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Biomedical subjects

Yuko Fujiwara

Publications and source records attributed to Yuko Fujiwara.

At least 19 recordsLinked to original sources
PubMed2006

Gata4 is necessary for normal pulmonary lobar development.

Mutations of Fog2 in mice result in a phenotype that includes pulmonary lobar defects. To determine whether formation of the accessory lobe bronchus is mediated by a Gata family cofactor, we evaluated embryonic lungs from mice carrying missense mutations that cause loss of FOG-GATA protein interaction. Lungs from embryos carrying a missense mutation in Gata6 were structurally normal, while lungs from embryos carrying mutations of either Gata4 or of both Gata4 and Gata6 had a structural phenotype that matched the Fog2 mutant phenotype. Expression analysis showed that Gata4 and Fog2 are expressed in the ventral and medial pulmonary mesenchyme during secondary budding. Although Gata4 has not previously been suspected as playing a role in lung development, we have found that a Fog2-Gata4 interaction is critical for the development of normal pulmonary lobar structure, and this phenotype is not influenced by the additional loss of Gata6 interaction. Fog2 and Gata4 in the early pulmonary mesenchyme participate in patterning the secondary bronchus of the accessory lobe.

Animals↗
PubMed2006

Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.

Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.

Animals↗
PubMed2006

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor.

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.

Binding Sites↗
PubMed2006

Extracellular proteome of human hepatoma cell, HepG2 analyzed using two-dimensional liquid chromatography coupled with tandem mass spectrometry.

Secreted proteins, which may be involved in the regulation of various biological processes, are the potential targets for diagnosis and treatment of diverse diseases. In this study, to identify the human hepatoma HepG2 cells-derived secreted proteins more extensively, we applied the protein sample preparations using the combinations of denaturation methods and molecular-mass cutoff via ultrafiltration to the two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS) analysis. We were able to identify a total of 86 proteins containing widely known secreted proteins of HepG2 such as alpha-fetoprotein, of which 73 proteins including 27 signal peptide-containing proteins have never been reported to be secreted from HepG2 cells in other proteomic studies. Among the identified signal peptide-containing proteins, ten proteins such as growth differentiation factor 15, osteopontin and stanniocalcin 2 were discovered as new secreted proteins of HepG2 cells. These observations suggest that the combinations of different sample preparation methods and 2D LC-MS/MS analysis are useful for identifying a wider range of low-abundance proteins and that the secreted proteins from HepG2 identified in this study may be useful as liver-specific biomarkers for diagnosis and treatment.

Carcinoma, Hepatocellular↗
PubMed2006

Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid.

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.

Animals↗
PubMed2006

Phosphonothioate and fluoromethylene phosphonate analogues of cyclic phosphatidic acid: Novel antagonists of lysophosphatidic acid receptors.

Isoform-selective antagonists of the lysophosphatidic acid (LPA) G-protein coupled receptors (GPCRs) have important potential uses in cell biology and clinical applications. Novel phosphonothioate and fluoromethylene phosphonate analogues of carbacyclic phosphatidic acid (ccPA) were prepared by chemical synthesis. The pKa values of these amphilic phosphonolipids and the parent cyclic phosphonate were measured titrimetrically using the Yasuda-Shedlovsky extrapolation. The pharmacological properties of these and other ccPA analogues were characterized for LPA receptor (LPAR) subtype-specific agonist and antagonist activity using Ca2+-mobilization assays in RH7777 cells expressing the individual EDG-family GPCRs. In particular, the phosphonothioate ccPA analogue inhibited Ca2+ release through LPA1/LPA3 activation and was an LPA1/LPA3 antagonist. The monofluoromethylene phosphonate ccPA analogue was also a potent LPA1/LPA3 antagonist. In contrast, the difluoromethylene phosphonate ccPA analogue was a weak LPAR agonist, while ccPA itself had neither agonist nor antagonist activity.

Animals↗
PubMed2006

Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis.

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

Antineoplastic Agents↗
PubMed2006

Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.

