The effect of repeated stress on KCC2 and NKCC1 immunoreactivity in the hippocampus of female mice.

K(+)-Cl(-) co-transporter (KCC2) and Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) are the main regulators of neuronal intracellular chloride concentration; altered expression patterns of KCC2 and NKCC1 have been reported in several neurodegenerative diseases. In this paper, we show the effect of repeated stress on KCC2, NKCC1, and serine 940 phosphorylated KCC2 (pKCC2(ser940)) immunoreactivity. The data were obtained from the hippocampus of female mice using single-plane confocal microscopy images. The mean fluorescence intensity of the perisomatic area of neurons, defined as raw fluorescence intensity (RFI) was calculated. Repeated stress (RS) resulted in a decrease in perisomatic area of immunoreactive (IR)-KCC2 and an increase of the IR-NKCC1. In addition, RS decreased perisomatic IR-pKCC2(ser940), corresponding to that of KCC2. The data in this article support the results of a previous study [1] and provide the details of immunohistological methods. Interpretation of the data in this article can be found in "Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice" by Tsukahara et al. [1].

A relation between osteoclastogenesis inhibition and membrane-type estrogen receptor GPR30.

Disruption of the cooperative balance between osteoblasts and osteoclasts causes various bone disorders, some of which are because of abnormal osteoclast recruitment. Osteoporosis, one of the bone disorders, is not effectively treated by currently available medicines. In addition to the development of novel drugs for palliative treatment, the exploitation of novel compounds for preventive treatment is important in an aging society. Quercetin, a major flavonoid found in many fruits and vegetables, has been expected to inhibit cancer and prevent several diseases because of its anti-inflammatory and estrogenic functions. It has been reported that quercetin has the potential to reduce bone resorption, but the mechanism by which this compound affects the differentiation of osteoclasts remains unknown. Here, using a bone marrow cell-based in vitro osteoclast differentiation system from bone marrow cells, we found that the ability of quercetin to inhibit osteoclastogenesis was related to its estrogenic activity. The inhibition was partially blocked by a specific antagonist for the nuclear receptor estrogen receptor α, but a specific antagonist of the membrane-type receptor GPR30 completely ablated this inhibition. Furthermore, quercetin suppressed the transient increase of Akt phosphorylation induced by the stimulation of macrophage colony-stimulating factor and receptor activator of NF-κB ligand with no effect on MAPK phosphorylation, suggesting exquisite crosstalk between cytokine receptor and G-protein coupled receptor signaling. These results indicate the important role of GPR30 in osteoclast differentiation and provide new insights to the development of new treatments for osteoporosis.

Repeated stress-induced expression pattern alterations of the hippocampal chloride transporters KCC2 and NKCC1 associated with behavioral abnormalities in female mice.

The balance of cation-chloride co-transporters, particularly KCC2 and NKCC1, is critical for GABAergic inhibitory signaling. However, KCC2/NKCC1 balance is disrupted in many neurodegenerative diseases. Moreover, correlations between chronic stress, KCC2 and NKCC1 in the hippocampus remain poorly understood. Despite the fact that emotional disorders in humans are far more prevalent in women, there have been relatively few studies about female subjects. Here we investigated behaviors and expression patterns of KCC2 and NKCC1 in the hippocampi of female mice under chronic stress. Repeated stress (RS) was induced in experimental mice by repeated forced water administration. Then, expression patterns of GABAergic signaling molecules were identified by immunohistochemical analysis and performance was assessed using several behavioral tests. The results of semi-quantitative analysis showed that RS decreased KCC2 expression and increased NKCC1 expression in membranes of granular and pyramidal cells in the hippocampus. The novel object recognition (NOR) test and sociability test revealed that RS induced cognitive and sociability deficits, whereas RS increased the time spent in the open arms of the elevated plus maze test and induced attention deficits in other tests. In summary, RS induced alterations in membrane KCC2/NKCC1 balance in the hippocampus of female mice, which may contribute to GABAergic disinhibition associated with cognitional, sociability and attention deficits.

Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion.

Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR(-/-)) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR(-/-) osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR(-/-) preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V(0) subunit d2 and dendritic cell-specific transmembrane protein in LDLR(-/-) plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR(-/-) mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.

Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.

Although extensive studies have done much to clarify the molecular mechanisms of osteoclastogenesis during the last ten years, there may still be unknown molecules associated with osteoclast differentiation. Thus, we used fluorescent differential display to screen for genes whose expression is induced by receptor activator of NF-κB ligand (RANKL), a crucial molecule for osteoclast formation. We identified caveolin-1 (Cav-1) as a RANKL-induced gene. Cav-1 is a major structural protein of caveolae and lipid rafts, cholesterol-enriched microdomains in the plasma membrane (PM). The RANKL-induced Cav-1 was immediately conveyed to lipid rafts. Conversely, expression of flotillin-1 (Flot-1), another scaffolding protein of lipid rafts, was reduced during osteoclastogenesis, indicating conversion of Flot-1-predominant rafts into Cav-1-enriched rafts. However, in vitro osteoclastogenesis of precursor cells from Cav-1-null mice was comparable to that of wild-type mice, while Cav-2 expression in the knockout osteoclasts was maintained. Conversely, Cav-2 gene silencing in Cav-1-null osteoclast precursors using siRNA for Cav-2 increased osteoclast formation, suggesting that the Cav-1/Cav-2 complex may act as a negative regulator for osteoclastogenesis. On the other hand, destruction of lipid rafts by removal of cholesterol from the PM by methyl-ß-cyclodextrin (MCD) treatment caused disordered signal transductions for osteoclastogenesis, such as hyperactivation of Erk1/2 and insensitivity of Akt to RANKL stimulus. The abnormal signaling was reproduced by deleting exogenous lipoproteins from the culture medium, which also resulted in reduced osteoclast formation. In addition, the deletion caused delayed expression of nuclear factor of activated T cells c1 (NFATc1), and depressed its activation in the cytosol and inhibited its translocation into nuclei. Simultaneously, the deletion reduced the level of FcRγ, a trigger protein for initiating the calcium signaling needed to activate NFATc1, and decreased Cav-1 in lipid rafts. These findings indicate that the molecular mechanisms of osteoclastogenesis are highly dependent on extracellular lipoprotein and the integrity of lipid rafts, and suggest possible involvement of cholesterol.

