P2Y receptor-mediated stimulation of Muller glial cell DNA synthesis: Dependence on EGF and PDGF receptor transactivation

PURPOSE. To determine whether P2Y receptor-evoked proliferation of Muller glial cells depends on transactivation of receptor tyrosine kinases. METHODS. Primary cultures of Muller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine (BrdU) immunoassay, and the phosphorylation states of the extracellular signal-regulated kinase (ERK1/2) and the p38 mitogenactivated protein kinase (P38 MAPK) were determined by Western blot analysis. RESULTS. In Muller cells, the mitogenic effect of P2Y receptor activation by extracellular adenosine triphosphate (ATP) depended on transactivation of both the platelet-derived growth factor (PDGF) and the epidermal growth factor (EGF) receptor tyrosine kinases, as suggested by the blocking effects of the tyrphostins AG1296 and AG1478 on the ATP-induced proliferation and phosphorylation of ERK1/2. Moreover, the PDGF-induced proliferation may depend on transactivation of the EGF receptor kinase. Antibodies against heparin-binding EGF (HB-EGF) or PDGF, as well as inhibition of matrix metalloproteinases (MMPs) blocked ATP-evoked proliferation. At least one metalloproteinase (MMP-9), was implicated in the signal transfer from P2Y to EGF receptors. In contrast, the mitogenic effect of fetal calf serum was independent of growth factor receptor activity. P2Y receptor activation stimulated Muller cell proliferation by activating the ERK1/2 and the phosphatidylinositol 3 (PI3) kinase signaling pathways, whereas the P38 MAPK pathway was not involved in mitogenic signaling. CONCLUSIONS. The present data suggest that P2Y-receptor-induced mitogenic signaling in Muller cells is mediated by transactivation of the PDGF and EGF receptor tyrosine kinases. The transactivation may be mediated by release of PDGF and MMP-dependent shedding of HB-EGF from the Muller cell matrix, respectively. The transactivation of the receptor tyrosine kinases may result in activation of ERK1/2 and PI3 kinase and an increase in the proliferation rate.