Masako Naito

Low-intensity pulsed ultrasound-induced ATP increases bone formation via the P2X7 receptor in osteoblast-like MC3T3-E1 cells.

Low‐intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dentistry. It has been shown that LIPUS induces the secretion of extracellular adenosine triphosphate (ATP), a key mediator of osteoblast response to mechanical stimuli. However, the detailed mechanism of LIPUS‐induced osteogenesis has been elusive. In this study, we investigated the role of the P2X7 receptor in LIPUS‐induced osteogenesis. LIPUS induced the release of extracellular ATP, differentiation of osteoblasts and osteogenesis via the P2X7 receptor, without affecting the activity of alkaline phosphatase (ALPase). These results suggest that LIPUS‐induced extracellular ATP promotes bone formation via the osteoblast P2X7 receptor independently of ALPase.

Inhibition of Wnt/β-catenin signaling by dexamethasone promotes adipocyte differentiation in mesenchymal progenitor cells, ROB-C26

Dexamethasone (Dex) stimulates the differentiation of mesenchymal progenitor cells into adipocytes and osteoblasts. However, the mechanisms underlying Dex-induced differentiation have not been clearly elucidated. We examined the effect of Dex on the expression and activity of Wnt/β-catenin signal-related molecules in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). Dex induced the mRNA expression of Wnt antagonists, dickkopf-1 (Dkk-1), and Wnt inhibitory factor (WIF)-1. Immunocytochemical analysis showed that the downregulation of β-catenin protein expression by Dex occured concomitantly with the increased expression of the PPARγ protein. Dex decreased phosphorylation of Ser9-GSK3β and expression of active β-catenin protein. To examine the effects of Dex on Wnt/β-catenin activity, we used immunocytochemistry to analyze TCF/LEF-mediated transcription during Dex-induced adipogenesis in Wnt indicator (TOPEGFP) C26 cells. Our results demonstrated that Dex repressed TCF/LEF-mediated transcription, but induced adipocyte differentiation. Treatment with a GSK3β inhibitor attenuated Dex-induced inhibition of TCF/LEF-mediated transcriptional activity, but suppressed Dex-induced adipocyte differentiation, indicating that adipocyte differentiation and inhibition of Wnt/β-catenin activity by Dex are mediated by GSK3β activity. Furthermore, β-catenin knockdown not only suppressed Dex-induced ALP-positive osteoblasts differentiation but also promoted Dex-induced adipocytes differentiation. These results suggest that inhibition of β-catenin expression by Dex promotes the differentiation of mesenchymal progenitor cells into adipocytes.

Suppression of lamin A/C by short hairpin RNAs promotes adipocyte lineage commitment in mesenchymal progenitor cell line, ROB-C26

Lamin A/C gene encodes a nuclear membrane protein, and mutations in this gene are associated with diverse degenerative diseases that are linked to premature aging. While lamin A/C is involved in the regulation of tissue homeostasis, the distinct expression patterns are poorly understood in the mesenchymal cells differentiating into adipocytes. Here, we examined the expression of lamin A/C in a rat mesenchymal progenitor cell-line, ROB-C26 (C26). Immunocytochemical analysis showed that lamin A/C was transiently down-regulated in immature adipocytes, but its expression increased with terminal differentiation. To elucidate the role of lamin A/C expression on mesenchymal cell differentiation, lamin A/C expression was suppressed using short hairpin RNA (shRNA) molecules in C26 cells. In the absence of adipogenic stimuli, lamin A/C shRNA decreased alkaline phosphatase (ALP) activity, but induced preadipocyte factor -1 (Pref-1) mRNA expression. In the presence of adipogenic stimuli, lamin A/C knockdown promotes adipocytes differentiation, as assessed by the detection of an increase in Oil Red O staining. RT-PCR analysis showed that lamin A/C shRNA resulted in increased mRNA expression of PPARγ2 and aP2 during adipocyte differentiation. These results suggest that decreased lamin A/C expression levels not only suppress osteoblast phenotypes but also promote adipocyte differentiation in C26 cells.

