Growth Factors Outside of the Platelet-derived Growth Factor (PDGF) Family Employ Reactive Oxygen Species/Src Family Kinases to Activate PDGF Receptor α and Thereby Promote Proliferation and Survival of Cells

Hetian Lei,Andrius Kazlauskas

doi:10.1074/jbc.m808426200

Abstract

The vitreous contains a plethora of growth factors that are strongly implicated in the formation of fibroproliferative diseases such as proliferative vitreoretinopathy. Although platelet-derived growth factors (PDGFs) are present in the vitreous, vitreal growth factors outside of the PDGF family activated the PDGF alpha receptor (PDGFRalpha) and promoted disease progression in a rabbit model of proliferative vitreoretinopathy (H. Lei, G. Velez, P. Hovland, T. Hirose, D. Gilbertson, and A. Kazlauskas (2008) submitted for publication.) In this report we investigated the mechanism by which non-PDGFs activated PDGFRalpha. We found that non-PDGFs increased the cellular level of reactive oxygen species (ROS) and that this event was necessary and sufficient for phosphorylation of PDGFRalpha. We speculated that the underlying mechanism was ROS-mediated inhibition of phosphotyrosine phosphatases, which antagonize receptor auto-phosphorylation. However, this did not appear to be the case. Non-PDGFs promoted tyrosine phosphorylation of catalytically inactive PDGFRalpha, and thereby indicated that at least one additional tyrosine kinase was involved. Indeed, preventing expression or blocking the kinase activity of Src family kinases suppressed non-PDGF-dependent tyrosine phosphorylation of PDGFRalpha. Thus non-PDGFs increased the level of ROS, which activated Src family kinases and resulted in phosphorylation of PDGFRalpha. Finally, although non-PDGFs induced only modest phosphorylation of PDGFRalpha, proliferation and survival of cells in response to non-PDGFs was significantly enhanced by expression of PDGFRalpha. These studies reveal a novel mechanism for activation of PDGFRalpha that appears capable of enhancing the responsiveness of cells to growth factors outside of the PDGF family.

Highlights

The findings described begin to elucidate intracellular events and identify effectors that are required for growth factors outside of the plateletderived growth factors (PDGFs) family to induce tyrosine phosphorylation of PDGF receptor (PDGFR)␣ (Fig. 7)
Non-PDGFs bind and activate their own receptors and thereby increase the cellular level of reactive oxygen species (ROS). This leads to a rise in Src family kinase (SFK) activity, which either directly or indirectly phosphorylates PDGFR␣
This series of events is associated with increased proliferation and survival of cells response to non-PDGFs such as basic fibroblast growth factor (bFGF)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—F, F␣, and F␣⌬X cells were previously described [4].3 Briefly, they are mouse embryo fibroblasts derived from mice null for both pdgfr genes. Cell Culture—F, F␣, and F␣⌬X cells were previously described [4].3 They are mouse embryo fibroblasts derived from mice null for both pdgfr genes. Non-PDGFs increased intracellular ROS, and this event was necessary for phosphorylation of PDGFR␣ by non-PDGFs. A, RPE19␣ cells were exposed for 10 min to bFGF (100 ng/ml) or rabbit vitreous (RV). Apoptosis Assay—Cells were seeded into 10 cm-dishes at a rosine phosphatases that dephosphorylate autophosphorylated density of 2 ϫ 105 cells per dish in DMEM plus 10% fetal bovine PDGFR␣ [49, 50] Statistics—All data were analyzed using the unpaired t test. p increased tyrosine phosphorylation of the wild-type, but not values of Ͻ0.05 were considered statistically significant

RESULTS

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DISCUSSION

Findings

This mechanism seems relevant to