Abstract
Purpose :
In primary avian and mammalian lens epithelial cell cultures, inhibitors of either ErbBs (EGF receptor family members) or ERK prevent TGFβ from inducing myofibroblast differentiation. In this study, we examine the signaling mechanisms that link these processes.
Methods :
Western blotting and immunofluorescent microscopy were used to assess the expression of markers of lens cell differentiation in serum-free primary cultures of embryonic chick lens epithelial cells grown on laminin (DCDMLs). Activation of ErbBs, Smad3, ERK, and p38 was assessed using phospho-specific antibodies.
Results :
Inhibitors of either ERK or ErbB signaling block TGFβ from upregulating both early (fibronectin) and late (αSMA) markers of myofibroblast differentiation, but do not prevent TGFβ from enhancing C-terminal phosphorylation of Smad3. Fibrosis-inducing levels of TGFβ increase the activation of ERK in DCDMLs within 1.5 h in a process that requires active TGFβ receptors and MEK. Kinase inhibitors of ErbBs, but not of other growth factor receptors active in lens cells, reduce the phosphoERK signal to below basal levels in either the absence or presence of TGFβ. This effect is attributable to constitutive ErbB activity (most likely mediated by endogenous ErbB ligand) playing a major role in regulating the tonic levels of ERK signaling. Activation of ERK by TGFβ does not appear to be mediated by rapid generation of ErbB ligand or by otherwise increasing the level of active (e.g., tyrosine autophosphorylated) ErbB on the cell surface. Upregulation of ERK by TGFβ is, however, sensitive to anti-oxidants. Conversely, subjecting DCDMLs to reactive oxygen species (ROS) is sufficient to enhance pERK levels. Antioxidants have previously been shown to block TGFβ-induced epithelial to myofibroblast transition (EMyT) in avian and mammalian lens systems.
Conclusions :
Our data are consistent with a model in which TGFβ-generated ROS serves to indirectly amplify ERK signaling downstream of tonically active ErbBs to mediate EMyT. Taken together with reports that ErbB inhibitors reduce posterior capsule coverage in human lens capsular bags, our findings support the potential of anti-ErbB therapies to block fibrotic PCO.
This is a 2020 ARVO Annual Meeting abstract.