We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on lipid and retinoid metabolism pathways, differentiation/dedifferentiation pathways, and protection against oxidative or inflammatory stress. The role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In addition, given the likely importance of noncoding RNAs (including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs)) as regulators of gene expression in the response of RPE cells to various signals, we are interested in determining changes in miRNAs and lncRNAs expression in RPE cells during differentiation, and due to agents with which they are treated in our experiments. In the past year we have made progress in the following areas: 1) In this reporting period we completed a study to investigate whether IFNgamma, TNFalpha and IL1beta have any adverse effect on the expression of genes essential for RPE function, employing RPE cell line ARPE-19 as a model system. Proinflammatory cytokines IFNgamma, TNFalpha and IL1beta secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways, which in turn may dysregulate cellular gene expression. ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. We found that proinflammatory cytokines (IFNgamma + TNFalpha + IL1beta) greatly increased the expression of chemokines and cytokines in these cells. However, this response was accompanied by markedly decreased expression of genes important for RPE function such as CDH1, RPE65, RDH5, RDH10, TYR and MERTK. This was associated with decreased expression of other genes highly expressed in RPE, MITF, TRPM1 and TRPM3, as well as microRNAs mir-204 and mir-211, which are known to regulate RPE-specific gene expression. The decreased expression of epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). Thus, RPE cells exposed to proinflammatory cytokines IFNgamma, TNFalpha and IL1beta showed decreased expression of key genes involved in visual cycle, epithelial morphology and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology. A manuscript describing these results was published in the reporting period. 2) The RPE cell line ARPE-19 provides a dependable and widely used alternative to native RPE. However, replication of the native RPE phenotype becomes more difficult because these cells lose their specialized phenotype after multiple passages. Compounding this problem is the widespread (and inappropriate) use of ARPE-19 cells in an undifferentiated state to attempt to model RPE functions. We wished to determine whether suitable culture conditions and differentiation could restore the RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. In our experiments, low passage ARPE-19 cells, at passages 9 to 12, grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum were differentiated for up to 4 months and developed the classic native RPE phenotype with heavy pigmentation. RPE-expressed genes, including RPE65, RDH5, and RDH10, as well as miR-204/211, were greatly increased in these cells maintained at confluence for 4 months. Total RNA extracted from cells cultured for either 4 days or 4 months was used for RNA sequencing (RNA-Seq) analysis to provide a comprehensive view of the relative abundance and differential expression of the genes in the differentiated ARPE-19 cells. Of the 16,757 genes with detectable signals, 1,681 genes were upregulated, and 1,629 genes were downregulated, with a fold change of 2.5 or more differences between 4 months and 4 days of culture. RNA-Seq Gene Ontology analysis showed that the upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors. The majority of the downregulated genes play a role in cell cycle and proliferation. Thus, the ARPE-19 cells cultured for 4 months developed a phenotype characteristic of native RPE and expressed proteins, mRNAs, and miRNAs characteristic of the RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of primary human fetal RPE, embryonic stem cell-derived RPE, and native human RPE cells to assess ARPE-19's inherent plasticity relative to the others. This comparison revealed an important overall similar expression ratio among all the models and native tissue. However, none of the cell culture models reached the absolute values in the native tissue. The results of this study demonstrated that low-passage ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated. A manuscript describing these results was published in the reporting period. 3) We continued a project studying noncoding RNAs, both miRNAs and lncRNAs. lncRNAs are involved in many aspects of regulation of gene transcription, post-transcriptional regulation (splicing, etc.), epigenetic regulation, and such programmatic functions as X-inactivation. Changes in expression of certain lncRNAs are associated with diseases such as various cancers and with Alzheimers disease. It is not unreasonable to expect that there will also be changes in lncRNAs associated with eye diseases such as AMD. Using RNAseq datasets we have identified lncRNAs that show changes (both up- and down-regulation) in our ARPE-19 differentiation model. Manipulation of expression of these lncRNAs results in changes to expression of a variety of important protein-coding genes. In connection with this, we found that a combination of IFNgamma, IL1beta and TNFalpha increased the expression of BANCR in ARPE-19 cells. BANCR is an lncRNA recently shown to regulate EMT and metastasis. IFNgamma by itself increased the expression of BANCR while the other two cytokines did not show any noticeable effect. The increase in BANCR expression by IFNgamma involved activation of STAT1. JAK Inhibitor I, which blocks JAK/STAT, effectively suppressed both the induction of BANCR and STAT1 activation. Thus, BANCR could potentially act as a link between RPE dysfunction and inflammatory response and, therefore, may play a role in the pathology of age-related macular degeneration (AMD). A manuscript describing these results was submitted for publication late in this reporting period. 4) We continued a project to examine the expression of secreted proteins (secretome) and exosomes in differentiated ARPE-19 cultured in DMEM with pyruvate for 4 months and exhibiting native-like RPE phenotype. We continue to collaborate with sections in the LRCMB and with other laboratories and sections (Molecular Structure and Functional Genomics, Laboratory of Immunology), as well as with extramural labs in the analysis (HPLC and mass spectrometry) of retinoid, lipids, and other compounds.
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