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The corpus callosum is a bundle of nerve fibers that connects the two cerebral hemispheres and is essential for coordinated transmission of information between them. Disruption of early stages of callosal development can cause agenesis of the corpus callosum (AgCC), including both complete and partial callosal absence, causing mild to severe cognitive impairment. Despite extensive studies, the etiology of AgCC remains to be clarified due to the complicated mechanism involved in generating AgCC. The biological function of PI3K signaling including phosphatidylinositol-3,4,5-trisphosphate is well established in diverse biochemical processes including axon and dendrite morphogenesis, but the function of the closely related phosphatidylinositol-3,4,-bisphosphate (PI(3,4)P2) signaling, particularly in the nervous system, is largely unknown. Here, we provide the first report on the role of inositol polyphosphate 4-phosphatase II (INPP4B), a PI(3,4)P2 metabolizing 4-phosphatase in the regulation of callosal axon formation. Depleting INPP4B by in utero electroporation suppressed medially directed callosal axon formation. Moreover, depletion of INPP4B significantly attenuated formation of Satb2-positive pyramidal neurons and axon polarization in cortical neurons during cortical development. Taken together, these data suggest that INPP4B plays a role in the regulating callosal axon formation by controlling axon polarization and the Satb2-positive pyramidal neuron population. Dysregulation of INPP4B during cortical development may be implicated in the generation of partial AgCC.

Lysophosphatidic acid (LPA) is a lipid growth factor that regulates diverse cell functions, including cell proliferation, survival and apoptosis. LPA has been demonstrated to be involved in the regulation of cortical neurogenesis by increasing the survival of neural precursors. Previously, we reported that LPA stimulated the inactivation of glycogen synthase kinase 3 (GSK3) via the G protein-coupled LPA1 and LPA2 receptors, by which apoptosis is suppressed in H19-7 cells [an embryonic hippocampal progenitor cell (HPC) line]. Increasing numbers of studies have demonstrated that certain G protein-coupled receptors activate β-catenin/T cell factor (TCF) signaling independently of Wnt, which is involved in cell fate determination, cell proliferation and cell survival. To determine whether LPA activates β-catenin-mediated transcriptional activation pathways and whether β-catenin/TCF signaling is involved in neurogenesis by controlling the survival of neural precursors, β-catenin/TCF signaling cascades induced by LPA were investigated in the HPCs. Activation of β-catenin/TCF signaling was determined by the nuclear translocation of β-catenin and the transcriptional activation of a TCF reporter gene. The activation of β-catenin/TCF signaling was blocked by pertussis toxin (PTX) and a protein kinase C (PKC) inhibitor. The expression of a constitutively active mutated form of GSK3β activated β-catenin/TCF signaling to comparable levels to those induced by LPA, and protected against apoptosis in differentiating H19-7 cells. These results showed that LPA activates β-catenin/TCF signaling in a PTX- and PKC-dependent manner, which contributes to LPA-induced cell survival in the HPCs. Activation of β-catenin/TCF signaling by LPA may be involved in neurogenesis by controlling the survival of neural precursors.

The mechanisms of secretory phospholipase A2 (sPLA2) action are not understood clearly. Previously, it was suggested that sPLA2s are internalized into cells for the targeting of sPLA2 to intracellular action sites. However, the mechanisms for sPLA2 internalization remain to be identified. The present study demonstrated for the first time that human group V sPLA2 (hVPLA2) is associated with lipid rafts and is internalized in a flotillin‑dependent pathway. The lipid raft association was probed by cholesterol‑sensitive enrichment of hVPLA2 in low‑density fractions and co‑patching of ganglioside GM1 rafts through cross‑linking of hVPLA2 in HEK293 and CHO cells. The hVPLA2 associated with lipid rafts was shown to be internalized into HEK293 cells at a relatively rapid rate (t1/2 =16 min) and this internalization was inhibited by the knockdown of flotillin‑1, but not by chlorpromazine, an inhibitor of clathrin‑mediated endocytosis. Moreover, internalized hVPLA2 was shown to be colocalized extensively with flotillin‑1 in a punctate structure, but not caveolin‑1. These data revealed that the internalization of hVPLA2 is mediated by flotillin‑1. Attenuation of arachidonic acid release from plasma membrane through the association of hVPLA2 with lipid rafts suggested that this association with lipid rafts may be important in protecting mammalian cells from excessive degradation of plasma membrane and trafficking hVPLA2 into intracellular targets.

