ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities.

ATP binding to the ϵ subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ϵ subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ϵ subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ϵ subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ϵ subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ϵ subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ϵ subunit was in its extended-state conformation. The present study reveals a novel role of the ϵ subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.

Bone morphogenetic protein-2 induces the differentiation of a mesenchymal progenitor cell line, ROB-C26, into mature osteoblasts and adipocytes.

AIMS: Osteoblasts and adipocytes originate from common precursor cells. We examined the effects of bone morphogenetic protein-2 (BMP-2) on the molecular mechanisms governing the diametric actions of BMP-2 on simultaneous mature osteoblast and adipocyte differentiation in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). MAIN METHODS: The present study using RT-PCR, Western blotting and ELISA investigated the effects of BMP-2 on transcription factors for osteoblasts (Runx2, Dlx5, Osterix, Msx2 and AJ18) and adipocytes (PPARgamma2), osteoblastic markers, alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC), and adipocyte differentiation-dependent protein, aP2 in C26 cells. KEY FINDINGS: BMP-2 increased not only mRNA and protein expressions of Dlx5 followed by inducing Osterix and BSP levels in a dose- and time-dependent manner with an increase in Runx2, Msx2, AJ18, ALP and OC levels, but also induced PPARgamma2 and aP2 levels. At the light microscopic level, BMP-2 induced mature osteoblastic and adipogenic differentiation in the C26 cultures not only by an increase in the number of ALP-positive osteoblasts, in their staining intensity and in the number of mature adipocytes with Oil Red O-positive lipid droplets, but also mineralized matrix formation of the cultures assessed by detecting an increase in calcium concentration and the formation of Alizarin Red S-positive mineralization nodules. SIGNIFICANCE: These results indicate that BMP-2 induces the differentiation of C26 mesenchymal progenitors into mature osteoblasts and adipocytes and the usefulness of this cell line for studying the regulatory mechanism of osteoblast and adipocyte differentiation from common mesenchymal progenitors.

Alignment control exercise changes lower extremity movement during stop movements in female basketball players.

This study was intended to evaluate a short-term intervention designed to improve the lower extremity alignment with neutral position during stop movements of female basketball players when performing a quick-stop jump shot. In this study, 20 healthy female college basketball players (mean age 20.5 years) participated. The authors monitored two groups for 4 weeks: one which trained (n=10) and one which did not train (n=10). The exercise program emphasized the neutral position of lower extremities for dynamic alignment control; the players avoided the valgus position with their knees. Kinematics analysis with a lower extremity angle in the stop action was performed using three digital video cameras and analyzed using software. Following the intervention exercise program, each subject was re-evaluated at two and four weeks to determine changes in movement patterns during the "sink shot" task. Two-way analysis of variance models were used to determine differences at pre-intervention, at mid-term, and post-intervention. After the 2-week intervention, the trained athletes group made basketball shots with greater change of the lower extremity motion pattern during the stop action than did non-trained athletes (p<0.05). However, no significant differences were found between other data of the 2-week and 4-week intervention groups. Results of this study show that two weeks of training can improve the dynamic alignment control of the lower extremities, as measured using the alignment angle of the coronal plane and the torsion angle of horizontal plane during a stop action.

Dexamethasone promotes DMP1 mRNA expression by inhibiting negative regulation of Runx2 in multipotential mesenchymal progenitor, ROB-C26.

Dentin matrix protein 1 (DMP1) is an acidic phosphorylated extracellular protein and essential for mineralization of dentin and bone; however, the precise mechanism regulating DMP1 expression is not fully understood. A synthetic glucocorticoid (GC), dexamethasone (Dex), promotes an early osteoblast differentiation of a mesenchymal progenitor, ROB-C26 (C26), in parallel with inductive expression of an osteoblast-specific transcription factor, Runx2, and other extracellular matrix proteins such as osteocalcin and bone sialoprotein (BSP). We have examined the effect of Dex on DMP1 expression via induction of Runx2 in C26 cells. Real time RT-PCR showed that Dex increases DMP1 mRNA expression levels at time- and dose-dependent manners and a GC antagonist, RU486, drastically inhibited DMP1 mRNA expression levels. Furthermore, Dex increased the luciferase activity of six-repeated osteoblast-specific cis-acting element 2 (6 x OSE2), which is the binding sequence of Runx2, suggesting that Dex stimulates DMP1 expression via activation of Runx2. However, unexpected results showed that overexpression of exogenous Runx2 depressed DMP1 mRNA expression level, even after cells had been treated with Dex, while downregulated expression of endogenous Runx2 enhanced Dex-induced DMP1 mRNA expression level. These results imply that large amounts of exogenous Runx2 inhibit DMP1 expression, whereas small amounts are more effective for Dex-induced DMP1 expression in C26 cells. Therefore, Dex may activate some factors that inhibit negative action of Runx2 on DMP1 expression. Since mitogen-activating protein kinase (MAPK) phosphatase-1 (MKP-1) has been reported to affect the Dex-induced osteoblast differentiation via decrease of Runx2-phosphorylation, we focus on the relationship between MKP-1 and DMP1 expression. Dex increases MKP-1 expression, and overexpression of exogenous MKP-1 showed significant increase of luciferase activity of 6 x OSE up to the level detected in Dex-treated C26 cells. However, no inductive DMP1 mRNA expression level was found in C26 cells unlike BSP and OPN. These results suggest that although MKP-1 increases DNA-binding activity of Runx2, DMP1 expression may require the collaboration of MKP-1 and additive factors to stimulate Runx2-mediated DMP1 expression in the post-transcriptional event of Dex-treated C26 cells.

Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP.

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.

Inductive effects of dexamethasone on the mineralization and the osteoblastic gene expressions in mature osteoblast-like ROS17/2.8 cells.

We examined the effects of dexamethasone (Dex), a synthetic glucocorticoid, on the formation of mineralized bone nodules and the gene expressions of the late osteoblastic markers, bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) in mature osteoblast ROS17/2.8 cells. Treatment of ROS17/2.8 cells with Dex resulted in the induction of mineralization accompanied with increasing BSP and OC expressions. Previous reports have demonstrated that BSP and OC expressions are regulated by Runx2. Then, we hypothesized that Dex might promote osteoblastic differentiation and mineralization on ROS17/2.8 by Runx2. In this study, no effect was observed in mRNA and protein expression of Runx2. However, the transcriptional activity of Runx2 was enhanced by Dex treatment. Furthermore, the Dex-induced BSP and OC expressions decreased after the transfection of Runx2 small-interfering RNAs (siRNAs). These results suggested that the enhancement of Runx2 transcriptional activity by Dex treatment may be followed by the activation of osteoblast marker genes, such as BSP and OC to thereby produce a bone-specific matrix that subsequently becomes mineralized.

Glucocorticoids induce the differentiation of a mesenchymal progenitor cell line, ROB-C26 into adipocytes and osteoblasts, but fail to induce terminal osteoblast differentiation.

To clarify the effects of glucocorticoids (GCs) on osteoblast and adipocyte differentiation, we investigated the effects of dexamethasone (Dex), a GC analogue on transcription factors for osteoblasts (Runx2, Dlx5 and Osterix) and adipocytes (C/EBPs such as C/EBPalpha, C/EBPbeta and C/EBPdelta, and PPARgamma2), late osteoblastic markers, bone sialoprotein (BSP) and osteocalcin (OC), and adipocyte differentiation-dependent protein, aP2 in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). C26 cells were dose- and time-dependently responsive to Dex in terms of an increase in not only mRNA and protein expressions of the C/EBPs, PPARgamma2 and aP2, but also Runx2, Dlx5, BSP and OC with no induction of Osterix, which is considered to act mainly on terminal osteoblast differentiation. Cycloheximide pretreatment indicated that Dex signaling immediately increases expressions of the C/EBPs and Dlx5, while expressions of the rest of the genes require de novo protein synthesis. Continuous Dex treatment stimulated adipocyte formation, but failed to induce Osterix expression and mineralized matrix formation. However, BMP-2 treatment of Dex-treated cells induced Osterix expression and subsequent mineralized matrix formation. These results indicate that Dex up-regulates the C/EBPs followed by increasing PPARgamma2 and aP2 expressions in C26 cells to induce adipocyte differentiation, while Dex enhances Dlx5 followed by increasing Runx2, BSP and OC expressions at gene and protein levels, but cannot induce Osterix expression, suggesting that Dex does not promote their terminal osteoblast differentiation.

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP.

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.

Autoregulatory mechanism of Runx2 through the expression of transcription factors and bone matrix proteins in multipotential mesenchymal cell line, ROB-C26.

Runx2 is essential for osteoblast differentiation and gene expression of bone matrix proteins, however, little is known about the mechanism regulating its activity. In this study, the role of Runx2 on gene expression of transcription factors, AJ18, Msx2, and Dlx5, was examined in vitro. It is known that AJ18 and Msx2 act as repressors to inhibit activity of Runx2, whereas Dlx5 promotes its activity. An expression vector inserted Runx2 cDNA was transiently overexpressed in a rat multipotential mesenchymal cell line, ROB-C26 (C26). Real time reverse transcription-PCR analysis showed that, in exogenous Runx2-overexpressing C26 cells (C26-Rx), AJ18 expression increased 1.8-fold, Msx2 expression increased 3.0-fold, and Dlx5 expression increased 2.7-fold compared to the cells transfected with vector alone (C26-Co). Luciferase assay also showed that, in C26-Rx, AJ18 promoter activity increased 2.1-fold compared to C26-Co. Furthermore, gene expression of alkaline phosphatase (ALP) and bone matrix proteins including type I collagen (Col1), osteocalcin (OC), osteopontin (OPN), and matrix Gla protein (MGP) was examined. In C26-Rx, MGP expression increased 1.8-fold, and OPN expression increased 1.4-fold compared to C26-Co. However, no significant difference in Col1, ALP, and OC expressions was detected between C26-Rx and C26-Co. These results suggest that the existence of autoregulatory feed back loops, which inhibit Runx2 activity through the interaction of AJ18, Dlx5, and Msx2 cooperating with that of MGP and OPN, interferes with the differentiation of C26 cells toward mature osteoblasts.