Dexamethasone inhibits chondrocyte differentiation by suppression of Wnt/β-catenin signaling in the chondrogenic cell line ATDC5

Glucocorticoids (GCs) regulate proliferation and differentiation in cultured mesenchymal cells through the modulation of various molecules. However, the relationship between growth factor signaling and GCs in differentiating chondrocytes has not been elucidated. In this study, we examined the effects of Wnt/β-catenin signaling on chondrocyte differentiation and the effects of a GC analogue, dexamethasone (Dex), on Wnt/β-catenin signaling activity by using a chondrocyte progenitor cell line ATDC5. Western blot analysis and TCF/LEF-optimized promoter EGFP (TOPEGFP) assay showed that both β-catenin protein levels and TCF/LEF transcription were up-regulated during insulin–transferrin–sodium selenite (ITS)-induced chondrogenic differentiation. Morphological analysis showed that TCF/LEF transcription activity was most prominent in cartilage nodule-like structures. Furthermore, a β-catenin mutant with constitutive transcriptional activity (ΔN90) showed increased Alcian blue staining intensity and mRNA expression of Sox9, Col2a, aggrecan, Col10, and alkaline phosphatase, even in the absence of ITS stimulation. In contrast, Dex suppressed formation of ITS-induced cartilage nodule-like structures, TCF/LEF-mediated transcription, and β-catenin protein levels. Real-time PCR analysis showed that Dex increased the mRNA expression levels of secreted frizzled-related protein 1 (sFRP1) and Axin2. Furthermore, treatment with a sFRP1 inhibitor or the ΔN90 β-catenin mutant transfection attenuated Dex-induced suppression of cartilage matrix production by increasing Sox9 mRNA levels. These results suggest that Dex inhibits chondrocyte differentiation via down-regulation of Wnt/β-catenin signaling, which promotes chondrocyte differentiation in ATDC5 cells.

Lamin A overexpression promotes osteoblast differentiation and calcification in the MC3T3-E1 preosteoblastic cell line

Lamin A/C is a component of the nuclear lamina, which is involved in cellular proliferation and differentiation. However, the mechanism by which lamin A regulates osteoblast differentiation is not well understood. In this study, we investigated lamin A/C expression during osteoblast differentiation in a preosteoblastic cell line, MC3T3-E1. Real-time PCR analysis showed that lamin A/C mRNA expression was upregulated during BMP-2 induced osteoblast differentiation. Treatment with the estrogen receptor antagonist, fulvestrant, inhibited osteoblast differentiation and the upregulation of lamin A/C mRNA and protein expressions in the presence of BMP-2. These results clearly demonstrated that lamin A/C expression correlates with osteoblast differentiation. To determine the roles of lamin A expression in osteoblast differentiation, MC3T3-E1 cells were transfected with a vector overexpressing lamin A. Results showed that lamin A overexpression promoted osteoblast differentiation and calcification by inducing the expression of alkaline phosphatase, type 1 collagen, BSP, osteocalcin, and DMP-1 in the presence of BMP-2. Furthermore, lamin A overexpression partially restored osteoblastic capacity in the presence of fulvestrant by increasing the expression of BSP, osteocalcin, and DMP-1. These results suggest that lamin A plays important roles in maintaining the osteoblast differentiation and function.

LIPUS suppressed LPS-induced IL-1α through the inhibition of NF-κB nuclear translocation via AT1-PLCβ pathway in MC3T3-E1 cells

Inflammatory cytokines, interleukin (IL)‐1, IL‐6, and TNF‐α, are involved in inflammatory bone diseases such as rheumatoid osteoarthritis and periodontal disease. Particularly, periodontal disease, which destroys alveolar bone, is stimulated by lipopolysaccharide (LPS). Low‐intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dental treatments. However, the mechanism underlying effects of LIPUS on LPS‐induced inflammatory cytokine are not well understood. We therefore aimed to investigate the role of LIPUS on LPS‐induced IL‐1α production.

Tetrahydrobiopterin Supplementation: Elevation of Tissue Biopterin Levels Accompanied by a Relative Increase in Dihydrobiopterin in the Blood and the Role of Probenecid-Sensitive Uptake in Scavenging Dihydrobiopterin in the Liver and Kidney of Rats

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase (NOS) and aromatic amino acid hydroxylases. BH4 and 7,8-dihydrobiopterin (BH2) are metabolically interchangeable at the expense of NADPH. Exogenously administered BH4 can be metabolized by the body, similar to vitamins. At present, synthetic BH4 is used as an orphan drug for patients with inherited diseases requiring BH4 supplementation. BH4 supplementation has also drawn attention as a means of treating certain cardiovascular symptoms, however, its application in human patients remains limited. Here, we tracked biopterin (BP) distribution in blood, bile, urine, liver, kidney and brain after BH4 administration (5 mg/kg rat, i.v.) with or without prior treatment with probenecid, a potent inhibitor of uptake transporters particularly including organic anion transporter families such as OTA1 and OAT3. The rapid excretion of BP in urine was driven by elevated blood concentrations and its elimination reached about 90% within 120 min. In the very early period, BP was taken up by the liver and kidney and gradually released back to the blood. BH4 administration caused a considerable decrease in the BH4% in blood BP as an inevitable compensatory process. Probenecid treatment slowed down the decrease in blood BP and simultaneously inhibited its initial rapid excretion in the kidney. At the same time, the BH4% was further lowered, suggesting that the probenecid-sensitive BP uptake played a crucial role in BH2 scavenging in vivo. This suggested that the overproduced BH2 was taken up by organs by means of the probenecid-sensitive process, and was then scavenged by counter-conversion to BH4 via the BH4 salvage pathway. Taken together, BH4 administration was effective at raising BP levels in organs over the course of hours but with extremely low efficiency. Since a high BH2 relative to BH4 causes NOS dysfunction, the lowering of the BH4% must be avoided in practice, otherwise the desired effect of the supplementation in ameliorating NOS dysfunction would be spoiled.

