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Osteoporosis increases the risk of bone fractures (e.g., the femur), reduces a person's activities of daily living (ADL) and increases the likelihood of being bedridden. Therapeutic drugs for osteoporosis include oral bisphosphonates and intravenous receptor activator of nuclear factor-κB ligand (RANKL) antibodies, both of which suppress osteoclast activity, as well as the subcutaneously administered recombinant human parathyroid hormone (PTH), which activates osteoblasts. However, there is currently no oral osteogenesis-promoting drug. In the present study, we found a low-molecular-weight compound, KY-273, with osteogenesis promoting effects. KY-273 induced osteoblast differentiation in ST2 cells and in rat bone marrow-derived mesenchymal stem cells at a dose of 0.1 μM. On the other hand, KY-273 did not clearly exert differentiation effects in osteoclasts, chondrocytes, adipocytes, or myoblasts. In ovariectomized rats, KY-273 clearly increased serum bone alkaline phosphatase (ALP) by at a dose of 3 mg/kg for 8 weeks, and increased both the cortical bone volume and medullary volume of the diaphyseal and epiphyseal regions of femoral bone, but did not affect trabecular bone. Although alendronate (used to decrease bone loss) increased trabecular bone, it did not have any significant effects on cortical bone. PTH increased epiphysis cortical and trabecular bone volume, and reduced medullary volume. KY-273 also displayed good oral absorption in rats. In conclusion, KY-273 is a promising candidate for use as an oral anti-osteoporosis drug with osteogenesis promoting effects.

In this study, we evaluated the role of the GABA(A) receptor (GABA(A)R), expressed by undifferentiated neural progenitors isolated from fetal mouse neocortex, in the mechanisms relevant to self-replication and differentiation toward neuronal and astroglial lineages. Round spheres were formed with clusters of proliferating cells within 2-4 days during culture with epidermal growth factor (EGF), whereas the size of these clusters was drastically increased in proportion to increasing durations up to 10 days. Sustained exposure to the GABA(A)R agonist muscimol at 100 microM led to significant increases in the size of neurospheres cultured for 6-10 days, with increased proliferative activity and unchanged lactate dehydrogenase release in a manner sensitive to the GABA(A)R antagonist bicuculline. Muscimol also significantly increased the incorporation of 5-bromo-2'-deoxyuridine in neurospheres in a bicuculline-sensitive manner, whereas both high potassium and nifedipine significantly decreased the neurosphere area with increased numbers of apoptotic cells. Prior activation of GABA(A)R significantly promoted the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage after the removal of EGF, with a concomitant decrease in neuronal marker protein expression. In neurospheres with GABA(A)R activation, a significant and predominant increase was seen in mRNA expression of CNTF receptors. These results suggest that prior tonic activation of GABA(A)R may preferentially promote the differentiation by CNTF of neural progenitor cells toward an astroglial lineage through selective up-regulation of CNTF receptor expression in the developing mouse brain., ((c) 2008 Wiley-Liss, Inc.)

Neural progenitor cell is a generic term for undifferentiated cell populations composed of neural stem, neuronal progenitor, and glial progenitor cells with abilities for self-renewal and multipotentiality. In this study, we have attempted to evaluate the possible functional expression of N-methyl-D-aspartate (NMDA) receptors by neural progenitor cells prepared from neocortex of 18-day-old embryonic rats. Cells were cultured in the presence of basic fibroblast growth factor (bFGF) for different periods up to 12 days under floating conditions. Reverse transcription-polymerase chain reaction and fluorescence imaging analyses revealed transient expression of functional NMDA receptors in neurospheres formed by clustered progenitors during the culture with bFGF. A similarly potent increase was seen in the fluorescence intensity after brief exposure to NMDA in cells differentiated after the removal of bFGF under adherent conditions, and an NMDA receptor antagonist invariably prevented these increases by NMDA. Moreover, sustained exposure to NMDA not only inhibited the formation of neurospheres when exposed for 10 days from day 2 to day 12 but also promoted spontaneous and induced differentiation of neurospheres to cells immunoreactive for a neuronal marker protein on immunocytochemistry and Western blotting analyses. These results suggest that functional NMDA receptors may be transiently expressed to play a role in mechanisms underlying the modulation of proliferation along with the determination of subsequent differentiation fate toward a neuronal lineage in neural progenitor cells of developing rat neocortex.

In this study, we have attempted to evaluate the possible role of metabotropic GABA(B) receptors (GABA(B)R) expressed by neural progenitor cells prepared from neocortex of embryonic Std-ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABA(A)R, GABA(B)R, and GABA(C)R subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration-dependent increases in the total area and proliferation activity of neurospheres at 10-300 microM, while the GABA(B)R agonist baclofen at 100 microM significantly increased the size of neurospheres expressing both GABA(B)R1 and GABA(B)R2 subunits in a manner sensitive to a GABA(B)R antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABA(B)R1 subunit. In neurospheres of GABA(B)R1-null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABA(B)R may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self-replication in the developing mouse brain., ((c) 2008 Wiley-Liss, Inc.)

We evaluated the possible functional expression of metabotropic glutamate receptors (mGluRs) by neural progenitors from embryonic mouse neocortex. Constitutive expression was seen with group I, II, and III mGluRs in undifferentiated cells and neurospheres formed by clustered cells during culture with epidermal growth factor. The group III mGluR agonist, L-2-amino-4-phosphonobutyrate, drastically reduced proliferation activity at 1-100 microM without inducing cell death, with group I and group II mGluR agonists being ineffective, in these neurospheres. Both forskolin and a group III mGluR antagonist significantly increased the proliferation alone, but significantly prevented the suppression by L-2-amino-4-phosphonobutyrate. Activation of group III mGluR significantly decreased mRNA expression of the cell cycle regulator cyclinD1, in addition to inhibiting the transactivation mediated by cAMP of cyclinD1 gene in the pluripotent P19 progenitor cells. Prior activation of group III mGluR led to a significant decrease in the number of cells immunoreactive for a neuronal marker, with an increase in that for an astroglial marker irrespective of differentiation inducers. These results suggest that group III mGluR may be functionally expressed to suppress self-renewal capacity through a mechanism related to cAMP formation with promotion of subsequent differentiation into astroglial lineage in neural progenitors.

Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors capable of self-replication and differentiation from discrete fetal rat brain structures. A reverse transcription-polymerase chain reaction analysis revealed the expression of particular GABA(A) receptor (GABA(A)R), GABA(B)R-1 and GABA(C)R, but not GABA(B)R-2, subunits in neocortical cells before commitment. Sustained exposure to the GABA(A)R agonist muscimol at 100 mumol/L led to significant increases in the mitochondrial activity and the total areas of neocortical neurospheres formed during the cultivation for 12 days in a manner sensitive to a GABA(A)R antagonist, with lactate dehydrogenase release being unchanged. Moreover, prior sustained exposure to muscimol significantly facilitated the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage, with a concomitant decrease in the neuronal marker protein expression, in an antagonist-sensitive manner on Western blotting analysis. However, muscimol failed to significantly affect the expression of both marker proteins in cells differentiated in either the presence or absence of all-trans-retinoic acid. These results suggest that prior activation of GABA(A)R may preferentially facilitate the commitment by CNTF of neural progenitor cells toward an astroglial lineage after simulation of the self-replication activity in the developing rat brain.