Protective protein/cathepsin A down-regulates osteoclastogenesis by associating with and degrading NF-kappaB p50/p65.

Disruption of the cooperative function balance between osteoblasts and osteoclasts causes various bone disorders, some of which are attributed to abnormal osteoclast recruitment. Osteoclast differentiation is dependent on the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) as well as the macrophage colony-stimulating factor. The osteoclast formation induced by cytokines requires activation of NF-kappaB, AP-1 and nuclear factor of activated T cells c1. However, osteoclasts are not the only cell types that express these transcription factors, suggesting that some unknown molecules specific for osteoclasts may associate with the transcription factors. Here, we explored the possibility of molecules binding directly to NF-kappaB and cloned protective protein/cathepsin A (PPCA) by yeast two-hybrid screening using a cDNA library of osteoclast precursors. Forced expression of PPCA with p50/p65 in HEK293 cells decreased both the level of p50/p65 proteins and the transcriptional activity. Abundant PPCA was detected in the lysosomes of the transfected HEK293 cells, but a small amount of this enzyme was also present in the cytosolic fraction. In addition, over-expression of PPCA caused the disappearance of p50/p65 in both the lysosomal and cytosolic fractions. PPCA was expressed throughout osteoclastogenesis, and the expression was slightly up-regulated by RANKL signaling. Knockdown of PPCA in osteoclast precursors with PPCA siRNA stimulated binding of nuclear proteins to oligonucleotides containing an NF-kappaB binding motif and increased osteoclastogenesis. Our present results indicate a novel role for PPCA in osteoclastogenesis via down-regulation of NF-kappaB activity and suggest a new function for PPCA as an NF-kappaB-degrading enzyme in addition to its known multifunctional properties.

PGC7/Stella protects against DNA demethylation in early embryogenesis.

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.

Production of IL-7 is increased in ovariectomized mice, but not RANKL mRNA expression by osteoblasts/stromal cells in bone, and IL-7 enhances generation of osteoclast precursors in vitro.

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220(+) cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less.

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.

GATA-2 and GATA-2/ER display opposing activities in the development and differentiation of blood progenitors.

GATA-2 is a zinc finger transcription factor essential for the development of hematopoiesis. While GATA-2 is generally considered to play an important role in the biology of hematopoietic stem and progenitor cells, its function within these compartments is not well understood. Here we have employed both conditional expression of GATA-2 and conditional activation of a GATA-2/estrogen receptor (ER) chimera to examine the effect of enforced GATA-2 expression in the development and differentiation of hematopoietic progenitors from murine embryonic stem cells. Consistent with the phenotype of GATA-2 null animals, conditional expression of GATA-2 from a tetracycline-inducible promoter enhanced the production of hematopoietic progenitors. Conditional activation of a GATA-2/ER chimera produced essentially opposite effects to those observed with conditional GATA-2 expression. GATA-2 and GATA-2/ER differ in their binding activities and transcriptional interactions from other hematopoietic-associated transcription factors such as c-Myb and PU.1. While we have exploited these differences in activity to explore the transcriptional networks underlying hematopoietic cell fate determination, our results suggest that care should be taken in interpreting results obtained using only chimeric proteins.

Novel arterial infusion chemotherapy using cisplatin, 5-fluorouracil, and leucovorin for patients with advanced hepatocellular carcinoma.

Advanced hepatocelluar carcinoma (HCC) has a poor prognosis. In this study, the authors evaluated the efficacy of chemotherapy using cisplatin (CDDP), 5-fluorouracil (5-FU), and leucovorin (LV), comparing our regimen with chemotherapy using CDDP and 5-FU. Nineteen patients with advanced HCC were treated by arterial infusion of a chemotherapeutic agent via a subcutaneously implanted injection port. In Group A (n=9), one course of chemotherapy consisted of the daily administration of CDDP (10 mg/1 h, on 5 days) and LV (12 mg/10 min, on 5 days) followed by 5-FU (250 mg/5 h, on 5 days). In Group B (n=10), except for the administration of LV, the same regimen was employed. This course was repeated each week for 4 weeks. In Group A, two patients showed a complete response (CR), and the other three showed a partial response (PR). In Group B, two patients showed PR. The response rate (CR+PR/all cases) in Group A was significantly higher than that in Group B (56 vs. 20%; P=0.022). The 1- and 2-year survival rates of Group A (66.7, 44.4%) were significantly higher than those of Group B (10, 0%) (P=0.033). These results suggest that our regimen may be useful in treating patients with advanced HCC.

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells.

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.