Hideyuki Yamamoto

The linoleic acid derivative DCP-LA selectively activates PKC-ϵ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM–100 μM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-ϵ. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-ϵ. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-ϵ, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-γ, a conventional PKC, but to a much lesser extent compared with that for PKC-ϵ, by a mechanism distinct from PKC-ϵ activation. Thus, DCP-LA serves as a selective activator of PKC-ϵ, possibly by binding to the phosphatidylserine binding site on PKC-ϵ. These results may provide fresh insight into lipid signaling in PKC activation.

Parp1-deficiency induces differentiation of ES cells into trophoblast derivatives

Embryonic stem (ES) cells deficient in the enzyme poly(ADP-ribose) polymerase (Parp1) develop into teratocarcinoma-like tumors when injected subcutaneously into nude mice that contain cells with giant cell-like morphology. We show here that these cells express genes characteristic of trophoblast giant cells and thus belong to the trophectoderm lineage. In addition, Parp1(-/-) tumors contained other trophoblast subtypes as revealed by expression of spongiotrophoblast-specific marker genes. The extent of giant cell differentiation was enhanced, however, as compared with spongiotrophoblast. A similar shift toward trophoblast giant cell differentiation was observed in cultures of Parp1-deficient ES cells and in placentae of Parp1(-/-) embryos. Analysis of other cell lineage markers demonstrated that Parp1 acts exclusively in trophoblast to suppress differentiation. Surprisingly, trophoblast derivatives were also detected in wildtype tumors and cultured ES cells, albeit at significantly lower frequency. These data show that wildtype ES cells contain a small population of cells with trophectoderm potential and that absence of Parp1 renders ES cells more susceptible to adopting a trophoblast phenotype.

Modulation of P2X receptors via adrenergic pathways in rat dorsal root ganglion neurons after sciatic nerve injury

Abstract The present study examined noradrenaline‐induced modulation of ATP‐evoked currents in dorsal root ganglion (DRG) neurons after sciatic nerve injury (transection). ATP (10 μM) generated fast/mixed type of whole‐cell membrane currents, possibly as mediated via P2X3/P2X3‐like receptors, and slow type of the currents, possibly as mediated via P2X2/3 receptors, in acutely dissociated L4/5 DRG neurons, without significant difference between sham and injury group. For sham group, noradrenaline (10 μM) enhanced fast/mixed type of ATP‐evoked currents in ipsilateral DRG neurons, that is not inhibited by H‐7, a broad inhibitor of protein kinases, but otherwise it had no effect on slow type of the currents. For injury group, noradrenaline (10 μM) significantly potentiated slow type of ATP‐evoked currents in ipsilateral DRG neurons, that is abolished by H‐7 or GF109203X, a selective inhibitor of protein kinase C (PKC), while it depressed fast/mixed type of the currents. In the analysis of real‐time reverse transcription‐polymerase chain reaction, an increase in the mRNAs for α1b, α2a, α2d, and β2 adrenergic receptors was found with the ipsilateral DRGs after sciatic nerve injury. Collectively, the results of the present study suggest that noradrenaline potentiates P2X2/3 receptor currents by activating PKC via α1 adrenergic receptors linked to Gq protein, perhaps dominantly α1b adrenergic receptors, in DRG neurons after sciatic nerve injury. This may account for a nociceptive pathway in response to noradrenergic sprouting after peripheral nerve injury.

Involvement of CD56brightCD11c+ Cells in IL-18–Mediated Expansion of Human γδ T Cells

γδ T cells are considered to be innate lymphocytes that play an important role in host defense against tumors and infections. We recently reported that IL-18 markedly amplified γδ T cell responses to zoledronate (ZOL)/IL-2. In an extension of this finding, we analyzed the mechanism underlying the IL-18–mediated expansion of γδ T cells. After incubation of PBMCs with ZOL/IL-2/IL-18, the majority of the cells expressed γδ TCR, and the rest mostly exhibited CD56brightCD11c+ under the conditions used in this study. CD56brightCD11c+ cells were derived from a culture of CD56intCD11c+ cells and CD14+ cells in the presence of IL-2 and IL-18 without the addition of ZOL. They expressed IL-18Rs, HLA-DR, CD25, CD80, CD83, CD86, and CD11a/CD18. In addition, they produced IFN-γ, TNF-α, but not IL-12, when treated with IL-2/IL-18, and they exerted cytotoxicity against K562 cells, thus exhibiting characteristics of both NK cells and dendritic cells. Incubation of purified γδ T cells with CD56brightCD11c+ cells in the presence of ZOL/IL-2/IL-18 resulted in the formation of massive cell clusters and led to the marked expansion of γδ T cells. However, both conventional CD56−/intCD11chigh dendritic cells induced by GM-CSF/IL-4 and CD56+CD11c− NK cells failed to support the expansion of γδ T cells. These results strongly suggest that CD56brightCD11c+ cells play a key role in the IL-18–mediated proliferation of γδ T cells.