The bone marrow and thymus, although both hemopoietic environments, induce very distinct differentiation outcomes. The former supports hemopoietic stem cell self-renewal and multiple hemopoietic lineages, while the latter supports T lymphopoiesis almost exclusively. This distinction suggests that the thymic environment acts to restrict the hemopoietic fates available to thymic immigrants. In this study, we demonstrate that the addition of the Notch ligand Delta-like-1 (Dll-1) to an in vitro system that otherwise supports myelopoiesis, greatly reduces the myelopoietic potential of stem cells or uncommitted progenitors. In contrast, committed myeloid progenitors mature regardless of the presence of Dll-1. The block in myelopoiesis is the direct result of Notch signaling within the hemopoietic progenitor, and Dll-1-induced signals cause a rapid increase in the expression of the zinc finger transcription factor GATA-2. Importantly, in the absence of GATA-2, Dll-1-induced signals fail to inhibit commitment to the myeloid fate. Taken together, our results support a role for GATA-2 in allowing Dll-1 to restrict non-T cell lineage differentiation outcomes.

Cells, Cultured↗
PubMed2006

Characterization of a megakaryocyte-specific enhancer of the key hemopoietic transcription factor GATA1.

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3' 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3'-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells.

Animals↗
PubMed2006

Phosphorothioate analogues of alkyl lysophosphatidic acid as LPA3 receptor-selective agonists.

The metabolically stabilized LPA analogue 1-oleoyl-2-O-methyl-rac-glycerophosphorothioate (OMPT) was recently shown to be a potent subtype-selective agonist for LPA3, a G-protein-coupled receptor (GPCR) in the endothelial differentiation gene (EDG) family. Further stabilization was achieved by replacing the sn-1 O-acyl group with an O-alkyl ether. A new synthetic route for the enantiospecific synthesis of the resulting alkyl LPA phosphorothioate analogues is described. The pharmacological properties of the alkyl OMPT analogues were characterized for subtype-specific agonist activity using Ca2+-mobilization assays in RH7777 cells expressing the individual EDG family LPA receptors. Alkyl OMPT analogues induced cell migration in cancer cells mediated through LPA1. Alkyl OMPT analogues also activated Ca2+ release through LPA2 activation but with less potency than sn-1-oleoyl LPA. In contrast, alkyl OMPT analogues were potent LPA3 agonists. The alkyl OMPTs 1 and 3 induced cell proliferation at submicromolar concentrations in 10T 1/2 fibroblasts. Interestingly, the absolute configuration of the sn-2 methoxy group of the alkyl OMPT analogues was not recognized by any of the LPA receptors in the EDG family. By using a reporter gene assay for the LPA-activated nuclear transcription factor PPARgamma, we demonstrated that phosphorothioate diesters have agonist activity that is independent of their ligand properties at the LPA-activated GPCRs. The availability of new alkyl LPA analogues expands the scope of structure-activity studies and will further refine the molecular nature of ligand-receptor interactions for this class of GPCRs.

Animals↗
PubMed2005

Cell migration activated by platelet-derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1-phosphate receptor-1.

We have previously identified a novel complex between the platelet-derived growth factor (PDGF)beta receptor and the sphingosine 1-phosphate receptor-1 (S1P1). The complex permits the utilization of active G-protein subunits (made available by constitutively active S1P1 receptor) by the PDGFbeta receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. Therefore, an inverse agonist of the S1P1 receptor is predicted to reduce signal transduction from PDGFbeta receptor tyrosine kinase by blocking the constitutive activity of the G-protein coupled receptor. SB649146 is a novel inverse agonist of the S1P1 receptor. First, SB649146 displaced the S1P1 receptor agonist dihydrosphingosine 1-phosphate from membranes expressing the recombinant S1P1 receptor. Second, SB649146 reduced basal recombinant S1P1 receptor-induced GTPgammaS binding and S1P-induced GTPgammaS binding in membranes. Third, SB649146 blocked the S1P-induced activation of p42/p44 MAPK in airway smooth muscle cells, a response that is mediated by the S1P1 receptor. We now report that inverse agonism of the S1P1 receptor with SB649146 reduced the endocytosis of the PDGFbeta receptor-S1P1 receptor complex and the stimulation of p42/p44 MAPK and cell migration in response to PDGF. These findings are the first to report that a GPCR inverse-agonist reduces growth factor-induced receptor tyrosine kinase signaling, fundamentally broadening their mechanism of action. The data obtained with SB649146 also suggest that the constitutively active endogenous S1P1 receptor enhances PDGF-induced cell migration.

