Abstract
Purpose: :
Hepatocyte growth factor (HGF) is a paracrine growth factor, secreted after corneal injury that acts through the c–Met receptor. It stimulates in corneal epithelial cells several signaling pathways that promote proliferation, migration and cell survival, (JBC, 278:21989, 2003; IOVS, 45:3492, 2004). Regulation of these activities could be modulated by protein tyrosine phosphatases (PTPs). We had previously reported that during corneal epithelial wound healing there are changes in the expression of two main PTPs, PTP1B and PTP1D (ARVO,2005). The purpose of this study was to investigate how the two PTPs regulate the activation of the HGF receptor and its downstream signals.
Methods: :
Rabbit corneal epithelial or human corneal epithelial cells were stimulated with HGF (20–40 ng/ml) or treated with the PTP inhibitor bpv(phen) (6–10 µM). In some experiments the extracts were immunoprecipitated with c–Met antibody or with phosphotyrosine or phosphoserine antibodies to determine specific phosphorylations. Samples were analyzed by Western blots for c–Met, PTP1B, PTP1D, p–Akt, p–ERK1/2, p–p70S6K and the p85 subunit of PI–3K.
Results: :
Phosphorylation of c–Met increase greatly when the activity of PTP1B and PTP1D were blocked with bpv(phen). There was also increase in HGF–stimulated phosphorylation of p85, the downstream kinases Akt–1 and p70S6K as well as in ERK1/2. HGF stimulation induces binding of both PTP1B and PTP1D to the c–Met receptor, that was maximal at 30 min. HGF stimulates the phosphorylation of tyrosine residues at 30 min in PTP1D and at 60 min in PTP1B.
Conclusions: :
HGF stimulation of corneal epithelial cells produce association of PTP1B and PTP1D to c–Met regulating its activity and avoiding excessive stimulation of PI–3K/Akt–1/p70S6K and ERK1/2 signaling. This is the first report that PTP1B bind to c–Met and regulates its function.
Keywords: cornea: epithelium • growth factors/growth factor receptors • signal transduction