Insulin-Like Growth Factor Binding Proteins (IGFBPs) are Differentially Expressed During Neuronal Differentiation of Human Retinal Pigment Epithelial (RPE) Cells Induced by N-(4-hydroxyphenyl)retinamide

Abstract

Purpose:: Insulin-like growth factors, IGF-I and IGF-II, play a major role in cellular proliferation, survival and differentiation. IGF-I is known to promote ocular neovascularization by regulating the expression of VEGF. The action of IGFs is regulated by a family of six conserved IGF binding proteins (IGFBPs). Earlier work from our laboratory has shown that N-(4-hydroxyphenyl)retinamide (4HPR), a retinoic acid derivative, induces neuronal differentiation of cultured human retinal pigment epithelial, ARPE-19, cells at low concentrations. In the present study we investigated the role of the IGFBPs in the 4HPR-induced neuronal differentiation of ARPE-19 cells.

Methods:: ARPE-19 cells in culture were treated with 4HPR (1 µM) for various time intervals in the presence or absence of AGN194301, an RARα-specific antagonist. Progression of neuronal differentiation of ARPE-19 cells was monitored using a phase-contrast microscope. RNA extracted from cells was used for measuring IGFBPs and calretinin transcripts by real-time RT-PCR. IGFBPs secreted into the culture medium were precipitated with trichloroacetic acid for western blot analysis.

Results:: 4HPR-induced neuronal differentiation of ARPE-19 cells was time-dependent as evidenced by morphological changes and increased expression of calretinin, a Ca²⁺-binding protein normally present in retinal ganglion cells and other retinal neurons. No significant change in the expression of IGPBP1 or -2 was observed during the neuronal differentiation. IGFBP4 mRNA was not detectable at any stage of differentiation. The expression of both IGFBP3 and -5 was decreased up to 90% in differentiating RPE cells treated with 4HPR for 72 h. In contrast, IGFBP6 mRNA expression was increased about 3-fold under similar conditions. The decrease in the expression of IGFBP3 and -5 as well as the increase in the expression of IGFBP6 associated with 4HPR-induced differentiation was effectively blocked when the cells were pretreated with AGN194301, an RARα antagonist.

Conclusions:: 4HPR-induced neuronal differentiation of RPE cells was associated with the differential expression of IGFBPs. Both neuronal differentiation and differential expression of IGFBPs were blocked by the RARα antagonist, AGN194301. These data suggest that IGFBPs, known regulators of the IGF signal transduction pathway, may play an important role in neuronal differentiation of RPE cells.

Keywords: retinal pigment epithelium • growth factors/growth factor receptors • differentiation