Animals↗
PubMed2005

Specification of astrocytes by bHLH protein SCL in a restricted region of the neural tube.

Astrocytes are the most abundant and functionally diverse glial population in the vertebrate central nervous system (CNS). However, the mechanisms underlying astrocyte specification are poorly understood. It is well established that cellular diversification of neurons in the embryo is generated by position-dependent extrinsic signals and combinatorial interactions of transcription factors that direct specific cell fates by suppressing alternative fates. It is unknown whether a comparable process determines embryonic astrocyte identity. Indeed, astrocyte development is generally thought to take place in a position-independent manner. Here we show multiple functions of Stem cell leukaemia (Scl, also known as Tal1), which encodes a basic helix-loop-helix (bHLH) transcription factor, in the regulation of both astrocyte versus oligodendrocyte cell fate acquisition and V2b versus V2a interneuron cell fate acquisition in the p2 domain of the developing vertebrate spinal cord. Our findings demonstrate a regionally restricted transcriptional programme necessary for astrocyte and V2b interneuron development, with striking parallels to the involvement of SCL in haematopoiesis. They further indicate that acquisition of embryonic glial subtype identity might be regulated by genetic interactions between SCL and the transcription factor Olig2 in the ventral neural tube.

Animals↗
PubMed2005

Identification of Darmstoff analogs as selective agonists and antagonists of lysophosphatidic acid receptors.

Darmstoff describes a family of gut smooth muscle-stimulating acetal phosphatidic acids initially isolated and characterized from the bath fluid of stimulated gut over 50 years ago. Despite similar structural and biological profiles, Darmstoff analogs have not previously been examined as potential LPA mimetics. Here, we report a facile method for the synthesis of potassium salts of Darmstoff analogs. To understand the effect of stereochemistry on lysophosphatidic acid mimetic activity, synthesis of optically pure stereoisomers of selected Darmstoff analogs was achieved starting with chiral methyl glycerates. Each Darmstoff analog was evaluated for subtype-specific LPA receptor agonist/antagonist activity, PPARgamma activation, and autotaxin inhibition. From this study we identified compound 12 as a pan-antagonist and several pan-agonists for the LPA(1-3) receptors. Introduction of an aromatic ring in the lipid chain such as analog 22 produced a subtype-specific LPA(3) agonist with an EC(50) of 692 nM. Interestingly, regardless of their LPA(1/2/3) ligand properties all of the Darmstoff analogs tested activated PPARgamma. However, these compounds are weak inhibitors of autotaxin. The results indicate that Darmstoff analogs constitute a novel class of lysophosphatidic acid mimetics.

Animals↗
PubMed2005

Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands.

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA(1) and LPA(3) receptor antagonists. Recently, we reported that the replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological evaluation at LPA GPCR, PPARgamma, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA(3) receptor subtype-selective agonist (EC(50)=3 nM), and 8b as a potent and selective LPA(3) receptor antagonist (K(i)=5 nM) and inhibitor of ATX (IC(50)=600 nM). Serinediamide phosphate 19b was identified as an LPA(3) receptor specific antagonist with no effect on LPA(1), LPA(2), and PPARgamma.

Animals↗
PubMed2005

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

Amino Acid Sequence↗
PubMed2005

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2.

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5a(desArg) is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.

Anaphylatoxins↗
PubMed2005

Lysophosphatidic acid inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells that line the airway, gut, and exocrine glands; it is well established that CFTR plays a pivotal role in cholera toxin (CTX)-induced secretory diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving gastrointestinal wound repair, apoptosis, inflammatory bowel disease, and diarrhea. Here we show, for the first time, that type 2 LPA receptors (LPA2) are expressed at the apical surface of intestinal epithelial cells, where they form a macromolecular complex with Na+/H+ exchanger regulatory factor-2 and CFTR through a PSD95/Dlg/ZO-1-based interaction. LPA inhibited CFTR-dependent iodide efflux through LPA2-mediated Gi pathway, and LPA inhibited CFTR-mediated short-circuit currents in a compartmentalized fashion. CFTR-dependent intestinal fluid secretion induced by CTX in mice was reduced substantially by LPA administration; disruption of this complex using a cell-permeant LPA2-specific peptide reversed LPA2-mediated inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea.

Analysis of Variance↗