In the past year we have made progress in the following areas: 1) Efforts to identify the down regulation of IGFBP5 expression in 4-HPR-induced neuronal differentiation of human RPE (ARPE-19) cells continued and we showed that the decrease in IGFBP5 expression appears to be due to neuronal differentiation and is not a prerequisite for the neuronal differentiation of human RPE cells (Samuel et al., J. Cell Physiol. 224:827-836, 2010). We found that IGFBP5 down regulation is inhibited by U0126, an inhibitor of MEK1/2, indicating the involvement of MAPK pathway. The overexpression of transcription factor C/EBP (CCAAT/enhancer binding proteins) inhibited the 4-HPR-induced down regulation of IGFBP5 expression and the neuronal differentiation of RPE cells. Interestingly, the binding of C/EBPbeta to the IGFBP5 promoter was decreased by the 4-HPR treatment in gel shift and chromatin immunoprecipitation analyses. Further, the deletion of C/EBP response element from human IGFBP5 promoter markedly decreased the basal promoter activity and abolished its responsiveness to 4HPR treatment in reporter assays suggesting that the expression of IGFBP5 is regulated by C/EBP. We also continued efforts to identify the role of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, in RPE cell death. SCD by its ability to regulate the ratio of saturated fatty acids to monounsaturated fatty acids is thought to control the structural integrity and fluidity of cell membranes, and thereby plays an important role in cell growth, differentiation and apoptosis. We observed that SCD protein expression and enzyme activity is decreased in human RPE cells during 4-HPR-induced apoptosis. We are investigating inhibition of 4HPR-induced apoptosis in human RPE cells by inhibitors of SCD by biochemical assays and microarray analysis of cultured RPE cells. We have modified and established HPLC and mass spectrometric assays for several lipid metabolites implicated in the SCD pathway and its role in cell growth, differentiation and apoptosis. 2) We have continued our work on the role of miRNA in regulating the inflammatory response of adult human retinal pigment epithelial (HRPE) cells in collaboration with Chandra Nagineni and John Hooks (NEI-LI). Our earlier work showed that the inflammatory cytokines (IFN-gamma + TNF-alpha + IL-1beta) can markedly increase the expression of miR-155 expression in HRPE cells. Although IFN-gamma is known to regulate gene expression by activating JAK/STAT signaling pathway, the role of this pathway in regulating the expression of miR-155 is not yet elucidated. Therefore, we studied the role of JAK/STAT pathway in mediating the regulation of miR-155 expression in HRPE cells by the inflammatory cytokines. We detected two putative STAT1 binding elements in the proximal region of BIC/miR-155 promoter. Electrophoretic mobility shift assays showed that increased protein binding to both these elements was present in nuclear extracts prepared from treated cells compared to control cells. The increase in miR-155 expression by the inflammatory cytokines was also associated with an increase in STAT1 activation. The increases in STAT1 activation, protein binding to STAT1 elements and miR-155 expression were effectively blocked by JAK inhibitor 1, an inhibitor of the JAK/STAT pathway. Thus, our results clearly show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by directly activating the JAK/STAT signaling pathway. Thus, the modulation of miR-155 expression by the JAK/STAT signaling pathway could play an important role in the inflammatory processes leading to AMD or other retinal degenerative diseases. 3) The conclusion of a multi-lab collaborative investigation characterizing the processes associated with pterygium was reflected in 2 papers published in the past year. Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. We used expressed sequence tag analysis of an unnormalized unamplified pterygium cDNA library to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. Expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells;some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth. We also found that doxycycline causes regression of murine models of pterygium.
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