(2000) Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins

(2000) Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins. J. disease mechanisms that involve excessive cell proliferation, in particular, tumor and proliferative glomerulopathies.Naini, S. M., Choukroun, G. J., Ryan, J. R., Hentschel, D. M., Shah, J. V., Bonventre, J. V. Cytosolic phospholipase A2 regulates G1 progression through modulating FOXO1 activity. assays and the zebrafish model for our studies. The zebrafish offers evolved like a facile model to study human being disease because many genes are highly conserved between the 2 vertebrate varieties, including cyclins, cyclin-dependent kinases (Cdks), and inhibitors of Cdks (15, 16). Manifestation profiles of cell cycle regulatory genes have shown that genes of major importance to G1 and S phases of the cell cycle, including orthologs of the retinoblastoma (pRb), cyclin D1, and cyclin E1, were expressed at very low levels early after fertilization and improved markedly between 3 and 6 h postfertilization (hpf), making zebrafish a suitable model to study early cell Artemisinin division, tissue-specific cellular proliferation, and more broadly, the part of cell cycle genes in development and disease (15). Here, we recognized the gene family in zebrafish, and we display a novel part for cPLA2 in the rules of G1 phase of the cell cycle. Lack of cPLA2 activity resulted in lower levels of cyclin D1, higher levels of p27Kip1, a designated decrease in kinase activity associated with Cdk4, and prolongation of G1 phase. This function of cPLA2 is dependent on its phospholipase activity and mediated through PGE2 signaling. MATERIALS AND METHODS Antibodies and chemicals The following antibodies were used: anti-cPLA2, anti-cPLA2 (Ser505), anti-AKT, -phospho-AKT (Ser473), anti-Forkhead package protein O1 (FOXO1), anti-phospho-FOXO1 (Ser256), and anti-phospho-ERK 1/2 (Tyr204) (from Cell Signaling Technology, Beverly, MA, USA). Anti–tubulin, anti-EGFP (enhanced green fluorescent protein), anti-cyclin D1, anti-cyclin E, anti-cyclin A, anti-p21Cip1, anti-p27Kip1, anti-Cdk2, anti-Cdk4, anti-ERK 1/2 anti-glyceraldehyde 3-phosphate dehydrogenase, and anti-lamin A/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-BrdU (5-bromo-2-deoxyuridine) was purchased from Abcam Integrated (Cambridge, MA, USA). Ionophore A23187 (operating concentration 10 M), BrdU (10 mM), platelet-derived growth element (PDGF; 10 mg/ml), PD9809 (100 M), Ly294002 (30 M), AA (39 pM), AS1842856 (0.1 M), and PGE2 (5 nM) were purchased from Sigma-Aldrich (St. Louis, MO, USA, USA). [3H]Thymidine (1 Ci/ml), [3H]AA (0.5 Ci/ml), [?32P]ATP (10 Ci), phosphatidylcholine 1-steratoyl-2-[1-14C]arachidonyl (0.5 nM), and methyltrienolone (R1881; 100 nM) were purchased from New England Nuclear (Boston, MA, USA). Prostaglandin E2 receptor 4 (EP4) antagonist (L-161982; 1 Rabbit polyclonal to GHSR M) and pyrrophenone (1 M) Artemisinin were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Zebrafish husbandry Wild-type (WT) zebrafish (hybridization hybridization antisense probes for zebrafish and were synthesized as explained previously (17). Digoxigenin-labeled Artemisinin antisense and sense RNA probes were generated from cDNAs of 24 hpf WT embryos using a digoxigenin-RNA labeling kit (Roche, Mannheim, Germany) according to the manufacturers instructions. Each experiment was carried out at least twice. Embryos were fixed in diluted formalin (1:2.7 in polybutylene terephthalate) at space temp for 1 h. Alkaline phosphatase-coupled anti-digoxigenin (Roche) was used to localize hybridized probes. NBT/BCIP (Roche) was used as the chromogenic substrate to produce blue precipitates. Microinjection of mRNA and morpholino oligonucleotides Antisense morpholino (MO) oligonucleotides (Gene Tools, Philomath, OR, USA) were designed to target the and translational start sites (ATG): MO (5-AGGTCAGGATGGCACCTTATTTCAA-3) and MO (5-CTCCTTTGGTGACATTTTCAGCCCG-3). MOs were resuspended in 1 Danieaus buffer [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, and 5.0 mM HEPES (pH 7.6)] with 0.1% phenol red (Sigma-Aldrich). Embryos from crosses of adult fish were injected in the 1- or 2-cell stage with an injection volume equal to 2.3 nl MOs per embryo. For the mRNA save experiment, human being cPLA2 cDNA was cloned into personal computers2+ vector and transcribed by using the SP6 mMESSAGE mMACHINE Kit (Ambion Corporation, Naugatuck, CT, USA)..