Isao Ishii

Lysophosphatidic acid as a novel cell survival/apoptotic factor

Lysophosphatidic acid (LPA) activates its cognate G protein-coupled receptors (GPCRs) LPA(1-3) to exert diverse cellular effects, including cell survival and apoptosis. The potent survival effect of LPA on Schwann cells (SCs) is mediated through the pertussis toxin (PTX)-sensitive G(i/o)/phosphoinositide 3-kinase (PI3K)/Akt signaling pathways and possibly enhanced by the activation of PTX-insensitive Rho-dependent pathways. LPA promotes survival of many other cell types mainly through PTX-sensitive G(i/o) proteins. Paradoxically, LPA also induces apoptosis in certain cells, such as myeloid progenitor cells, hippocampal neurons, and PC12 cells, in which the activation of the Rho-dependent pathways and caspase cascades has been implicated. The effects of LPA on both cell survival and apoptosis underscore important roles for this lipid in normal development and pathological processes.

In vivo roles of lysophospholipid receptors revealed by gene targeting studies in mice

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are extracellular ligands for a family of G protein-coupled receptors (GPCRs), LPA1/2/3 and S1P1/2/3/4/5. Through coupling to multiple classes of G proteins and activating multiple signaling pathways, LPA/S1P receptors have been shown to be integral players for many essential cellular and physiological processes. Generation and analysis of mice deficient in each of LPA1, LPA2, S1P1, S1P2, and S1P3 have provided valuable information on the in vivo roles of these receptors. This review is focussed on expression patterns of each receptor gene in wild-type mice, targeted deletion approaches for generating mutant animals, main phenotypes of receptor-null mice, and alterations in signaling characteristics in receptor-deficient primary cells. Altogether, these data give insights to the importance of LPA/S1P receptors at the cellular and organismal level.

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

AIMSnSphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype.nnnMETHODS AND RESULTSnSPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice.nnnCONCLUSIONnThese results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.

Genetic background conversion ameliorates semi-lethality and permits behavioral analyses in cystathionine β-synthase-deficient mice, an animal model for hyperhomocysteinemia

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.

Neurobiology of receptor-mediated lysophospholipid signaling. From the first lysophospholipid receptor to roles in nervous system function and development

Abstract: Identification of the first lysophospholipid receptor, LPA1/Vzg‐1, cloned by way of neurobiological analyses on the embryonic cerebral cortex, has led to the realization and demonstration that there exist multiple, homologous LP receptors, including those encoded by a number of orphan receptor genes known as “Edg,” all of which are members of the G‐protein‐coupled receptor (GPCR) superfamily. These receptors interact with apparent high affinity for lysophosphatidic acid (LPA) or sphingosine‐1‐phosphate (S1P or SPP), and are referred to based upon their functional identity as lysophospholipid receptors: LPA and LPB receptors, respectively, with the expectation that additional subgroups will be identified (i.e., LPC, etc.). Here an update is provided on insights gained from analyses of these receptor genes as they relate to the nervous system, particularly the cerebral cortex, and myelinating cells (oligodendrocytes and Schwann cells).

Increased expression of the lysosomal protease cathepsin S in hippocampal microglia following kainate-induced seizures.

To examine lesions caused by seizures in the developing brain, seizures were induced by the intraperitoneal injection of kainate and nicotine into juvenile mice. After a week, whole brain sections were examined using histochemistry and the gene expression profiles in the neocortices and hippocampi were analyzed using a DNA microarray. Propidium iodide and Fluoro-Jade C staining revealed that kainate but not nicotine-induced degeneration of the hippocampal pyramidal neurons. Comparative analyses of 12,488 probe sets on the microarray chip revealed the differential expression of 208 and 1243 probe sets in the neocortices and hippocampi of kainate-injected mice, respectively, as well as that of 535 and 436 probe sets in the neocortices and hippocampi of nicotine-injected mice, respectively, the patterns of change were largely drug-specific and region-specific. Among a variety of kainate-modified genes including those representing neurodegeneration and astrogliosis, we identified an increased gene expression of the lysosomal cysteine protease cathepsin S in the hippocampi of kainate-injected mice. Western blot analysis of the hippocampal homogenates revealed that kainate induced a 3.3-fold increase in cathepsin S expression. Immunohistochemistry using cell type-specific markers showed that cathepsin S was induced in microglia, especially those surrounding degenerating pyramidal neurons, but not in neurons themselves or astroglia, in the hippocampal CA1 region of kainate-injected mice. These results indicate that seizures induced by kainate elicit neurodegeneration, astrogliosis, and microglial activation accompanied by the expression of cathepsin S while those induced by nicotine do not.