PEDF Regulates a Group of Six Interferon–Inducible Genes That Are Highly Expressed in Actively Dividing RPE Cells via the p38MAP Kinase Pathway

Abstract

Abstract: : Purpose: To study the transcription of genes in actively dividing (pre–confluent) and non–dividing (post–confluent) RPE cells and to evaluate how PEDF alters the expression of genes in each growth phase. Methods: Human ARPE–19 cells were placed in serum free medium 24 hr prior to treatment with 100ng/mL of PEDF. Cell proteins were extracted after 30 min from PEDF treated and non–treated cultures and phosphoprotein changes analyzed by western blot of 40 signaling molecules. 24 and 48 hrs after treatment with either PEDF or SF–medium alone total RNA was isolated by TRIzol. RNA with 280/260 ratio >1.9 was used to construct Cy3 and Cy5 labeled probes(Genisphere, PA). Probes were hybridized to human Operon 27K, 70–mer oligo arrays overnight. Thresholds were > 2–fold gene expression changes in at least two arrays. Results: PEDF decreased phosphorylation of p38 MAP kinase in mitotic RPE cells. Treatment of dividing RPE cells with PEDF for 24 and 48 hrs increased expression of 43 and 125 genes and decreased expression of 74 and 48 genes, respectively. Treatment of quiescent RPE cells with PEDF for 24 and 48 hrs resulted in an increase in 1994 and 72 genes and a decrease in 79 and 81 genes, respectively. PEDF induced changes in expression of groups of genes including apoptosis (33 genes), growth factors and their receptors (21 genes), cytoskeleton (47 genes), angiogenesis (3 genes), and cell cycle (33 genes). Many of these changes varied with confluence. Most notable, however, was the strong increase in expression of a distinct subset of interferon inducible genes at 50% confluence; genes that were almost undetectable at 90% confluence. These changes were confirmed by RT–PCR. Conclusions: The specific subsets of genes changed by PEDF varied with growth phase of RPE cultures, emphasizing that effects of factors such as PEDF are dependent upon the state of the cells. The induction of a specific set of interferon inducible genes in actively growing RPE cells suggests that the RPE may play a novel role in the regulation of intraocular inflammatory responses.

Keywords: gene/expression • gene microarray • retinal pigment epithelium