Glutamate Stimulates RPE Cell Proliferation Through the ERK 1/2 Pathway

Abstract

Purpose:: Retinal Pigment epithelium (RPE) cells are major components of the epiretinal membranes characteristic in proliferative vitreoretinopathy (PVR). These membranes derive from the proliferation, epithelial-mesenchymal transformation and migration of RPE cells onto both retinal surfaces and the vitreous, thus inducing retinal detachment and loss of vision. Glutamate, the major excitatory neurotransmitter in the vertebrate retina, has been found to be excessively released from ischemic retina, and also to be elevated in the vitreous of PVR patients. Hence, this compound could be involved in the morphological and physiological changes in RPE cells, leading to abnormal wound healing. Glutamate has been shown to induce RPE cell proliferation, process in which the ERK1/2 MAP kinase (MAPK) pathway plays a key role. Additionally serum, which contains high glutamate concentrations, activates the MAPK pathway in RPE cells. However, a possible effect of glutamate in the induction of RPE cell proliferation and trans-differentiation through the ERK1/2 pathway has not been explored. The aim of this work was to first, determine the presence and subtypes of glutamate receptors in rat RPE cells in culture, and to further determine the outcome of the specific stimulation of these receptors on cell proliferation, ERK activation and morphological trans-differentiation of RPE cells.

Methods:: RPE was dissected from Long Evans rats, dissociated and cultured for different time periods. Receptors were characterized using a radioligand-binding assay. Cell proliferation was assessed using a colorimetric MTS reduction assay, and ERK phosphorylation was measured by Western blot analysis. Cell transformation was visualized by immunocytochemistry and Western blot densitometric analysis for alpha-SMA.

Results:: Our results show that 1) ionotropic and metabotropic glutamate receptors present in RPE cells are predominantly of the NMDA and Group I metabotropic subtype. 2) Glutamate activation of AMPA and NMDA receptors induced a dose-dependent increase in cell proliferation, concomitant to ERK1/2 activation. 3) Both, glutamate-induced proliferation and ERK activation are cause-effect related, since the MEK inhibitor PD98056 inhibited both effects. 4) Glutamate induced a decrease in alpha-SMA fibers, which is also mediated by the ERK 1/2 pathway.

Conclusions:: We conclude that glutamate induces RPE cell proliferation through ERK1/2 activation, but does not participate in the epithelial-mesenchymal transition in these cells.

Keywords: excitatory neurotransmitters • retinal pigment epithelium • proliferation