Hiromu Murofushi

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.

Rapid generation of specific antibodies by enhanced homologous recombination

In the chicken immune system, gene conversion, a type of homologous recombination, primarily contributes to diversification of the immunoglobulin gene. Here, we report on the rapid generation of specific monoclonal antibodies using the chicken DT40 B-cell line undergoing gene conversion. We discovered that the gene conversion frequency at the immunoglobulin locus is increased by treating DT40 cells with a histone deacetylase inhibitor, trichostatin A (TSA), thereby generating diversity at the immunoglobulin locus in the majority of treated cells. This indicates that TSA treatment accelerates the autonomous diversification of surface IgMs on DT40 cells. We took advantage of this effect to select DT40 cells producing specific antibodies with antigen-conjugated magnetic beads. This autonomously diversifying library (ADLib) selection system enables the quick establishment (∼1 week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design.

The Histone Chaperone Facilitates Chromatin Transcription (FACT) Protein Maintains Normal Replication Fork Rates

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.

Antinociceptive effect of cyclic phosphatidic acid and its derivative on animal models of acute and chronic pain

1. AbstractBackgroundCyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain.Results(1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aβ. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treatments with 2ccPA significantly attenuated thermal hyperalgesia and mechanical allodynia following partial ligation of the sciatic nerve. (5) In rats, repeated post-treatments with 2ccPA also significantly attenuated thermal hyperalgesia and mechanical allodynia following chronic sciatic nerve constriction.ConclusionsOur results suggest that cPA and its stable analog 2ccPA inhibit chronic and acute inflammation-induced C-fiber stimulation, and that the central effects of 2ccPA following repeated treatments attenuate neuropathic pain.

Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.

MOV10 as a novel telomerase-associated protein

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.

2- O -Carba-oleoyl cyclic phosphatidic acid induces glial proliferation through the activation of lysophosphatidic acid receptor

Lysophosphatidic acid (LPA) and cyclic phosphatidic acid (cPA) are one of the lipid mediators regulating cell proliferation and differentiation through the activation of LPA receptors. An LPA receptor-mediated signal is important for the development of the central nervous system, while it has been demonstrated that LPA caused microglial activation and astroglial dysfunction. Previously, we have reported that cPA and carba analog of cPA, 2-O-carba-cPA (2ccPA), protected neural damage caused by transient ischemia. However, little is known about the target cell of cPA/2ccPA in the central nervous systems. Here, we examined the effect of 2ccPA on glial proliferation and differentiation using the primary astrocytes and oligodendrocyte precursor cells (OPCs) cultures. 2ccPA increased the DNA synthesis of astrocytes and OPCs, but it did not reduce the formazan production in the mitochondria. Further, 2ccPA increased the cell number and cell survival against oxidative stress. The inhibition of LPA receptors by ki16425 abolished 2ccPA-induced DNA synthesis. Extracellular signal-regulated kinase (ERK) was activated by 2ccPA, which contributed to the astroglial DNA synthesis. These results suggest that 2ccPA is a beneficial regulator of glial population through the activation of LPA receptor without reduction of mitochondrial activity.

Qualitative and quantitative comparison of cyclic phosphatidic acid and its related lipid species in rat serum using hydrophilic interaction liquid chromatography with tandem-mass spectrometry

Cyclic phosphatidic acid (cPA) is a simple lipid containing a fatty acid attached at the sn-1 position and a cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. The pharmacological effects of cPA have been demonstrated in several diseases such as cancer and neuropathic pain; however, the composition of the molecular species of cPA in relative to other lipid species in biological samples is still unclear. Recently, hydrophilic interaction liquid chromatography (HILIC) has demonstrated the ability to perform lipidomic analyses of biological samples. In the present study, we developed the quantitative measurement of cPA and its related lipid species, such as lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC), in rat serum using HILIC equipped with tandem-mass spectrometry (MS/MS). The lipid analysis using HILIC-MS/MS system demonstrated high linearity and reproducibility. The modified Bligh and Dyer method using citric acid was showed high efficiency on the extraction of cPA and LPA without contamination of artificial products. In rat serum, cPA and LPC contained more saturated fatty acids such as palmitic acid and stearic acid than unsaturated fatty acids, whereas LPA and phosphatidylcholine more contained unsaturated fatty acids than saturated fatty acids. The analytical methods for measuring cPA and its related lipid species in the present study will aid the analysis of their metabolism.