Abstract
Abstract: :
Purpose: It has been observed in non–ocular systems that EGF antagonizes TGFß signaling. In addition, we have observed that the expression of p15INK4B, a cell cycle inhibitor, is upregulated in corneal epithelial cells by TGFß, but blunted by EGF. Since Smad is a major pathway in TGFß signaling, we examined the effects of TGFß and EGF on Smad 2 and 4–activation (translocation). Methods: Primary human corneal epithelial cells were plated in four–well chamber slides, grown to approximately 70% confluence, and then serum starved overnight. Fresh media containing either EGF (0.5 or 5 ng/ml) or no growth factor was applied to the cells for 15 minutes. After 15 minutes, the media was removed and fresh media containing either EGF (0.5 or 5 ng/ml), TGF–ß1, –ß2, or –ß3 (0.2 or 2 ng/ml), a combination of EGF and TGFß, or no growth factors (control) was applied for 30 minutes. The media was then removed, cells were fixed with methanol, and indirect immunofluorescence was performed with antibodies against Smad 2 and Smad 4. Results: Upon stimulating the cells with TGF–ß1, –ß2 or –ß3 only, Smads 2 and 4 were activated; however, upon EGF stimulation, neither Smad translocated. When TGF–ß1 was combined with EGF, both Smad 2 and 4 translocated into the nucleus. This was also observed when TGF–ß2 or –ß3 high concentration was combined with either EGF concentration. However, when TGF–ß2 or –ß3 low concentration was combined with either EGF concentration, the amount of translocation of both Smad 2 and 4 was greatly diminished. Conclusions: EGF does appear to blunt TGFß signaling. Surprisingly, there appears to be more of an effect on TGF–ß2 and TGF–ß3 than there is on TGF–ß1. We hypothesize that relative levels of EGF and TGFß isoforms released in corneal wounds may influence the wound response.
Keywords: growth factors/growth factor receptors • cornea: epithelium • cornea: basic science