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Purpose: The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines., Methods: Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting., Results: Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1β, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic., Conclusions: Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1β). The induction of miR-146a showed a dependency on IL-1β, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.

In a microarray analysis of human retinal pigment epithelial cells (HRPE) treated with TGF-β, in addition to the alteration of a number of known Extracellular matrix (ECM)-related genes regulated by TGF-β, we found a significant increase in the expression of Kallmann Syndrome (KAL)-1 gene, that codes for the protein anosmin-1. Enhanced expression of KAL-1 by TGF-β was validated by real-time PCR analysis. In in vitro experiments, TGF-β receptor inhibitor abolished TGF-β-induced expression of KAL-1. Immunofluorescence staining showed increased presence of anosmin-1 in TGF-β treated HRPE cells, with distinct localization at the intercellular junctions. Treatment of HRPE cells with TGF-β enhanced secretion of anosmin-1 and the release of anosmin-1 was further augmented by heparin sulfate. Enhanced secretion of anosmin-1 in the presence of TGF-β and heparin was also observed in other ocular cells such as corneal epithelial and corneal fibroblast cultures. The role of anosmin-1, a protein with adhesion functions, in retinal structure, function and pathology has not been known and remains to be investigated., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To investigate whether conjunctival epithelial cells express transport processes for opioid peptides., Methods: We monitored the uptake of [(3)H]deltorphin II and [(3)H]DADLE, two hydrolysis-resistant synthetic opioid peptides, in the rabbit conjunctival epithelial cell line CJVE and elucidated the characteristics of the uptake process., Results: CJVE cells express robust uptake activity for deltorphin II and DADLE. Both opioid peptides compete with each other for transport. Several endogenous and synthetic opioid peptides, but not non-peptide opioid antagonists, are recognized by the transport process. Though various peptides inhibit the uptake of deltorphin II and DADLE in a similar manner, the uptake of deltorphin II is partly Na(+)-dependent whereas that of DADLE mostly Na(+)-independent. The transport process shows high affinity for many endogenous/synthetic opioid peptides. Functional features reveal that this transport process may be distinct from the opioid peptide transport system described in the retinal pigment epithelial cell line ARPE-19 and also from the organic anion transporting polypeptides, which are known to transport opioid peptides., Conclusions: CJVE cells express a novel, hitherto unknown transport process for endogenous/synthetic opioid peptides. This new transport process may offer an effective delivery route for opioid peptide drugs to the posterior segment of the eye.

Retinal pigment epithelium (RPE) and transforming growth factor-beta (TGF-beta) have been shown to be involved in various retinal diseases. We have studied the role of inflammatory cytokines on the expression and secretion of TGF-beta in human RPE cells (HRPE). Confluent cultures of HRPE derived from donor eyes were used. RT-PCR analyses showed that TNF-alpha and IL-1beta increased the mRNA levels of both TGF-beta1 and TGF-beta2. IFN-gamma enhanced constitutively expressed, as well as, TNF-alpha-and IL-1beta-induced TGF-beta1 mRNA levels but decreased TGF-beta2 mRNA. The effects of these cytokines on TGF-beta1 and TGF-beta2 secretion correlated with the mRNA levels. TGF-beta1 was always produced as the latent form while 21-31% of TGF-beta2 was in the active form. IFN-gamma reduced the production of active form of TGF-beta2 to 4-9%. TGF-beta3 secretion was not detectable under any of the conditions. The Real-Time PCR analysis of TGF-beta mRNAs confirmed the observed results. The TGF-beta1 and TGF-beta2 secretion was induced by TGF-beta2 and TGF-beta1, respectively. Under these conditions, the contrasting effects of IFN-gamma on TGF-beta1 and TGF-beta2 secretion were also observed. JAK inhibitor selectively inhibited IFN-gamma induced TGF-beta1 secretion and mRNA levels while reversing the inhibitory effects of IFN-gamma on TGF-beta2. Analyses of transcription factor activity strongly indicated the role of STAT-1 but not NFkappaB, C-Myc, C-Jun, SP-1, MEF-2. Our data demonstrate that IFN-gamma differentially regulates constitutively expressed, as well as, cytokine-induced TGF-beta1 and TGF-beta2 mRNA levels and secretion of TGF-betas by HRPE.