Lysophosphatidic acid (LPA) is a lipid growth factor known to regulate diverse cell functions, including cell proliferation, survival, and apoptosis. Tight regulation of cell survival in neuronal precursor is essential during neurogenesis in both developing and adult brain. Increasing data show that diverse external factors including LPA play roles in controlling cell survival and apoptosis in early developing neurons. However, the underlying control mechanism remains unclear. To explore how LPA regulates cell survival or apoptosis in a developing neuron, mechanisms for cell survival and signaling cascades by LPA were investigated in H19-7 hippocampal progenitor cells. Here, we showed that LPA promotes cell survival by protection from apoptosis. Mcl-1 was demonstrated to be crucial in LPA-induced cell survival by transfection of the siRNA specific for Mcl-1 and overexpression of Mcl-1. LPA-induced cell survival was critically mediated by the upregulation of Mcl-1 which was regulated not only through a post-translational control but a transcriptional control. Mcl-1 stabilization by LPA-induced inhibitory phosphorylation of GSK-3 contributed predominantly to the Mcl-1 upregulation. Both LPA-induced cell survival and the GSK-3 phosphorylation were attenuated by PTX and by siRNA specific for LPA1 or LPA2 receptor. Taken together, these results showed that Mcl-1 stabilization by inhibitory phosphorylation of GSK-3 through Gi/o coupling of the LPA1 and LPA2 receptors following Mcl-1 upregulation plays a critical role in LPA-induced survival of H19-7 cells. In developing neurons, modulation of Mcl-1 levels may constitute a crucial mechanism for controlling their fates.

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA(2). "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA(2) inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism of cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activity was determined. The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA(2) and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA(2). ANX-I-(1-274) did not interact with cPLA(2). Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2). These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.

Lipocortin 1 (LC1) is a 37 kDa member of the annexin family of proteins. It has been proposed to act as a mediator of some of the actions of glucocorticoids in anti-inflammatory and immune suppressive functions. LC1 has been shown to play a role in cell proliferation, apoptosis, and differentiation. However, the exact biological functions of LC1 still remain obscure. Here it is shown that LC1 displays a chaperone-like function. Stoichiometric amounts of LC1 suppressed thermally induced inactivation and aggregation of the test enzymes citrate synthase and glutamate dehydrogenase. LC1 was also effective in refolding guanine hydrochloride-denatured glutamate dehydrogenase, as judged by circular dichroism spectroscopy.

Rhodobacter sphaeroides is a purple nonsulfur photosynthetic bacterium that is considered a possible source of H 2 production. R. sphaeroides KD131, which was isolated from sea mud in South Korea, was found to produce high levels of H 2 . Here we report the complete and annotated genome sequence of R. sphaeroides KD131.

A gene encoding mature human insulin-like growth factor II (IGF-II) was constructed from the modified IGF-II cDNA sequence and two double-stranded synthetic oligodeoxynucleotide linkers. It was fused to a truncated lacZ gene such that IGF-II was expressed as part of C-terminus of beta-galactosidase. This fused lacZ'-IGF-II gene was under the control of tac promoter and we overproduced the beta-galactosidase-IGF-II fusion protein in the Escherichia coli. The fusion protein formed inclusion bodies inside the cells. The fusion protein was purified from the isolated inclusion bodies and IGF-II protein was obtained from their fusion protein by CNBr cleavage. The released IGF-II was confirmed by its molecular weight as determined by SDS-PAGE and by its ability to bind anti-IGF antibody.

cAMP response element-binding protein (CREB) has been known to play a pivotal role in neuronal differentiation and neuronal plasticity. Lysophosphatidic acid (LPA) was reported to activate CREB in Rat2 fibroblast cells. To study the roles of LPA in neuronal differentiation, we determined whether LPA activates CREB in H19-7, hippocampal progenitor cells. LPA induced three-fold increase in cAMP level in a pertussis toxin-independent manner. Moreover, LPA stimulated CREB phosphorylation, which was inhibited by not only H89 but also Rp-cAMP. In H19-7 cells, high-level expression of lpa1 and moderate-level expression of lpa4 were detected, whereas any detectible expression of lpa2 or lpa3 was not detected by reverse transcription polymerase chain reaction. Together, these data suggested that LPA potentiates cAMP accumulation through activating Gs, and thereby, LPA can stimulate cAMP-CREB signaling cascade.

Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251–255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1–113) and ANX-I(34–346) were fully active, whereas ANX-I(114–346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.