During this period, a study identifying a functional ligand binding site of PEDF-R was completed. PEDF bound the PEDF-R ectodomain L4 with affinity similar to the full-length PEDF-R, and selectively bound E5b and P1 peptides among peptides spanning L4. Recombinant C-terminal truncated PEDF-R4 (1-232) and internally truncated PEDF-R and PEDF-R4 (203-232) retained phospholipase activity, however without the 203-232 region, PEDF-R lost the PEDF affinity that stimulated enzymatic activity. PEDF-R was detected in the surface of retina R28 cells. Its requirement for PEDF-mediated cell survival and antiapoptotic activities was demonstrated using bromoenolactone to inhibit PLA2 activity and siRNA to selectively knockdown Pnpla2 in R28 cells. P1 and E5b peptides blocked PEDF binding to both recombinant PEDF-R and PEDF-R in intact R28 cells, and prevented the PEDF:PEDF-R-mediated retina cell survival and retinal ganglion cell axon growth activities. Our findings established that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation. Following this study, the enzymatic stimulation of PEDF-R by PEDF was examined by determining fatty acid levels in PEDF-treated R28 cells by mass spectrometry. The blocking effect of P1 on PEDF antiapoptotic activity was tested in the inherited mouse model of retina degeneration rd1. PEDF reduced the number of TUNEL positive photoreceptors in the outer nuclear layer and P1 attenuated this effect. Antiapoptotic effects were observed also in PEDF-treated retina explants from rd10 mice. PEDF levels were determined in Ccl2-/-/Cx3cr1-/- on C57BL/6N Crb1rd8 (DKO rd8) mice, a model for progressive focal retinal degeneration. Serpinf1 transcripts and PEDF protein levels were significantly reduced in RPE and retina and in the culturing media of RPE cells from these animals, 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. Investigations of the PEDF survival activity on photoreceptors continued 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 on the search 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 that could be regulated at a posttranslational level. Toward the creation of a conditional Pnpla2 null mice, a construct containing LoxP sites flanking the Pnpla2 gene was designed and generated. Mouse embryonic stem cells containing the construct were successfully integrated in early stage mouse embryos to obtain agouti founder mice. Animal Study Proposals for the studies of the conditional Pnpla2 null mice were developed and approved by the animal committee. The founder mice were transferred to our animal facility and backcrossed for several generations to C57Bl/6 background mice. Primer pairs were designed to genotype and select heterozygous positive for the presence of the LoxP sites at each backcrossing step. Studies on the structure-function relationship between PEDF and PEDF-R continued to identify structural components necessary for both binding to PEDF-R and the antiapoptotic activity of PEDF. 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, namely a 17-mer, shares neurotrophic activity with PEDF and binds P1. The antiapoptotic effects of 17-mer peptides with alterations of residues to alanine with varying affinities for P1 were assessed. The binding affinity correlated with efficacy in preventing retinal cell apoptosis. Expression plasmids with full length SERPINF1 containing mutations to altered single amino acids within the 17-mer region of PEDF were designed and engineered. The effects of the PEDF-derived peptides were examined in cell-based assays. Peptides 34-mer and 44-mer were tested for protecting the RPE barrier from VEGF-E induced dysfunction. Transepithelial resistance was measured on monolayers of APRE-19 cells. Given the potential involvement of VEGF receptors in the PEDF signaling cascade leading to increased RPE transepithelial resistance, binding of PEDF to recombinant VEGF factor receptor type 1 and type 2 soluble fragments was tested. Because PEDF can elevate the levels of certain cytokines, the production of tumor necrosis factor alpha by RAW264.7 cells treated with PEDF, 34-mer or 44-mer was also explored. PEDF in conditioned media from cells expressing full length SERPINF1 has several isoforms that can be isolated by ion exchange column chromatography. Purified PEDF-1 and PEDF-2 were characterized for ubiquitination, sumoylation, sulfation, binding to P1 and by their survival effects on 661W cells exposed to light. Potential cleavage sites for several proteases and chemicals were identified on the PEDF amino acid sequence and CNBr cleavage was performed with the PEDF isoforms. A novel inhibitor of lipoxygenase was discovered from 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. P1 and E5b peptides protected ARPE-19 cells from oxidative damage. Soybean lipoxygenase V specifically bound to E5b and P1 by peptide affinity chromatography. E5b or P1 peptides inhibited soybean lipoxygenase V activity, and this inhibition was blocked by fluoresceinated-PEDF as a capturing protein. Human 5-lipoxygenase bound to P1 peptide and PEDF-R by affinity chromatography and pull-down assays, respectively. Effects of P1 on human 5-lipoxygenase activity were followed by measuring the 5-lipoxygenase products in oxidative damaged ARPE-19 cells. Recessive null mutations in SERPINF1 cause type VI osteogenesis imperfecta, a heritable bone dysplasia characterized by easy susceptibility to fracture, growth deficiency and defects in bone mineralization. PEDF in serum of type V osteogenesis imperfecta patients was analyzed by western blotting and its levels measured using ELISA. PEDF levels are regulated also in tumors. The levels of secreted PEDF and cell membrane PEDF-R and ATP-synthase were measured in colon cancer cells expressing either the normal PCPH protein or the mt-PCPH oncoprotein.
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