Effects of combined treatment with fermented soybean (natto) intake and exercise on bone metabolism in ovariectomized rats.

OBJECTIVES: Using ovariectomized rats, we examined the influence of combined exercise tolerance and natto intake on the bone loss inhibitory effect. METHODS: We divided female Wistar rats into the following groups: Ovariectomy, Ovariectomy + Exercise, Ovariectomy + Natto Intake, Ovariectomy + Exercise + Natto Intake, and Pseudo-operative (Sham group). After conducting experiments on each group, we collected the tissues and performed morphological and molecular biological analyses. RESULTS: In comparison with the Ovariectomy group, only in the Ovariectomy + Exercise group was there a significant bone loss inhibitory effect in the femoral cancellous bone. Although there was a tendency toward this trend seen in the Natto Intake and Exercise + Natto Intake groups, these differences were not significant. The increase in messenger RNA expression levels of alkaline phosphatase (osteoblast marker) in the bone marrow caused by ovariectomy was suppressed by individual factors, and by those in combination. However, messenger RNA expression levels of estrogen receptor alpha in the bone marrow showed a decreasing tendency with each factor, and decreased significantly with the combination, similar to the Sham group. CONCLUSION: This suggests that natto intake and exercise maintain bone mass by different molecular mechanisms and that these two factors do not simply act synergistically in combination to maintain bone mass.

Peroxisome proliferator-activated receptor α mediates di-(2-ethylhexyl) phthalate transgenerational repression of ovarian Esr1 expression in female mice.

Di-(2-ethylhexyl)-phthalate (DEHP) is a phthalate ester that binds peroxisome proliferator-activated receptor α (PPARα) to induce proliferation of peroxisomes and regulate the expression of specific target genes. The question of whether the effect of DEHP on female reproductive processes is mediated via PPARα-dependent signaling is controversial. In this study, we investigated the effect of exposure to DEHP on ovarian expression of estrogen receptor α (Esr1) and aromatase (Cyp19a1) in three generations of Sv/129 wild-type (WT, +/+) and PPARα (-/-) knockout mice. Compared with untreated controls, ovarian expression of Esr1 decreased in response to DEHP treatment in the F0 (0.56-fold, P=0.19), F1 (0.45-fold, P=0.023), and F2 (0.35-fold, P=0.014) generations of WT mice, but not PPARα-null mice. Our data indicate that transgenerational repression by DEHP of ovarian Esr1 gene expression is mediated by PPARα-dependent pathways. Further studies are required to elucidate the mechanisms underlying crosstalk between PPARα and Esr1 signaling in reproductive processes.

A rhythmic change of vesicular glutamate transporter (VGLUT) 2 expression in the rat pineal gland.

The pineal gland secretes melatonin under circadian control via nocturnal noradrenergic stimulation, and expresses vesicular glutamate transporter (VGLUT) 1, VGLUT2 and a VGLUT1 splice variant (VGLUT1v). Although we previously reported that VGLUT2 mRNA level of rat pineal gland at postnatal day 21 is higher in the nighttime than in daytime, questions remained as to the time of postnatal onset of this phenomenon and a 24-h change in the mRNA or protein level at postnatal days. The day-night difference in VGLUT2 mRNA level was evident 14 days after birth. In the adult, VGLUT2 mRNA and protein levels increased in the dark phase, with the protein level showing a 6-h delay. The nocturnal elevation in VGLUT2 mRNA level diminished under the constant light condition but persisted under the constant dark condition. The present data suggest that VGLUT2 in the rat pineal gland is involved in some nocturnal glutamatergic function.

SH3P2 is a negative regulator of cell motility whose function is inhibited by ribosomal S6 kinase-mediated phosphorylation.

Although the extracellular signal-regulated kinase (ERK) pathway functions downstream of Ras in induction of the cell motility response, the detailed molecular mechanism by which this pathway regulates cell motility has remained elusive. The application of a functional expression cloning strategy to discover proteins that regulate cell motility has resulted in the identification of an SH3 domain-containing protein, SH3P2. Overexpression of SH3P2 in HeLa S3 cells inhibited cell motility, whereas RNA interference-mediated depletion of SH3P2 enhanced motility in various tumor cell lines, suggesting that SH3P2 functions as a negative regulator of cell motility. The expression level of SH3P2 alone did not correlate well with the motility of tumor cells, however. SH3P2 was phosphorylated on Ser(202) by ribosomal S6 kinase (RSK) in an ERK pathway-dependent manner, and such phosphorylation inhibited the ability of SH3P2 to suppress cell motility. The RSK inhibitor BI-D1870 suppressed SH3P2 phosphorylation and tumor cell motility as effectively as did the MEK inhibitor PD184352. Furthermore, expression of the unphosphorylatable SH3P2 mutant SH3P2(S202A) inhibited tumor cell motility, indicating that phosphorylation of SH3P2 at Ser(202) is a key determinant of such motility. These results suggest that SH3P2 is an essential molecule that functions downstream of the ERK pathway to modulate cell motility.

