Kyoko Nakamura

Role of Krüppel-like Factor 15 (KLF15) in Transcriptional Regulation of Adipogenesis

Krüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor γ (PPARγ) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein β (C/EBPβ) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARγ in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPβ, C/EBPδ, or C/EBPα in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPα acted synergistically to increase the activity of the PPARγ2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR γ expression.

Receptor-Selective Diffusion Barrier Enhances Sensitivity of Astrocytic Processes to Metabotropic Glutamate Receptor Stimulation

An mGluR5-selective diffusion barrier enriches mGluR5 in astrocytic processes, enabling compartmentalized calcium signaling. Keeping Calcium Signals in the Processes Although astrocytes, the most numerous form of glial cell in the brain, are electrically inexcitable, their ability to release chemical messengers and respond to such messengers with propagated calcium signals allows them to participate actively in the regulation of local blood flow and of synaptic efficacy. Here, Arizono et al. expressed a genetically encoded calcium indicator in neuron-astrocyte cocultures and hippocampal slices and found that, compared to the soma, astrocyte processes showed enhanced calcium responses to stimulation of the metabotropic glutamate receptor (mGluR). The enhanced calcium response observed in processes resulted from an increased density of mGluRs, rather than from differences in the distribution or sensitivity of the calcium release machinery. Analysis of the movement of single mGluR5s revealed a membrane barrier that selectively blocked the movement of mGluR5 between astrocyte somata and their processes. Noting that various neurological disorders are associated with abnormal calcium signaling in astrocytes, the authors speculate that the existence of this barrier—and thereby of compartmentalized calcium signals—could allow individual processes to regulate associated partners (synapses or blood vessels) independently, in the absence of a somatic calcium signal. Metabotropic glutamate receptor (mGluR)–dependent calcium ion (Ca2+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca2+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca2+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca2+ responses. Immunocytochemistry indicated that these polarized Ca2+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca2+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca2+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

PDK1 Regulates Cell Proliferation and Cell Cycle Progression through Control of Cyclin D1 and p27Kip1 Expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a “floxed” allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1lox/lox MEFs but not in Pdk1+/+ MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G0-G1 phase to the S phase was delayed, and cell cycle progression at G2-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27Kip1 expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G0-G1 to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G2-M progression in these cells required both overexpression of cyclin D1 and depletion of p27Kip1 by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27Kip1.

Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation

In a previous study, we demonstrated that β1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc3Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5−/−) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc3Cer synthase activity was detected in the tissues of these mice. These results demonstrate that β3GnT5 is the sole enzyme synthesizing Lc3Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5−/− B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5−/− resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5−/− B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5−/− B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5−/− B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.

Mouse liver gangliosides.

The major gangliosides from mouse liver were purified and characterized by t.l.c., g.l.c., sialidase treatment, and a methylation study. GM3(NeuAc), GM3(NeuGc), GM2(NeuGc), GM1(NeuGc), and GDla(NeuGc, NeuGc) were identified. The structural identification of three of the gangliosides, GM2(NeuGc), GM1(NeuGc), and GDla(NeuGc, NeuGc), was supported by the results of 1H-n.m.r. analysis, and the structures of GM3(NeuGc), GM2(NeuGc), and GM1(NeuGc) were further confirmed by negative-ion fast-atom bombardment mass spectrometry. Ganglioside mapping showed that there was polymorphic variation of gangliosides in the liver of inbred strains of mice and that the major gangliosides were GM3(NeuGc) in WHT/Ht, GM2(NeuGc) in BALB/c and C3H/He, and GM2(NeuGc), GM1(NeuGc), and GDla(NeuGc, NeuGc) in ICR mice. Gangliosides containing N-acetylneuraminic acid, except for GM3(NeuAc), were not detected as major gangliosides in the strains of mice we analyzed.

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

Significance Reversible and repetitive structural changes are essential for ligand-gated ion channels to mediate biological signaling. The inositol 1,4,5-trisphosphate receptor (IP3R) assembles ligand-gated ion channels that mediate calcium signaling. IP3 activates channels at a distance by reversible allosteric changes in the IP3R tetramer. Here we show a new mode of posttranslational modification that irreversibly blocks allosteric changes in the IP3R. We identified an IP3R-modifying enzyme as tissue transglutaminase that inhibits IP3R function by locking subunit configurations. This modification chronically impaired calcium signaling and autophagy regulation in living cells, and up-regulated modification was observed in Huntington disease models. To our knowledge, this is the first demonstration of transglutaminase-catalyzed posttranslational modification in ligand-gated channel allostery and provides a new framework for enzymatic regulation of allostery. The inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP3R type 1 (IP3R1) and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.