Effect of IL-18 on Expansion of γδ T Cells Stimulated by Zoledronate and IL-2

Zoledronate (Zol) has recently been shown to expand γδ T cells that play important roles in host defenses against infection and tumors. In this study, we examined effects of interleukin-18 (IL-18) on expansion of γδ T cells in human peripheral blood mononuclear cells (PBMCs) stimulated by Zol and IL-2. The expansion of γδ T cells stimulated by Zol and IL-2 was strongly promoted by exogenous IL-18, and to the contrary, inhibited by neutralizing anti-IL-18 receptor antibody. The γδ T cells that expanded in the presence of Zol, IL-2, and IL-18 exhibited the phenotype of effector memory cells characterized by CD44 (+), CD27 (−), and CD45RA (−). In addition, they expressed NKG2D, perforin, CD94, CD25, and CD122, and 15% to 40% of them were positive for CD56. Incubation of γδ T cells in the presence with IL-18 produced GM-CSF, IFN-γ, and TNF-α at much higher levels than those incubated without IL-18. They showed strong cytotoxicity against tumor cells including mesothelioma cells and inhibited growth of xenograft of mesothelioma in mice. These observations indicate that IL-18 can efficiently promote expansion of γδ T cells with potent antitumor activity.

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors.

Nicotinic acetylcholine (ACh) receptors, such as α7, α3β4 and α4β2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8‐[2‐(2‐Pentyl‐cyclopropylmethyl)‐cyclopropyl]‐octanoic acid (DCP‐LA) (100 nm), a linoleic acid derivative, potentiated responses of α7, α3β4 and α4β2 ACh receptors expressed in Xenopus oocytes that are blocked by 3‐(1‐[dimethylaminopropyl] indol‐3‐yl)‐4‐[indol‐3‐yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for α3β4 ACh receptors. DCP‐LA enhanced the nicotine‐triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell‐shaped dose‐dependent manner at concentrations ranging from 10 nm to 10 µm, although DCP‐LA by itself had no effect on GABA release. The DCP‐LA action was inhibited by GF109203X or α‐bungarotoxin, an inhibitor of α7 ACh receptors, but not by mecamylamine or dihydro‐β‐erithroidine, an inhibitor of α3β4 and α4β2 ACh receptors. A similar effect on GABA release was obtained with 12‐O‐tetradecanoylphorbol 13‐acetate, a PKC activator. DCP‐LA (100 nm) also enhanced GABA release triggered by choline, an agonist of α7 ACh receptors, but not 3‐[2(s)‐azetidinylmethoxy] pyridine, an agonist of α4β2 ACh receptors. In addition, DCP‐LA (100 nm) increased the rate of nicotine‐triggered GABAA receptor‐mediated miniature inhibitory post‐synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or α‐bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP‐LA stimulates GABA release by enhancing activity of pre‐synaptic α7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.

Modulation of innate immunity by IL-18

IL-18 is expressed in various reproductive organs and its expression in the uterus fluctuates in linkage with menstrual cycle, implantation, pregnancy and delivery. However, the roles of this cytokine in reproduction remain obscure. IL-18 is a pleiotropic cytokine and exerts apparently complicated and sometimes paradoxical functions in immune and inflammatory responses, and a consensus understanding of its action has not been attained. Recent investigations reveal that IL-18 activates anti-apoptotic signals and promotes both survival and proliferation of activated lymphocytes as well as various cells exposed to different stressors. Especially, IL-18 enhances the expansion of NK and gammadelta T cells isolated from the circulation and stimulated in various ways. The expansion of gammadelta T cells, stimulated by zoledronate and IL-2, was strongly promoted by exogenous IL-18 and was inhibited by neutralizing anti-IL-18 receptor antibody. The expansion of gammadelta T cells was coincident with an increased number of CD11c+ cells. The gammadelta T cells that expanded in the presence of zoledronate, IL-2 and IL-18 exhibited the phenotype of effector memory cells characterized as CD44+ CD27- CD45RA- cells. In addition, they expressed NKG2D, perforin, CD94, CD25 and CD122, and 30-40% of them were positive for CD56. Incubation of expanded gammadelta T cells with IL-18 induced production of GM-CSF, IFNgamma and TNFalpha at much higher levels than those incubated without IL-18. They showed strong cytotoxicity against tumor cells, including mesothelioma cells, and inhibited growth of mesothelioma xenografts in mice. These observations suggest that IL-18 can efficiently promote expansion of gammadelta T cells with potent cytotoxicity.

