequencing was performed to examine global gene expression changes. The volume of the subcapsular opacity was determined using whole-mount immunofluorescence of lens anterior capsules. Lentivirus was utilized to create cell lines with Smurf1 knockdown or overexpression in SRA01/04. Protein levels were assessed by Simple Western. Lens epithelial cell (LEC) proliferation was evaluated by CCK8 and EdU assays. LEC migration was measured by Transwell and wound healing assays.Results: The mRNA levels of genes associated with cell proliferation, migration, epithelial-mesenchymal transition (EMT), TGF-ß/BMP pathway and UPS, including Smurf1, were upregulated in ASC model. The mRNA expression of Smurf1 was also upregulated in anterior lens capsules of age-related cataract patients. Anterior chamber injection of A01 inhibited ASC formation and EMT. In vitro knockdown of Smurf1 resulted in reduced proliferation, TGF-ß2-induced migration and EMT of LECs. Smurf1 inhibition upregulated Smad1, Smad5 and pSmad1/5. Conversely, Smurf1 overexpression displayed the opposite phenotypes.Conclusion: Smurf1 regulated the progression of fibrotic cataract by influencing the proliferation, migration and EMT of LECs through regulation of Smad signaling pathway, offering a novel target for the treatment of fibrotic cataract. Overall design: To investigate the function of Smurf1 in the regulation of the biological behavior of lens epithelial cells, we established SRA01/04 cell lines in which Smurf1 has been knocked down  by shRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq of Smurf1 knockdown and negative control cell lines treated with TGF-ß2  for 48 hours.Each group has four biological replicates.