Comparative effects of TGF-β2/Smad2 and TGF-β2/Smad3 signaling pathways on proliferation, migration, and extracellular matrix production in a human lens cell line

The signaling pathway of transforming growth factor β2 (TGF-β2)/Smad plays an important role in the pathological process in posterior capsule opacification (PCO) after cataract surgery. Smad2 and Smad3 are both receptor-regulated Smads (R-Smads) of the TGF-β2 signaling pathway. We aim to find which among Smad2, Smad3, and Smad2&3 plays a key role in PCO pathology. The signal characteristics of TGF-β2 and Smad proteins in the human lens cell line HLE-B3 were investigated. Smad2, Smad3, or Smad2&3 were silenced using small interfering RNA. We then tested cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell growth curve assays, migration by transwell and wound-healing assays, and extracellular matrix production including α-smooth muscle actin (αSMA), fibronectin, and type I collagen by real-time PCR assay, with and without TGF-β2 exposure. Silencing Smad3 blocked the effect of TGF-β2 on cell proliferation and production of fibronectin and type I collagen. Silencing Smad2 blocked the effect of TGF-β2 on cell migration and production of αSMA. Smad2 depletion enhanced Smad3 activity in cell proliferation and ECM production, whereas Smad3 depletion enhanced Smad2 activity in migration and αSMA expression. Silencing Smad2 and Smad3 efficiently blocked the effect of TGF-β2on cell proliferation, migration, and extracellular matrix production. Smad2 and Smad3 are both key in the TGF-β2 signaling pathway. We can prevent the development of PCO following cataract surgery by blocking the TGF-β2/Smad2&3 signaling pathway.

Research highlights
► Smad2 mediates the effect of TGF-β2 on cell migration and production of αSMA.
► Smad3 mediates TGF-β2’s effect on cell proliferation and production of ECM.
► Smad2 depletion enhances Smad3 activity, and vice versa.
► Silencing Smad2 and Smad3 efficiently blocks the effect of TGF-β2.