Genetic analysis of genes causing hypertension and stroke in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) are frequently used as model rats not only in studies of essential hypertension and stroke, but also in studies of attention deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto rats (WKY) are normally used as controls in these studies. In this study, using these rats, we aimed to identify the genes causing hypertension and stroke, as well as the genes involved in ADHD. Since adrenal gland products can directly influence cardiovascular, endocrine and sympathetic nervous system functions, gene expression profiles in the adrenal glands of the 3 rat strains were examined using genome-wide microarray technology when the rats were 3 and 6 weeks of age, a period in which the rats are considered to be in a pre-hypertensive state. Gene expression profiles were compared between SHR and WKY and between SHRSP and SHR. A total of 353 genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated and candidate genes were selected as significantly enriched genes. SHR-specific genes isolated when the rats were 3 weeks of age contained 12 enriched genes related to transcriptional regulatory activity and those isolated when the rats were 6 weeks of age contained 6 enriched genes related to the regulation of blood pressure. SHRSP-specific genes isolated when the rats were 3 weeks of age contained 4 enriched genes related to the regulation of blood pressure and those isolated when the rats were 6 weeks of age contained 4 enriched genes related to the response to steroid hormone stimulus. Ingenuity pathway analysis of enriched SHR-specific genes revealed that 2 transcriptional regulators, cAMP responsive element modulator (Crem) and Fos-like antigen 1 (Fosl1), interact with blood pressure-regulating genes, such as neurotensin (Nts), apelin (Apln) and epoxide hydrolase 2, cytoplasmic (Ephx2). Similar analyses of SHRSP-specific genes revealed that angiotensinogen (Agt), one of the blood pressure-regulating genes, plays pivotal roles among SHRSP-specific genes. Moreover, genes associated with ADHD, such as low density lipoprotein receptor (Ldlr) and Crem, are discussed.

Cisplatin-induced acute renal failure in mice is mediated by chymase-activated angiotensin-aldosterone system and interleukin-18.

Mechanism(s) of cisplatin-induced acute renal failure, as manifested by increases in blood urea nitrogen and creatinine, was evaluated in relation to production and activation of endogenous mediator(s) in mice. In interleukin (IL)-18-deficient (IL-18KO) mice, cisplatin failed to induce acute renal failure. Administration of recombinant IL-18 prior to cisplatin restored acute renal failure in IL-18KO mice. Accumulation of cisplatin in the kidney was not different in IL-18KO and wild-type (WT) mice, but, clearance of cisplatin was more rapid in IL-18KO mice than in WT mice. Cisplatin increased serum levels of aldosterone and angiotensin II in WT mice, but only angiotensin II levels in IL-18 KO mice. Administration of IL-18 augmented plasma levels of aldosterone and angiotensin II in WT mice. Eplerenone, an aldosterone receptor blocker, TY-51469, a chymase inhibitor and PD123319, a selective angiotensin II type 2 (AT2) receptor antagonist, but not benazepril, an angiotensin-converting enzyme inhibitor, and candesartan, a selective angiotensin II type 1 (AT1) receptor antagonist improved acute renal failure caused by cisplatin, confirming involvement of IL-18, aldosterone and angiotensin II in cisplatin-induced, chymase-dependent acute renal failure in mice. These results show that IL-18, aldosterone and angiotensin II synergistically act to prolong the accumulation of cisplatin in the kidney, leading to acute renal failure. Combined therapy with inhibitors for chymase and aldosterone receptors or AT2 receptors might reduce acute renal failure induced by cisplatin.

Chymase inhibition improves vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats

Objective: To clarify the role of chymase in hypertension, we evaluated the effect of a chymase inhibitor, TY-51469, on vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats (SHR-SP). Methods: SHR-SP were treated with TY-51469 (1 mg/kg per day) or placebo from 4 to 12 weeks old or until death. Wistar–Kyoto rats were used as a normal group. Results: SBP was significantly higher in both the placebo and TY-51469 groups than in the normal group, but there was no significant difference between the two treatment groups. Plasma renin, angiotensin-converting enzyme activity and angiotensin II levels were not different between the placebo and TY-51469 groups. In contrast, vascular chymase-like activity was significantly higher in the placebo than in the normal group, but it was reduced by TY-51469. Acetylcholine-induced vascular relaxation was significantly higher in the TY-51469 group than in the placebo group. There was significant augmentation of the number of monocytes/macrophages and matrix metalloproteinase-9 activity in aortic tissue from the placebo group compared with the normal group, and these changes were attenuated by TY-51469. There were also significant increases in mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-&agr; in the placebo group that were attenuated by TY-51469. Cumulative survival was significantly prolonged in the TY-51469 group compared with the placebo group. Conclusion: Chymase might play an important role in vascular dysfunction via augmentation both of matrix metalloproteinase-9 activity and monocyte/macrophage accumulation in SHR-SP, and its inhibition may be useful for preventing vascular remodeling and prolonging survival.