The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a Cterminal CAAX motif. The 3.9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.The inositol-polyphosphate 5-phosphatases (5-phosphatases) 1 are a large family of enzymes that remove the 5-position phosphate from the inositol ring of phosphatidylinositols including phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), and the inositol phosphates, inositol 1,4,5-trisphosphate (Ins(1,4,5)-P 3 ) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ). Nine distinct mammalian 5-phosphatases have been identified and characterized, while four 5-phosphatases have been described in Saccharomyces cerevisiae. All 5-phosphatases are defined by the presence of a conserved 300-amino acid domain, which contains two signature motifs, proposed to mediate substrate binding and catalysis (1, 2). Although all 5-phosphatase enzymes contain these signature motifs, there is considerable diversity in the substrate specificity of 5-phosphatase isoforms. The enzyme family has been subclassified on this basis into four types, I-IV. The type I enzymes, characterized by the 43-kDa 5-phosphatase (also called 5-phosphatase I), hydrolyze Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 but not any of the 5-position phosphoinositide substrates; the type II 5-phosphatases, including synaptojanin, 5-phosphatase II (originally designated the 75-kDa 5-phosphatase), and the protein product of the oculocerebrorenal syndrome (OCRL) gene, hydrolyze PtdIns-(4,5)P 2 , PtdIns(3,4,5)P 3 , Ins(1,3,4,5)P 4 ...
Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.
Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.
Macrophages phagocytose particles to resolve infections and remove apoptotic cells. Phosphoinositide 3-kinase generates phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] is restricted to the phagocytic cup, promoting phagocytosis. The PtdIns(3,4,5)P 3 5-phosphatase (5-ptase) Src homology 2 (SH2) domaincontaining inositol-5-phosphatase 1 (SHIP1) inhibits phagocytosis. We report here that another PtdIns(3,4,5)P 3 -5-ptase, the 72-kDa-5-phosphatase (72-5ptase), inhibits Fc␥ receptor (Fc␥R)-but not complement receptor 3 (CR3)-mediated phagocytosis, affecting pseudopod extension and phagosome closure. In contrast, SHIP1 inhibited Fc␥R and CR3 phagocytosis with greater effects on CR3-stimulated phagocytosis. The 72-5ptase and SHIP1 were both dynamically recruited to Fc␥R-stimulated phagocytic cups, but only SHIP1 was recruited to CR3-stimulated phagocytic cups. To determine whether 5-ptases focally degrade PtdIns(3,4,5)P 3 at the phagocytic cup after specific stimuli, time-lapse imaging of specific biosensors was performed. Transfection of dominant-negative 72-5ptase or 72-5ptase small interfering RNA (siRNA) resulted in amplified and prolonged PtdIns(3,4,5)P 3 at the phagocytic cup in response to Fc␥R-but not CR3-stimulation. In contrast, macrophages from Ship1 Ϫ/Ϫ /AktPH-GFP transgenic mice exhibited increased and sustained PtdIns(3,4,5)P 3 at the cup in response to CR3 activation, with minimal changes to Fc␥R activation. Therefore, 72-5ptase and SHIP1 exhibit specificity in regulating Fc␥R-versus CR3-stimulated phagocytosis by controlling the amplitude and duration of PtdIns IntroductionMacrophages engulf large particles (Ͼ 1.0 m), including invading pathogens and apoptotic cells, by phagocytosis to resolve infections and regulate tissue remodeling. Phagocytosis is a dynamic process triggered by particle binding to cell surface receptors including Fc ␥ receptor (Fc␥R) and complement receptor 3 (CR3), 1 which initiate a complex series of events including actin and membrane remodeling, leading to the extension of pseudopods, phagosome closure, and particle engulfment. A critical event in phagocytosis is the generation of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] at the phagocytic cup. PtdIns(3,4,5)P 3 is synthesized by the class I phosphoinositide-3 (PI3)-kinase after clustering of Fc␥Rs in response to immunoglobulin G (IgG)-opsonized particle binding or CR3 activation 2-4 and promotes pseudopod extension and phagosome closure. Macrophages with reduced PtdIns(3,4,5)P 3 , as consequence of targeted deletion of the p85 adapter subunit of the PI3-kinase, exhibit decreased phagocytic ability. 5 PI3-kinase inhibition prevents the complete phagocytosis of erythrocytes coated with IgG (eIgG) or complement component C3bi (eC3bi), 6,7 with this defect specific for large but not small particles. 7 PtdIns(3,4,5)P 3 recruits pleckstrin homology (PH)-domain-containing effectors including phospholipase C-␥ and myosin X, which hydrolyze PtdIns(4,5)P 2 and drive pseudopod extension, respectively. 8,9 P...
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