Overexpression of KLF15 transcription factor in adipocytes of mice results in down-regulation of SCD1 protein expression in adipocytes and consequent enhancement of glucose-induced insulin secretion.

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic β cells mediated through modulation of oxidative stress.

Effects of repeated electroconvulsive seizure on cell proliferation in the rat hippocampus.

Electroconvulsive therapy (ECT) is known as a successful treatment for severe depression. Despite great efforts, the biological mechanisms underlying the beneficial effects of ECT remain largely unclear. In this study, animals received a single, 10, or 20 applications of electroconvulsive seizure (ECS), and then cell proliferation and apoptosis were investigated in the subgranular zone (SGZ) of the dentate gyrus. We analyzed whether a series of ECSs could induce changes in the dentate gyrus in a dose‐response fashion. A single‐ECS seizure significantly increased cell proliferation in the SGZ by ∼2.3‐fold compared to sham treatment. After 10 ECSs, a significant increase in cell proliferation was observed in the SGZ by ∼2.4‐fold compared to sham treatment. Moreover, 10 ECSs induced a significant increase in cell proliferation by 1.3‐fold compared to a single‐ECS group. However, cell proliferation did not differ between the group with 20 ECSs and sham group. In addition, a significant increase in the number of apoptotic cells was found in the group with 10 ECSs, whereas no significant change in it was found in either a single ECS or 20 ECSs group compared to sham treatment. These findings indicate that the optimal number of treatments and duration of stimulation requires investigation. Further studies are needed to elucidate the intracellular mechanisms underlying both effective and excessive ECT. Synapse 64:814–821, 2010.

Effects of single and repeated electroconvulsive stimulation on hippocampal cell proliferation and spontaneous behaviors in the rat.

Electroconvulsive therapy (ECT) has therapeutic effects on refractory depression and schizophrenia, although its biological mechanisms are still unclear. Recent studies in rodents suggest that electroconvulsive stimulation-induced seizures (ECSs) influence hippocampal adult neurogenesis, which has gained considerable traction as a possible cellular substrate for the treatment of depression. The aim of this study is to explore alteration of neurogenesis in the hippocampus following ECSs and the relationship between neurogenesis and behavior in rats. In the present study, we administered a single or 10-repeated application of electroconvulsive stimulations that reliably resulted in seizure (an animal model of electroconvulsive therapy) to rats. Then cell proliferation of newborn cells in the subgranular zone (SGZ) of the dentate gyrus (DG) was investigated 3 and 14 days after ECS treatments. Cell differentiation was also examined 4 weeks after newly formed cells were confirmed. As a result, ECS-induced cell proliferation in the hippocampus showed biphasic changes after ECS. The amount of cell proliferation at 3 days after the last ECS increased twice as much as the sham group. However, the number of proliferating cells at 14 days later decreased to a half of the sham level. Differentiation of newly formed cells was not influenced in ECS-treated groups compared with sham-treated groups. In addition, we investigated the effects of ECS on behavioral changes in rats by measuring locomotor activity in an open field test and spontaneous alteration behavior in a Y-maze test. Spontaneous behavior and memory function were not influenced by repeated ECSs. These results suggest that repeated ECSs affect progenitors that have a limited ability for cell proliferation, like amplifying progenitors, to increase newly generated neurons without negative behavioral change.

Application of a Multivalent Glycoprobe: Characterization of Sugar-Binding Specificity of Siglec Family Proteins

Publisher Summary This chapter describes how the glycoprobe was prepared and then applied to the characterization of the binding specificity of sialic acid-binding Ig-like lectins (Siglecs). Siglecs () are a family of lectins expressed mainly on hematopoietic cells. The binding of Siglecs to specific sialoglycoconjugates is associated with their specific functions, for example, cellular attachment or recognition. The interaction of Siglecs with sialoglycoconjugates on the cell surface may comprise an initial step in the process of cellular attachment or recognition. The interaction is believed to occur in a multivalent manner that is, an array of Siglecs on the cell surface binds multiple sialoglycoconjugates on the recognized cells. To characterize the multivalent interaction of Siglecs, a polyvalent “glycoprobe” that carries more than 100 oligosaccharides was developed. Glycochain structures of gangliosides and oligosaccharides and their recognition by Siglecs are tabulated. The chapter discusses the preparation and polymerization of oligosaccharyl streptavidin, the assay for adherent cells, and suspension assay of Siglecs.