We completed studies on the comparison of PEDF with another serpin, protease nexin-1 (PN-1), which has demonstrable neurotrophic properties in the brain and antiangiogenic activity in the retina. However, the PN-1 retinoprotective properties have not yet been evaluated. While PN-1 has serine protease inhibitory activity, the neurotrophic and antiangiogenic PEDF lacks protease inhibitory properties. Sequence comparisons performed using computer bioinformatics programs, showed that PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. Mouse and bovine eyes, native retina and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage from bovine eyes was also used. Gene expression and protein levels were evaluated by reverse-transcription PCR and western blotting, respectively. The retina and RPE expressed the SERPINE2 gene and contained PN-1 protein. The interphotoreceptor matrix also contained PN-1 protein. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. The addition of recombinant human PN-1, a version of PN-1 referred to as PN-1R346A lacking serine protease inhibitory activity, or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, like PEDF. Bcl2 levels were measured by RT-PCR. PN-1, PN-1R346A, and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. The survival activity was measured in the presence of the selective and competitive catalytic inhibitor of PEDF-R, atglistatin. We evaluated the protective effects of proteins and peptides in R28 cells against death induced by serum starvation using the IncuCyte Zoom system for live-cell monitoring in real time. Cell growth and proliferation after PEDF and PN-1 treatment was monitored every 4 hours for a total of 48 hours to generate time lapse videos and quantifications of cell density to evaluate cultured cells. Apoptosis was quantitated using Annexin V conjugated to cyanine-based fluorescent dyes, which bound to the extracellularly exposed phosphatidylserine (PS) in cells committed to apoptosis. Cytotoxicity was quantitated using DNA binding dyes that enter cells with compromised plasma membrane. These experiments were performed in serum-starved R28 cells, treated or untreated with retinoprotective molecules such as, PEDF, PEDF 17mer, PEDF 44mer, PN-1, PN-1R346A, and PN-1 17mer. We examined the regulation of selected intracellular molecules that may respond to the PEDF/PEDF-R interactions in retina cells. RT-PCR and Western blotting were performed for Sirtuin-1 and HDAC-1 with R28 cells treated with PEDF. A microarray set for the identification of genes involved in retina cell death signaling pathways triggered by PEDF and PEDF-R was used. We assayed enzymatic and metabolic products lysophosphatidylcholine, lysophosphatidic acid and beta-hydroxybutyrate of PEDF-R activity in R28 cell cultures treated with. Subcellular localization of PEDF-R was investigated using antibodies to the predicted extracellular and intracellular domains of polypeptide under permeabilized and non-permeabilized conditions in R28 and ARPE-19 cells. The role of PEDF as a survival factor in mouse photoreceptor cell-line 661W upon treatment with zaprinast and with calcium ionophore, A23187 was explored. An in vitro model to mimic loss of function of phosphodiesterase 6 (Pde6) was developed using 661W cells treated with zaprinast, a PDE6b enzymatic inhibitor. The levels of cyclic guanosine monophosphate (cGMP) were measured by ELISA. The effect of PEDF on the increased cGMP level was evaluated. The intracellular Ca2+ was measured with calcium indicator Fluo-4AM using a microplate reader. The effects of PEDF on regulation of intracellular calcium levels by zaprinast and ionophore A23187 treatments were also tested. Cell death and viability in response to zaprinast was quantified using ethidium homodimer and ATP-dependent cell TiterGlo, respectively, in the absence and presence of PEDF. We measured the beta-hydroxybutyrate production upon addition of photoreceptor outer segments to polarized ARPE-19 cells, in the presence and absence of PEDF. Silencing and overexpressing vectors for PNPLA2 were used to genetically regulate this gene in ARPE-19 cells. Expression vectors for full-length human PEDF versions with single point alterations at R99A and H105A as well as versions lacking the ability to bind heparin, collagen, or hylauronan were used to transfect HEK.Ebna 293 cells to create stable cell lines. HEK.Ebna cells were cycled between serum and serum free media. Milligram amounts of recombinant protein were produced. Serum free media was collected from cells expressing each variant and used to purified the secreted recombinant PEDF protein via cation exchange chromatography. The variants were characterized by limited proteolysis, collagen binding, and binding to PEDF-R. Expression vectors with human PNPLA2 versions were transformed into bacterial cells. Expression vectors with human and mouse PNPLA2 were transformed into DH10Bac cells for generation of recombinant bacmids to transfect into sf9 insect cells. The expression of the heterologous genes was optimized and the recombinant proteins analyzed by SDS-PAGE, western blotting and enzymatic phospholipase assays. Secreted PEDF and SERPINF1 expression were determined in media of fibroblasts containing deletions in the MITF gene.
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