Up-regulation of Axin2 by dexamethasone promotes adipocyte differentiation in ROB-C26 mesenchymal progenitor cells

Dexamethasone (Dex) regulates osteoblastic and adipocytic differentiation in mesenchymal progenitor cells through regulation of Wnt/β-catenin signaling. To elucidate the regulatory mechanisms underlying the effects of Dex, we examine the expression of Axin2, which is an intracellular inhibitor of Wnt/β-catenin signaling, in ROB-C26 clonal mesenchymal progenitor cells (C26). We observed the induction of Axin2 mRNA in C26 cells in response to Dex treatment. Treatment with a glucocorticoid receptor (GR) antagonist, mifepristone, showed that Dex-induced up-regulation of Axin2 is mediated by the GR. In the absence of Dex, gene silencing by using Axin2-targeted short hairpin RNA increased the number of alkaline phosphatase (ALP)-positive and nuclear β-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear β-catenin-positive cells. Furthermore, Axin2 knockdown in Dex-treated cells suppressed adipocyte differentiation (as determined by reduced Oil Red O staining), reduced the number of PPARγ-positive and aP2-positive cells and decreased the mRNA expression of PPARγ2 and aP2. These results suggest that Axin2 plays a key role in adipocyte and osteoblastic differentiation by controlling β-catenin expression.

Promyelocytic leukemia zinc finger mediates glucocorticoid-induced cell cycle arrest in the chondroprogenitor cell line ATDC5.

Glucocorticoids (GCs) affect the proliferation of growth plate chondrocytes. In this study, we investigated the role of the GC-inducible promyelocytic leukemia zinc finger (PLZF) gene in chondrocyte differentiation by using the chondrogenic cell line ATDC5. PLZF overexpression suppressed cell cycle progression (p < 0.01) and promoted differentiation into hypertrophic chondrocytes by inducing mRNA expression of alkaline phosphatase (p < 0.01), and the cyclin-dependent kinase (CDK) inhibitor p21 (p < 0.01). In contrast, PLZF knockdown impaired differentiation into hypertrophic chondrocytes and promoted cell cycle progression (p < 0.01). Treatment with the GC analogue dexamethasone (10(-6) M) suppressed cell cycle progression in ATDC5 cells. PLZF shRNA attenuated dexamethasone-induced cell cycle arrest (p < 0.01) by downregulating the mRNA expression of the CDK inhibitors p21 and p57 (p < 0.01). These results clearly indicated that PLZF promoted differentiation into hypertrophic chondrocytes and mediated dexamethasone-induced cell cycle arrest by regulating CDK inhibitors.

Influence of estrogen deficiency on guided bone augmentation: investigation of rat calvarial model and osteoblast-like MC3T3-E1 cells

The effect of estrogen deficiency in bone augmentation, and the mechanisms by which estrogen deficiency impedes osteoblast differentiation and collagen matrix production, were examined. Twenty female Jcl:Wistar rats were divided into two groups: ovariectomized rats; and control rats. Guided bone augmentation was performed by positioning plastic caps in the calvarium of all animals at 8 wk after ovariectomy or sham surgery. Micro-computed tomography and histological sections were used to determine the amount of bone augmentation within the plastic caps. At 8 wk, there was statistically significantly less newly formed bone volume in ovariectomized rats. Immunohistological staining revealed the rare alignment of runt-related transcription factor 2-positive osteoblast-like cells and collagen I-positive bundle fibers in ovariectomized rats. In cell culture experiments, pre-osteoblast-like cells, MC3T3-E1, were treated with the estrogen receptor antagonist, fulvestrant. In treated cells, alkaline phosphatase activity remained high, whereas Alizarin Red staining was completely inhibited. Extracellular staining intensity of collagen I was decreased after fulvestrant treatment. Consistent with these observations, gene-expression analysis confirmed that fulvestrant treatment led to weaker expression of mRNA for osteogenic transcription factors and bone matrix protein-related genes. The results demonstrate that estrogen deficiency suppresses osteoblast differentiation and collagen matrix production in bone augmentation.