Abstract
Abstract: :
Purpose: TGFß1 has been observed to transduce its signal through Smad proteins as well as by activating the p38 pathway in non–corneal tissues. We have previously observed that TGFß1 stimulates the translocation of Smad proteins in primary human corneal epithelium, and in the current investigation, we examined if TGFß1 also stimulates the p38 pathway. Methods: Primary human corneal epithelial cells were cultured in T75 flasks in Keratinocyte–SFM with EGF and pituitary extract until they reached ∼70% confluence. Upon reaching 70% confluence, the cells were rinsed with PBS and cultured overnight in serum–free media (Keratinocyte–SFM without EGF and pituitary extract = basic medium). The next day, the cells were incubated with basic medium plus 2ng/ml of TGFß1 for 5, 10, 15, or 30 minutes, 1, 2, 4, 8, or 24 hours. Cells in basic medium only served as a control. After the appropriate time, the cells were harvested and lysed for western blotting. Equal amounts of protein per sample were loaded onto a 12% SDS–PAGE, electrophoresed and transferred to a membrane. Western blotting was performed with anti–p38 and anti–phospho(p)–p38. Results: After treating primary human corneal epithelial cells with TGFß1, the levels of p38 remain unchanged. However, the active form of p38 (p–p38) appears to increase within 5 minutes of treatment with TGFß1 (5.3 fold enhancement). The level of p–p38 then decreases by 15 minutes, increases again by 1 hour, and continues to increase until 24 hours (10.0 fold enhancement). Conclusions: TGFß1 appears to stimulate the p38 pathway with both an immediate and long–term response. Thus, TGFß1 may activate both the Smad and p38 pathways in human corneal epithelial cells.
Keywords: growth factors/growth factor receptors • cornea: epithelium • signal transduction