Particular subpopulations of midbrain and hypothalamic dopamine neurons express vesicular glutamate transporter 2 in the rat brain.

Vesicular glutamate transporters (VGLUT1, -2, and -3) mediate the accumulation of transmitter glutamate into synaptic vesicles in glutamatergic neurons. VGLUT1 and VGLUT2 are more reliable glutamatergic neuron markers, since VGLUT3 also exists in other neuron types. To study whether the dopaminergic neuron uses glutamate as a cotransmitter, we analyzed VGLUTs expression in dopamine neurons of adult male rats by in situ hybridization and immunohistochemistry. In the ventral midbrain, in situ hybridization analysis revealed no VGLUT1 mRNA expression, a widespread but discrete pattern of VGLUT2 mRNA expression, and a highly limited expression of VGLUT3 mRNA. Reverse-transcriptase polymerase chain reaction analysis detected full-length VGLUT2 gene transcripts in the ventral midbrain. Using in situ hybridization combined with tyrosine hydroxylase (TH) immunostaining, only VGLUT2 signals were detectable in some TH-labeled neurons of A10 dopamine neuron groups, with the highest incidence (20%) in the rostral linear nucleus of the ventral tegmental area. In the forebrain, VGLUT2 signals were demonstrated in half of the A11 TH-labeled neurons in the hypothalamus. Double-label immunostaining for VGLUT2 and vesicular monoamine transporter 2 or TH showed that double-labeled varicosities are rarely observed in any target regions examined of A10 and A11 dopamine neuron groups. These results indicate that VGLUT2 is expressed in subsets of A10 and A11 dopamine neurons, which might release dopamine and glutamate separately from different varicosities in the majority of their single axons.

Up-regulation of VGLUT2 expression in hypothalamic-neurohypophysial neurons of the rat following osmotic challenge.

A second vesicular glutamate transporter (VGLUT2) has been reported to be expressed in neurosecretory neurons of the hypothalamic-neurohypophysial system. To study its role in the neurosecretory neurons, we evaluated the expression of the VGLUT2 gene in the paraventricular (PVN) and supraoptic (SON) nuclei as well as the immunoreactivity in the neurohypophysis under euhydrated and chronic hyperosmotic conditions with in situ hybridization and immunohistochemistry. The intensity of hybridization signals in the PVN, SON and thalamus of rats subjected to water deprivation for 7 days, or drinking 2% NaCl for 4 or 7 days, was compared with that of euhydrated rats (control). The overall intensity in the entire PVN or SON, but not the thalamus, was higher in osmotically stimulated rats than in controls. Within the PVN, a significantly higher intensity of signals than that of controls was found only in the dorsolateral posterior magnocellular region in 4-day salt-loaded rats and in all subregions in water-deprived or 7-day salt-loaded rats. The intensity in the SON was higher in the stimulated rats than in controls, regardless of subregions. In the neurohypophysis, VGLUT2 staining was frequently localized in vasopressin terminals of control rats and was apparently reduced in stimulated rats. These results indicate that VGLUT2 is principally expressed in magnocellular vasopressin neurons, suggesting some local effect of intrinsic glutamate on neurohypophysial hormone secretion.

Expression and immunohistochemical localization of vesicular glutamate transporter 2 in the migratory pathway from the rat olfactory placode.

The localization of vesicular glutamate transporter 2 (VGLUT2) was examined by immunohistochemistry and in situ hybridization histochemistry in the developing rat olfactory region with special relation to the spatiotemporal location of NCAM, a neural cell adhesion molecule expressed in differentiated neurons, and the calcium-binding protein calbindin D-28k, a marker of neurons migrating from the vomeronasal organ anlage (Y. Toba et al. (2001) J. Neuroendocrinol., 13, 683-694). Both VGLUT2 and NCAM immunoreactivities were first detected at embryonic day 11.5 (E11.5) in the neuronal cell mass beneath the telencephalic vesicle. After E12.5, VGLUT2-immunoreactive cells were detected in the migratory pathways from both medial and lateral olfactory pits, anlagen of the vomeronasal organ and olfactory epithelium. Between E15.5 and E19.5, moderate to intense VGLUT2 immunoreactivity was observed in cell clusters situated along NCAM-bearing vomeronasal nerves, and frequently colocalized with calbindin D-28k immunoreactivity. Using in situ hybridization histochemistry, VGLUT2 mRNA signals were detected in the clustered cells as well as in cells of the vomeronasal and olfactory epithelium. After E20.5, migrating cells gradually decreased in number and VGLUT2 immunoreactivity attenuated in the clustered cells, although calbindin D-28k immunoreactivity in these residual cells was still intense. The presence of intense VGLUT2 immunoreactivity in neurons actively migrating from the olfactory placode suggests that this transporter is involved in the migratory process of these neurons.