Age-related macular degeneration (AMD) is a sight threating retinal eye disease that affects millions of aging individuals world-wide. Choroid-retinal pigment epithelium (RPE)-neuroretina axis in the posterior compartment of the eye is the primary site of AMD pathology. There are compelling evidence to indicate association of vascular endothelial growth factors (VEGF) to AMD. Here, we report the inhiβitory actions of resveratrol (RSV) on inflammatory cytokine, TGF-β and hypoxia induced VEGF secretion βy human retinal pigment epithelial cells (HRPE). HRPE cultures prepared from aged human donor eyes were used for the studies in this report. HRPE secreted βoth VEGF-A and VEGF-C in small quantities constitutively. Stimulation with a mixture of inflammatory cytokines (IFN-γ, TNF-α, IL-1β), significantly increased the secretion of βoth VEGF-A and VEGF-C. RSV, in a dose dependent (10-50 uM) manner, suppressed VEGF-A and VEGF-C secretion induced βy inflammatory cytokines significantly. RT-PCR analysis indicated that effects of RSV on VEGF secretion were possiβly due to decreased mRNA levels. TGF-β and coβalt chloride (hypoxia mimic) also upregulated HRPE cell production of VEGF-A, and this was inhiβited βy RSV. In contrast, RSV had no effect on anti-angiogenic molecules, endostatin and pigment epithelial derived factor secretion. Studies using an in vitro scratch assay revealed that wound closure was also inhiβited βy RSV. These results demonstrate that RSV can suppress VEGF secretion induced βy inflammatory cytokines, TGF-β and hypoxia. Under pathological conditions, over expression of VEGF is known to worsen AMD. Therefore, RSV may βe useful as nutraceutical in controlling pathological choroidal neovascularization processes in AMD. [ABSTRACT FROM AUTHOR]

Chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in vision loss. Human retinal pigment epithelial cell (HRPE) layer and choroidal tissue are the primary pathological sites in AMD. Pathological and therapeutic evidences have strongly indicated the vascular endothelial growth factor (VEGF) molecules as critical components in CNV pathogenesis. In these studies, we used human primary HRPE and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IFN-γ + TNF-α + IL-1β) mix (ICM) on global gene expression profiles in HRPE cells, revealed 10- and 9-fold increase in VEGF-A and VEGF-C expression, respectively. The microarray results were validated by quantitative RT-PCR and secretion of VEGFs proteins. IL-1β is the most potent in inducing VEGFs secretion followed by IFN-γ and TNF-α, and the secretion was more effective in the presence of 2 and 3 cytokines. NF-κB and JAK-STAT pathway, but not HIF-1α, Sp-1, Sp-3, and STAT-3, transcription factors were upregulated and translocated to nucleus by ICM treatment. The mRNA levels of VEGF-A and VEGF-C and secretion of these proteins were also significantly enhanced by ICM in HCHF cells. The secretion of other angiogenic molecules, PEDF, SDF-1α, endostatin, and angiopoietins was not affected by ICM. Our results show that the inflammatory cytokines enhance secretion of VEGF-A and VEGF-C by HRPE and HCHF cells. These studies indicate that VEGFs secreted by these cells initiate and promote pathological choroidal and retinal noevascularization processes in AMD. J. Cell. Physiol. 227: 116-126, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-κB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p <0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses. [Copyright &y& Elsevier]

ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5α andABCB 5β) of the ABC transporterABCB 5 in human melanoma cells. The deducedABCB 5α protein appears to be an altered splice variant containing only a putative ABC, whereas theABCB 5β isoform shares approximately 70% similarity withABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of theABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles ofABCB 5α/β. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed bothABCB 5α andABCB 5β. However,ABCB 5α/β expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile ofABCB 5α/β in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. NeitherABCB 5α norABCB 5β expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta.ABCB 5α/β mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting thatABCB 5α/β expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression ofABCB 5α/β might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]