We continued working on the characterization of a PEDF receptor termed PEDF-R. PEDF-R is a patatin-like phospholipase PNPLA2 with high affinity for PEDF. We optimized conditions for in vitro expression of full-length PEDF-R, and both internally and C-terminal truncated polypeptides (PEDFRE5b, PEDFR4 and PEDFR4E5b) using nanolipoprotein particles. The recombinant proteins partitioned as soluble proteins, and the purified proteins were assayed for phospholipase activity. The four recombinant proteins exhibited phospholipase activities but only the activities of PEDFR and PEDFR4 were stimulated by PEDF in a concentration-dependent fashion. His-tag pull down assays were performed and showed that PEDFR-His tag bound PEDF, but PEDFRE5b-His tag did not. The results demonstrated that while the C-terminal half region of PEDF-R was dispensable for PLA activity and PEDF-mediated stimulation, the E5b region of PEDFR was critical for PEDF binding and PEDF-mediated stimulation of its PLA activity. To investigate the cell signaling pathway triggered by the PEDF:PEDF-R interactions, Bcl-2 expression levels were determined in rat retinal precursor R28 cells. Serum starvation of R28 cells resulted in the decrease of Bcl-2 levels leading to cell death. However, upon treatment with PEDF there was an increase in the levels of Bcl-2 with cell survival. Commercially purchased PEDF-R siRNA was tested in R28 cells. RT-PCR was performed and revealed a decrease in the PEDF-R transcripts levels. Western blots of biochemically fractionated plasma membrane proteins were performed to detect PEDF-R levels after silencing. To explore the dependence of the PEDF binding region (E5b and P1) of PEDFR for survival signaling triggered by PEDF, the levels of Bcl-2 in R28 cells treated with PEDF in the absence and presence of PEDF binding peptides (E5b and P1) were determined. We performed western blots and enzymatic assays to detect PEDF-R in retina 661W cells, a retinoblastoma derived cell line with characteristics of cone photoreceptor cells (in collaboration with Dr. Muayyad R Al-ubaidi). To assay the effects of PEDF on cell survival, the 661W cells were exposed to high intensity light or were serum-starved in the presence of PEDF protein. Upon light damage, 661W cells underwent cell death. Cultures treated with PEDF increased cell viability relative to the controls without PEDF. Surprisingly, with serum starved cultures of cells in later passages, PEDF treatments resulted in cell death. We continued investigating PEDF protein isoforms to elucidate mechanisms that confer the multimodal functionality to this protein. Recombinant human PEDF subjected to anion-exchange column chromatography reveals at least two PEDF protein versions, PEDF1 eluting with lower ionic strength, higher pI, and lower apparent molecular weight, and PEDF2 eluting with higher ionic strength, lower pI and higher apparent molecular weight. In collaboration with the laboratory of Dr. Larry Benowitz, the proteins were assayed for axon growth and survival of retinal ganglion cells (RGCs). Both versions protected retina cells against death due to serum-starvation and promoted RGC axon growth, being PEDF2 more potent than PEDF-1. A PEDF-binding peptide derived from PEDF-R was used as an effective blocking agent for the PEDF axon growth activities. We also examined whether the PEDF isoforms affect the activity of p38 in breast tumor cells, which is known to play an important role in survival, apoptosis, and cell migration (in collaboration with Dr. Vicente Notario). We also examined whether the PEDF isoforms exhibit antimigratory effects using tumor cells and ARPE-19 cells. We did not observe p38 activation by either isoform, but PEDF2, rather than PEDF1 exhibited a potent antimigratory activity on both cells types tested. We continued to examine the effects of glycosaminoglycans on PEDF binding to PEDF-R-derived peptides, given that heparin can increase the binding affinity of PEDF for cell-surface receptors and can induce conformational changes in the PEDF protein. Using protein chemistry and peptide affinity chromatography, we observed an increased in affinity between PEDF and PEDF-R peptides with additions of heparin. Cell media containing the secreted recombinant PEDF proteins with alterations at amino acid positions that are critical for binding to glycosaminoglycans and collagen were used to develop purification protocols. Expression plasmids containing mutated PEDF cDNAs were used to transfect tumor cells. Following transfection, invasion through matrigel and collagen I, and cell adhesion assays were performed in collaboration with Dr. Crispin Dass. A study to evaluate the effects of docosahexaenoic acid (DHA), a major omega-3-polyunsaturated fatty acid (ω-3-PUFAs), in the development of experimental choroidal neovascularization (CNV) in rodents was completed. Experimental second generation Long Evans rats fed with diets of varying ω-3-PUFA content designed to produce significantly different retinal DHA levels were used in our studies. A transgenic mouse model (fat-1) engineered to over-produce DHA was also studied. CNV was induced by rupture of Bruchs membrane using laser photocoagulation. At 7 days after induction, animals were euthanatized, and eyes were collected. RPE/choroid flatmounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and high-performance 3D imaging software. The median of CNV complex volumes of animals with DHA-adequate diets was lower by 63% relative to that of animals with DHA-deficient diets. The median of CNV complex volumes in fat-1 transgenic mice was decreased by 59% relative to that of wild type controls. Dietary intake or genetic manipulation to increase the sources of DHA significantly diminished the volume of induced CNV lesions in rodents. We continued to investigate the effects of PEDF on cell surface ATP synthase of endothelial and tumor cell lines. Cell authentication confirmed the identity of the bladder tumor T24 cells (in collaboration with Dr. Vicente Notario). We performed western blots and detected the beta subunit of F1 ATP synthase in T24 plasma membrane protein extracts. The T24 cells exhibited cell-surface ATP synthase activity. The effects of PEDF and its derived peptides, 34-mer and 44-mer, on tumor T24 cell viability and cell-surface ATP synthesis were evaluated. The observations led to conclude that the 34-mer region contains structural determinants that confer antitumorigenic activity and cell-surface ATP synthase inhibitory properties to the PEDF polypeptide. Analyses of ATP supplementation to tumor cell viability and endothelial tube formation (in collaboration with Dr. Luigi Notari) assays in the presence of PEDF were performed. ATP additions attenuated both the PEDF-mediated cell death viability and antiangiogenic activity. In collaboration with the laboratory of Dr. Patricia Steeg, the study on opposing effects of PEDF on breast cancer cell versus neuronal survival: Implications for brain metastasis and metastasis-induced brain damage was completed. PEDF protein was altered to include two-tandem FLAG-epitope tags on the C-terminus of the protein. We found that FLAG-tagged PEDF also exhibited biological activity by inducing R28 cell survival. The role of PEDF on photoreceptor survival and neuroprotective effects has been reported in vivo in retinal degeneration animal models rd and rds, and in light-induced photoreceptor degeneration. In collaboration with the laboratory of Dr. Chi Chao Chan, human recombinant PEDF protein was injected intravitreally and subconjunctivally on Ccl2−/−/Cx3cr1−/−mice, a murine model of age-related macular degeneration. Retinal histologic analyses and A2E levels determinations were performed.
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