Kimiko Murakami-Murofushi

Molecular Cloning and Characterization of a Novel Human G-protein-coupled Receptor, EDG7, for Lysophosphatidic Acid

Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca2+ mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca2+] i of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca2+response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca2+] i of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca2+ response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca2+ response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca2+ response, modulation of adenylyl cyclase, and MAP kinase activation.

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination

In Saccharomyces cerevisiae, Mre11 protein is involved in both double‐strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA‐binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+‐dependent nuclease activities. A point mutation in the N‐terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild‐type level of nuclease activity was detected in a C‐terminal truncated protein (Mre11ΔC49), although it had reduced DNA‐binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate‐sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11ΔC49 mutant exhibited almost wild‐type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11ΔC49 mutant. These results indicate that Mre11 has two separable functional domains: the N‐terminal nuclease domain required for DSB repair, and the C‐terminal dsDNA‐binding domain essential to its meiotic functions such as chromatin modification and DSB formation.

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.

A Novel Membrane Protein, Ros3p, Is Required for Phospholipid Translocation across the Plasma Membrane in Saccharomyces cerevisiae

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3(Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the geneROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of theROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of theROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Δ cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.

Phospholipase D2-dependent Inhibition of the Nuclear Hormone Receptor PPARγ by Cyclic Phosphatidic Acid

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of natures simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

Inhibition of tumor invasion and metastasis by a novel lysophosphatidic acid (cyclic LPA).

Fetal calf serum (FCS) and 1‐oleoyl lysophosphatidic acid (LPA) were previously found to be potent inducers of invasion (transcellular migration) in an in vitro system. A novel LPA, composed of cyclic phosphate and cyclopropane‐containing hexadecanoic acid (PHYLPA), first isolated from myxoamoebae of Physarum polycephalum, and its synthetic derivatives (cLPA) were tested for their ability to inhibit tumor cell invasion and metastasis. Amoung these, Pal‐cLPA, which has a palmitoyl moiety, was most potent in inhibiting invasion, with 93.8% inhibition at the concentration of 25 μM. Invasion in vitro by mouse melanoma cells (B16), human pancreatic adenocarcinoma cells (PSN‐1), human lung cancer cells (OC‐10) and human fibrosarcoma cells (HT‐1080) was also inhibited by Pal‐cLPA. The stimulation of MM1 cells with LPA triggered F‐actin formation, which was impaired by the addition of Pal‐cLPA at invasion‐inhibitory concentration. Pal‐cLPA induced a rapid increase in adenosine 3′,5′‐cyclic monophosphate (cAMP) concentration in MM1 cells. The addition of dibutyryl cAMP significantly abrogated LPA‐induced invasion by MM1 cells and actin polymerization in the cells. The inhibition of MM1 cell invasion by Pal‐cLPA may be ascribed to an increased level of cAMP. Pal‐cLPA also suppressed invasion in vitro by MM1 cells induced by FCS dose dependently, without affecting proliferation. It also suppressed the pulmonary metastasis of B16 mouse melanoma cells injected into the tail vein of C57BL/6 mice. Thus, Pal‐cLPA is effective in inhibiting invasion and metastasis of a variety of tumor cells. Int. J. Cancer 81:918–922, 1999.

Hyperosmotic stress‐induced reorganization of actin bundles in Dictyostelium cells over‐expressing cofilin

Cofilin is a low‐molecular weight actin‐modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin‐interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature‐sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known.

Early weaning induces anxiety and precocious myelination in the anterior part of the basolateral amygdala of male Balb/c mice.

Weaning is one of the most important events that occur during the early stages of life. For example, precocious weaning is known to increase anxiety-related behaviors in rodents. Here, we demonstrate that in addition to increasing anxiety, early-weaning manipulations alter the accumulation of galactosylceramide, a specific myelin constituent, and the axonal structure of myelinated fibers in the amygdala of male Balb/c mice. We found that early-weaned male mice entered the open arms of an elevated plus-maze less frequently than normally weaned mice at 3 and 5 weeks of age, which indicates persistently higher anxiety levels. However, early-weaned females exhibited fewer entries into the open arms only at 5 weeks of age. Lipid analysis of mice amygdalas showed the early accumulation of galactosylceramide in early-weaned male, but not female, mice at 5 weeks. The precocious accumulation of galactosylceramide was observed only in the amygdala; galactosylceramide accumulation was not observed in the prefrontal cortex or hippocampus of early-weaned male mice. Electron microscopy showed an increase in the number and a decrease in the diameter of myelinated axons in the anterior part of the basolateral amygdala in early-weaned male mice at 5 weeks. These results suggest that the higher anxiety levels observed in early-weaned male mice could be related to precocious myelin formation in the anterior part of the basolateral amygdala.

Pertuzumab, a novel HER dimerization inhibitor, inhibits the growth of human lung cancer cells mediated by the HER3 signaling pathway

A humanized anti‐HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2‐expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti‐tumor activity of pertuzumab against eight non‐small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)‐α. Pertuzumab significantly inhibited the HRG‐α‐stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG‐α‐stimulated phosphorylation of HER3, mitogen‐activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)‐stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG‐α‐stimulated HER2/HER3 heterodimer formation. HRG‐α‐stimulated HER3 phosphorylation was also observed in the PC‐9 cells co‐overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG‐α nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG‐α‐dependent cell growth in lung cancer cells through inhibition of HRG‐α‐stimulated HER2/HER3 signaling. (Cancer Sci 2007; 98: 1498–1503)

Biological functions of a novel lipid mediator, cyclic phosphatidic acid.

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was isolated originally from myxoamoebae of a true slime mold, Physarum polycephalum, and has now been detected in a wide range of organisms from slime molds to humans. It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. This substance shows specific biological functions, including antimitogenic regulation of the cell cycle, regulation of actin stress fiber formation and rearrangement, inhibition of cancer cell invasion and metastasis, regulation of differentiation and viability of neuronal cells, and mobilization of intracellular calcium. Although the structure of cPA is similar to that of lysophosphatidic acid (LPA), its biological activities are apparently distinct from those of LPA. In the present review, we focus mainly on the enzymatic formation of cPA, the antimitogenic regulation of the cell cycle, the inhibition of cancer cell invasion and metastasis, and the neurotrophic effect of cPA.