BMP7/ActRIIB regulates estrogen-dependent apoptosis: new biomarkers for environmental estrogens.

A ligand-receptor pair, bone morphogenetic protein-7 (BMP7) and activin receptor IIB (actRIIB), was identified from a pool of DNA fragments recovered from MCF7 cells treated with 17beta-estradiol (E2) by chromatin immunoprecipitation with antiestrogen receptor-alphaantibody. The E2 responsiveness of both genes was confirmed in MCF cells and in the mouse uterus. Repeated treatment with E2 resulted in decreased expression of both actRIIB and BMP7 mRNA in the uteri of ovariectomized mice. A single oral administration of bisphenol A (BPA), an environmental estrogen, inhibited actRIIB and BMP7 expression and apoptosis in the luminal epithelium of the mouse uterus at diestrus (or early proestrus). This decrease, due to BPA administration, was restored by an estrogen receptor (ER) antagonist suggesting that it is mediated through ERs. These results suggest that E2 and BPA suppress estrogen-dependent apoptosis of epithelial cells of the endometrium through down-regulation of actRIIB and BMP7. Thus, we propose that BMP7 and actRIIB, a ligand-receptor pair, are involved in regulation of the apoptotic signaling pathway and might therefore be new biomarkers of the effects of environmental estrogens on the female reproductive tract.

Over-expression of corticotropin-releasing factor mRNA in inferior olivary neurons of rolling mouse Nagoya.

Expression of corticotropin-releasing factor (CRF) mRNA was examined in the inferior olivary nucleus (ION) of an ataxic mutant, rolling mouse Nagoya (RMN) by semi-quantitative in situ hybridization. The most marked difference in the level of CRF mRNA signals between RMN and non-ataxic littermates (control mice) was observed in the beta-subnucleus and ventrolateral protrusion of the ION. The level of signals in these subnuclei was about twofold higher in RMN than in the controls. Signal levels in the dorsal nucleus, principal nucleus and subnucleus A were slightly but significantly higher in RMN than in the controls. In the other subnuclei, there were no differences in signal level between RMN and controls. These results suggest a region-related over-expression of CRF mRNA in the ION of RMN. This may be responsible for the increased sensitivity of some Purkinje cells to glutamate, resulting in ataxic symptoms of RMN.

Expression of inorganic phosphate/vesicular glutamate transporters (BNPI/VGLUT1 and DNPI/VGLUT2) in the cerebellum and precerebellar nuclei of the rat.

Expression of inorganic phosphate/vesicular glutamate transporters (BNPI/VGLUT1 and DNPI/VGLUT2) was studied in the cerebellum and precerebellar nuclei of rats using immunohistochemistry and in situ hybridization. DNPI/VGLUT2-stained mossy fibers were principally seen in the vermis (lobules I and VIII-X) and flocculus, whereas BNPI/VGLUT1-stained mossy fibers were localized throughout the cortex. Some vermal and floccular mossy fibers were stained for both transporters. High levels of DNPI/VGLUT2 mRNA hybridization signals were demonstrated in many neurons throughout the vestibular nuclear complex as well as the lateral reticular, external cuneate, inferior olivary and deep cerebellar nuclei. Significant BNPI/VGLUT1 mRNA signals were demonstrated in the lateral reticular nucleus and vestibular nuclear complex but not in the inferior olivary nucleus, indicating that climbing fibers have DNPI/VGLUT2 only. These results show that DNPI/VGLUT2 is expressed preferentially to vestibulo-, reticulo- and cuneocerebellar neurons, some of which also possess BNPI/VGLUT1, suggesting some differential and co-operative functions between DNPI/VGLUT2 and BNPI/VGLUT1 in the cerebellum.

Differential expression of two distinct vesicular glutamate transporters in the rat retina.

Expression and cellular localization of vesicular glutamate transporters (BNPI and DNPI) were studied in the rat retina. RT-PCR showed expression of both transporter mRNAs. hybridization demonstrated BNPI mRNA signals in the inner segments of photoreceptors and the inner nuclear layer, whereas DNPI mRNA signals were confined to the ganglion cell layer. Punctate BNPI immunoreactivity was localized in the inner and outer plexiform layers, and weak DNPI immunoreactivity was detectable only in some cells and fibers of the ganglion cell layer. The present study suggests that BNPI exists in photoreceptors and bipolar cells, while DNPI is present in ganglion cells, as specific systems in distinct glutamatergic neurons of the retina.