TBR31-2 is one of the bone marrow stromal cell lines. Differentiation toward osteogenic cells and calcification was observed when TBR31-2 cells were cultured for 4 weeks. Bone morphogenetic protein-2 (BMP-2) stimulated alkaline phosphatase (ALP) activity in a dose-and time-dependent manner. On the other hand, troglitazone increased oil droplet accumulation in a dose-dependent manner. In the presence of BMP-2, an increase of expression in osteogenic cell differentiation marker genes and a decrease of expression in adipocyte differentiation marker genes were observed with the exception of the induced expression of peroxisome proliferator-activated receptor g g (PPARg g), however, troglitazone, a ligand of PPARg g treatment exhibited the opposite tendency. Interestingly, treatment with both BMP-2 and troglitazone resulted in a decrease of ALP activity and an increase of oil droplet accumulation. Reverse tanscription-polymerase chain reaction (RT-PCR) analysis also indicated that osteogenic differentiation markers decreased and that adipocyte differentiation markers increased. Thus, when the cells were cultured with BMP-2, osteogenic differentiation was enhanced while the expression of PPARg g was maintained, and the addition of troglitazone caused a significant number of differentiated cells into adipocytes. These findings indicate that BMP-2 enhanced osteogenic differentiation and the expression of adipogenic transcription factor (PPARg g) followed by osteogenic differentiation without activation of PPARg g by its ligand.

The development of osteoblasts is regulated by various hormones including parathyroid hormone (PTH), 1) 1a,25-dihydroxyvitamin D 3 (1a,25(OH)D 3 ), 2) estrogen 3) and glucocorticoids 4) as well as other local factors, such as bone morphogenetic proteins (BMPs), hedgehogs and the transcription factor, core-binding factor a-1 (Cbfa1). 5) In recent years, extracellular nucleotides have also been considered to be regulating factors in bone cells; for example, the release of ATP from osteoblasts into the extracellular space after stimulation by mechanical strain [6][7][8][9] and stimulating factors, 6) the presence of functional P2 receptors in osteoblasts, 6,10) or the role of extracellular nucleotides in autocrine/paracrine signaling in the cell. 6,[10][11][12] It is generally accepted that immortalization of various cell types by simian virus (SV) 40 T-antigen could at least lead to some stabilization of cell-type specific functions and that the growth and differentiation of cell lines established by the temperature-sensitive (ts) mutant of SV 40 T-antigen gene could be controlled by temperature shifts. [13][14][15][16] Yanai et al. tried to establish many stromal cell lines from the bone marrow of transgenic mice harboring ts SV 40 T-antigen gene. [17][18][19][20] TBR31-2 is one of those stromal cell lines, and Negishi et al. reported that TBR31-2 cells have characteristics of undifferentiated cells that have the potential to express the multipotential cell lineages, including osteoblasts during longterm culture. 21) In addition, as their differentiation is strictly controlled by the temperature shift and culture medium, they are advantageous when examining the molecular mechanism separating the proliferative phase from differentiation phase.The role of the increase in intracellular calcium ion level ([Ca 2ϩ ] i ) induced by ATP in regulating TBR31-2 cells has not yet been investigated. Therefore, we investigated the effects of extracellular ATP on [Ca 2ϩ ] i in TBR31-2 cells induced to differentiate toward osteoblasts. MATERIALS AND METHODSMaterials RITC80-7 medium and plastic tissue culture flasks were obtained from Asahi Techno Glass (Tokyo, Japan), and other materials were obtained from the following sources: fetal bovine serum (FBS) and alpha-modified minimum essential medium (a-MEM) from Gibco-BRL Laboratories (Great Island, NY, U.S.A.); human recombinant epidermal growth factor (EGF) from Wakunaga Pharmaceutical (Tokyo, Japan); human recombinant BMP-2 and L-ascorbic acid from Wako Pure Chemicals (Osaka, Japan); bicinchoninic acid (BCA) protein assay reagent from Pierce (Rockford, IL, U.S.A.); ATP from Oriental Yeast Co., Ltd. (Tokyo, Japan); uridine 5Ј-triphosphate (UTP), ADP, a,bmethylene ATP (a,b-mATP), MRS2179 (a selective P2Y 1 receptor antagonist), U73122 (a phospholipase C inhibitor), and thapsigargin (TSG, a calcium pump inhibitor) from Sigma Chemical Co. (St. Louis, MO, U.S.A.); pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulphonic acid (PPADS, a non-specific P2 receptor antagonist) from Tocri...

1. High-blood pressure or diabetes may be related to the loss of bone mass or the development of osteoporosis. We examined the mechanical bone properties of the SHR/NDmcr-cp (SHR-cp) rat, an obese strain that develops hypertension, hyperlipidaemia and insulin-independent diabetes.2. The mechanical properties of the femur of 22-week-old Wistar-Kyoto (WKY) and SHR-cp rats were measured by Peng's three-point bending procedure modified by Shintani. Femurs were then defatted and dried. After weighing, the dried bones were ashed and the ash was weighed. The values of the dry weight, ash weight and ash weight/dry weight (%) were used as a description of the physical parameters of the bone.3. All values of stiffness, strength, toughness and ductility in SHR-cp were significantly lower than those of WKY rats (P < 0.05). The value of ash weight/dry weight (%) was lower in SHR-cp rats (P < 0.01). These results showed that bone fragility was greater in SHR-cp rats, indicative of osteopenia.