We continued studies on the structure-function relationship of PEDF to identify additional structural components necessary for its antiapoptotic activity. PEDF has two biologically active regions: an antiangiogenic 34-mer peptide and a neurotrophic 44-mer peptide. A smaller peptide derived from the solvent exposed region of the 44-mer, a particular 17-mer, shares neurotrophic activity with PEDF and binds P1, a peptide derived from the PEDF binding region of PEDF-R. P1 blocks both the PEDF-PEDF-R interactions and the antiapoptotic activity of PEDF. A set of alanine scanning peptides derived from the 17mer region had varying affinities for P1 and antiapoptotic effects on retina cells. The blocking effects of P1 inhibiting the cytoprotective activities of these 17mer peptides were assessed in retina cells under serum starvation in culture as well as in in the inherited mouse model of retina degeneration rd1/rd1 in vivo. PEDF reduced the number of TUNEL positive photoreceptors in the outer nuclear layer and the blocking P1 peptide attenuated the antiapoptotic effect. To evaluate the PEDF-R binding affinities of full length PEDF protein modified as in the 17mer peptides, expression plasmids containing the SERPINF1 gene with mutations altering single amino acids within the 17-mer region of PEDF were individually designed and constructed. BHK cells were transfected with these plasmids to overexpress the mutated SERPINF1 and obtain recombinant proteins. Purification protocols were developed for the recombinant altered PEDF proteins from the conditioned media of the stably transfected BHK cells containing PEDF expression vectors. We completed a study evaluating the effects of PEDF in Ccl2-/-/Cx3cr1-/- on C57BL/6N Crb1rd8 (DKO rd8) mice, a model for progressive focal retinal degeneration. Serpinf1 transcripts in the RPE and retina, and PEDF protein levels in the culturing media of RPE cells from these animals were significantly reduced, respectively. PEDF administration significantly attenuated focal photoreceptor degeneration associated with apoptotic and inflammatory pathways, as well as lowered vascular endothelial growth factor (VEGF) expression in DKO rd8 eyes. We also completed a study investigating PEDF survival activity on photoreceptors using 661W cells, a cell line derived from mouse cone photoreceptors. PEDF increased the number of 661W cells exposed to light in the presence of 9-cis retinal. PEDF-R was immunodetected in 661W plasma membrane fractions. PEDF increased the phosphorylated Akt to total Akt ratio in these cells. Another study searching for alternatively Pnpla2 spliced variants was completed. Ensembl and other expressed sequence tag databases reveal putative alternative splice variants in mouse and rat for Pnpla2. To obtain experimental evidence for Pnpla2 splice variants polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-length Pnpla2 transcript and enhance amplification of low abundant splice variants. Recombinant plasmids with human full-length PNPLA2 or PNPLA2 cDNAs lacking exon 5b were used to validate the techniques. PCR products for Pnpla2 transcripts resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. Even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, immunoblots of 661W cells revealed PEDF-R isoforms. The data provide evidence for the existence of a single, full-length Pnpla2 transcript for PEDF-R likely regulated at the posttranslational level. We continued the studies on a novel inhibitor of lipoxygenase discovered fromm a PEDF-R region. Lipoxygenases are enzymes responsible for the metabolism of arachidonic acid and other polyunsaturated fatty acids, thereby contributing to the generation of reactive oxygen species under oxidative stress. Two peptides from the PEDF-binding region of PEDF-R protected ARPE-19 cells from oxidative damage and specifically bound and inhibited soybean lipoxygenase V activity. Human 5-lipoxygenase also bound to P1 peptide and PEDF-R by affinity chromatography and pull-down assays, respectively. Proximity ligation assays were also performed to evaluate the interactions between PEDF-R and 5-lipoxygenase in ARPE-19 cells. Downstream effects of P1 on human 5-lipoxygenase activity in oxidative damaged RPE cells were followed by measuring the 5-lipoxygenase products (LTB4 and 5-HETE) by ELISA and LC/MS. Expression of the PNPLA2 and 5-LOX genes was measured in RPE cells under oxidative stress with hydrogen peroxide treatments by RT-PCR. Primary cultures of RPE cells from pig eyes were developed and optimized. The cytoprotective effects of peptides from the PEDF-R ectodomain on these cells under oxidative stress were evaluated. Silencing vectors were used to ablate the PNPLA2 and 5-LOX expression in ARPE-19 cells. A PNPLA2 expression vector was used to increase the levels of PEDF-R in ARPE-19 cells. RT-PCR and western blots were performed to confirm the changes in the levels of each gene. The effects of silencing and overexpression were then evaluated and compared in the cells undergoing oxidative stress by cell death and viability assays. Experiments to establish the precise apparent molecular weight of PEDF-R were performed. Expression vectors with the human and mouse PNPLA2 cDNAs were constructed. The recombinant proteins were obtained in bacterial transcription/translation systems and mammalian cells transfected with the plasmids. Proteins from in vitro transcription/translation reactions and total lysates of transfected cells were analyzed by western blotting with specific antibodies using gels with three different concentrations of polyacrylamide. The apparent molecular weights of the recombinant proteins were interpolated from a plot generated from logarithm of molecular weight as a function of relative migration of known unlabeled protein molecular weight standards. We continued investigating the downstream effects of PEDF-R interactions in cells. The enzymatic stimulation of PEDF-R by PEDF was quantified by determining fatty acid levels in PEDF-treated R28 cells by mass spectrometry. Recessive null mutations in SERPINF1 cause type VI osteogenesis imperfecta, a heritable bone dysplasia characterized by high susceptibility to fracture, growth deficiency and defects in bone mineralization. Serum levels of PEDF are significantly decreased in type VI OI patients. Dominant mutations in IFITM5, encoding BRIL, cause type V osteogenesis imperfecta. In a collaborative study investigating the role of PEDF in type V OI with clinical characteristics of type VI OI, we found that although its serum levels were not decreased in patients, SERPINF1 expression was decreased in proband osteoblasts, and PEDF protein was barely secreted from proband cells in culture. The data suggested that BRIL can regulate PEDF in osteoblasts. The expression of IFITM5 in human, pig and mouse RPE cells was evaluated by RT-PCR. Broncopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. In a collaborative study evaluating the role of PEDF in lung vascular development in neonatal hypoxia, it was found that PEDF levels increase in hyperoxia compared to room air-exposed lungs. The levels of PEDF were positively correlated with reduced vasculogenesis and alveolarization in neonatal hyperoxia, implying that PEDF mediates impaired lung vascular development in neonatal hyperoxia.
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