Abstract
Purpose: :
We have previously shown that both the EGF and TGFß signaling pathways are activated during corneal wound repair. The goal of the present study was to determine if activation of the EGF pathway affects TGFß signaling. To examine this question, the effects of EGF on expression of TGFß receptor II (TRII) and TGFß target protein p15INK4b (p15) were assayed.
Methods: :
To examine the effect of EGF on TRII in situ, 3mm debridement wounds were made in rat corneas and allowed to heal in organ culture for 18 hours in the presence or absence of the EGFR kinase inhibitor AG1478 (30µM). Frozen sections were prepared and TRII was localized using standard immunofluorescence techniques. To determine the effect of EGF on TGFß signaling in human cells, human corneal epithelial cells were isolated from limbal rims and treated for 6 hours with 1) basal medium, 2) EGF (5ng/ml), 3) TGFß1 (2ng/ml), 4) MEK inhibitor (U0126, 10µM), or 5) EGF plus MEK inhibitor. Cells were then harvested and TRII and p15 were assayed using RT–PCR and Western blotting.
Results: :
Corneas allowed to heal in the presence of AG1478 expressed TRII in fewer cells than untreated corneas. In cell culture experiments, EGF stimulated TRII mRNA expression by 4.0–fold and protein by 1.4–fold. The MEK inhibitor decreased the EGF stimulation by 4.4–fold. TGFß stimulated the expression of p15 by 3.6–fold. EGF blunted this expression by 51.6%.
Conclusions: :
EGF appears to have a dual affect on TGFß signaling in that it both stimulates the expression of TRII, which is necessary for propagation of signaling, and attenuates the expression of the target protein p15. This dual regulation most likely is under temporal and spatial control.
Keywords: growth factors/growth factor receptors • cornea: epithelium • signal transduction