Abstract
Purpose :
Posterior capsular opacification and anterior subcapsular cataract are attributed to transforming growth factor-β (TGFβ) induced epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs). Previous studies from our lab have identified matrix metalloproteinase-9 (MMP9) as a potential regulator of cytoskeletal reorganization during EMT of LECs. Here, we studied the effect of MMP9 inhibition on TGFβ activation of proteins involved in actin reorganization and cell migration.
Methods :
A NanoString gene expression analysis was performed on LEC explants from 1.5-2 month-old wild-type (WT) mice, transgenic mice overexpressing TGFβ in the lens (TGtg) or TGtg mice on a MMP9 knockout background (TGtg:MMP9KO). A cytoskeletal protein array was conducted using similar samples as above. For validation, rat LEC explants were obtained from 17-19 days old Wistar rat pups, and a novel MMP9-specific inhibitor, JNJ0966, was used. These explants were either treated with <5% dimethyl sulfoxide (RatD), 6ng TGFβ (RatTG), 20μM JNJ0966 (RatJNJ) for 48 hrs or pretreated with 20μM JNJ0966 for 2 hours followed by 6ng TGFβ for 48 hours (RatTG:JNJ). Immunofluorescence and Western Blot analyses were performed using antibodies specific to proteins identified from the protein array and imaged for analysis.
Results :
Gene expression analyses revealed significant decreases in alpha-smooth muscle actin (αSMA)(p<0.01) and F-actin formation (p<0.01) in TGtg:MMP9KO LECs when compared to TGtg LECs. Furthermore, the protein array identified fold changes of 0.41 for cortactin, 0.93 for phosphorylated lim-kinase 1 (LIMK1) and 0.64 for phosphorylated focal adhesion kinase (FAK) in TGtg:MMP9KO LECs when compared to TGtg LECs. RatTG:JNJ explants showed cortical staining of E-cadherin and minimal staining of αSMA polymerization when compared to RatTG explants. Immunofluorescence and Western Blot analyses further revealed a marked decrease in phosphorylated FAK (Y397) and LIMK1 (T508) in RatTG:JNJ explants when compared to RatTG explants.
Conclusions :
The protein array and its validation indicate that MMP9 deficiency results in lowered levels of activated LIMK1 and FAK, proteins known to be involved in TGFβ-induced actin polymerization and cell migration. Further analyses should be performed to understand the mechanism by which MMP9 regulates the activity of these proteins during TGFβ-induced EMT.
This is a 2021 ARVO Annual Meeting abstract.