Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ + TNF-α + IL-1β). Microarray analysis revealed ~11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Stearoyl-CoA desaturase (SCD) is a regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate. The regulation of SCD is of physiological importance because the ratio of saturated fatty acids to unsaturated fatty acids is thought to modulate membrane fluidity. Differential display analysis of retinal pigment epithelial (ARPE-19) cells identified SCD as a gene regulated by retinoic acid. Two SCD transcripts of 3.9 and 5.2 kilobases in size were found to be expressed in these cells by Northern blot analysis. All-trans-retinoic acid (all-trans-RA) increased SCD mRNA expression in a dose-and time-dependent manner; a ϳ7-fold increase was observed with 1 M all-trans-RA at 48 h. SCD mRNA expression was also increased by 9-cis-retinoic acid (9-cis-RA) as well as 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TT-NPB), a retinoic acid receptor (RAR)-specific agonist. AGN194301, a RAR␣-specific antagonist, suppressed the SCD expression induced by all-trans-RA, TTNPB, and 9-cis-RA. These results indicate the involvement of RAR␣ in the induction of SCD expression by retinoic acid. However, AGN194204, a RXR (retinoid X receptor) pan agonist, also increased SCD mRNA expression. This increase was not blocked by AGN194301, suggesting that an RARindependent mechanism may also be involved. Thus, SCD expression in retinal pigment epithelial cells is regulated by retinoic acid, and the regulation appears to be mediated through RAR and RXR.Stearoyl-CoA desaturase (SCD, EC 1.14.99.5), 1 a microsomal enzyme, catalyzes the initial desaturation of long chain saturated fatty acids into monounsaturated fatty acids. Palmitate and stearate are the preferred substrates for this enzyme. They are converted to palmitoleate and oleate, respectively (1, 2). This oxidative reaction also requires the participation of 0 2 , NADPH, cytochrome b 5 , and cytochrome b 5 reductase. Two SCD genes, SCD1 and SCD2, characterized from both rat and mouse, encode functionally active proteins that share Ͼ80% sequence homology (3, 4). SCD1 and SCD2 show different tissue-specific expression patterns, possibly because of marked differences in the promoter sequences of their genes (5). There are also two loci for SCD genes in the human genome, one on chromosome 10 and another on chromosome 17 (6). However, the gene on chromosome 17 appears to be a transcriptionally inactive pseudogene. The gene on chromosome 10 encodes the functionally active 359-amino acid SCD protein. This gene yields two transcripts, 5.2 and 3.9 kb in size, which differ in the length of the 3Ј-untranslated region.The regulation of SCD by dietary factors, hormones, and peroxisomal proliferators has been studied in mouse and rat (7-12). The regulation of SCD is of physiological importance because changes in this enzyme activity could lead to changes in cell membrane phospholipid composition (8). Palmitoleic and oleic acids are the predominant unsaturated fatty acids present in fat depots and membrane phospholipids (9...