Yoshikazu Mikami

CD271/p75(NTR) inhibits the differentiation of mesenchymal stem cells into osteogenic, adipogenic, chondrogenic, and myogenic lineages.

We describe a novel role for CD271 in the differentiation of mesenchymal stem cells (MSCs), including deciduous dental pulp stem cells (DDPSCs) and murine multipotent MSCs (C3H10T1/2 cells). The CD271(+) subpopulation of deciduous dental pulp cells (CD271(+)/DDPSCs) and the forced expression of CD271 in C3H10T1/2 (10T271) were analyzed by fluorescence-activated cell sorting. CD271 expression was detected in DDPSCs that expressed both CD44 and CD90, simultaneously, and the clonogenic capacity of the CD271(+)/DDPSCs was higher than that of the CD271(-)/DDPSCs that expressed both CD44 and CD90. Further, the differentiation of CD271(+)/DDPSCs into osteoblasts and adipocytes was inhibited although CD271(-)/DDPSCs were capable of differentiating into osteoblasts and adipocytes. CD271 was overexpressed in C3H10T1/2 cells, which have the potential to differentiate into osteoblasts, adipocytes, chondrocytes, and myocytes. CD271 inhibited the differentiation of C3H10T1/2 cells into any of these lineages. These results indicate a role for CD271 in inhibiting the differentiation of MSCs.

Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells

Alkaline phosphatase (ALP) is generally believed to be a faithful marker of osteoblast differentiation, and its expression is induced by bone morphogenetic protein‐2 (BMP‐2) and dexamethasone (Dex). However, the effects of combined administration of BMP‐2 and Dex on ALP transcription have not been extensively examined. In this study, we found that BMP‐2 and Dex synergistically increase ALP levels in mouse C3H10T1/2 pluripotent stem cells. However, switching from one inducer to the other, by adding BMP‐2 or Dex to cell cultures at different times, was no more effective than continuous treatment with either inducer alone. A significant induction of ALP mRNA expression was observed only in cells continuously treated with both inducers. This result suggests that both BMP‐2 and Dex may act in the same pathway or at the same stage of differentiation. A luciferase assay using ALP promoter deletion constructs showed that a region of the promoter containing a putative signal transducer and activator of transcription 3 (STAT3) response element (SRE) responds to treatment with a combination of BMP‐2 and Dex. Furthermore, a ChIP assay indicated that STAT3 bound to the SRE. In addition, a STAT3 siRNA suppressed the synergistic effect of BMP‐2 and Dex on ALP levels. These results indicate that STAT3 may play an important role in regulating ALP expression. To our knowledge, this is the first time that STAT3 has been implicated in the regulation of ALP expression by BMP‐2 and Dex. These findings raise the possibility of developing new strategies for the enhancement of bone formation using a combination of BMPs and Dex. J. Cell. Physiol. 223: 123–133, 2010.

Dexamethasone modulates osteogenesis and adipogenesis with regulation of osterix expression in rat calvaria-derived cells†

Osteoblasts and adipocytes originate from common mesenchymal progenitor cells and although a number of compounds can induce osteoblastic and adipogenic differentiation from progenitor cells, the underlying mechanisms have not been elucidated. The present study examined the synergistic effects of dexamethasone (Dex) and bone morphogenetic protein (BMP)‐2 on the differentiation of clonal mesenchymal progenitor cells isolated from rat calvaria into osteoblasts and adipocytes, as well as the effects of the timing of treatment. Cells were cultured for various periods of time in the presence of Dex and/or BMP‐2. When cells were treated with Dex + BMP‐2 during the early phase of differentiation, they differentiated into adipocytes. However, when cells were treated with Dex + BMP‐2 during the late phase of differentiation, a synergistic effect on in vitro matrix mineralization was observed. To examine differences between the early and late phases of differentiation, ALP activity was measured in the presence of BMP‐2. ALP activity increased markedly on Day 9, corresponding to the onset of the synergistic effect of Dex. Dex treatment inhibited osterix (OSX) expression in cells committed to adipogenic differentiation, but not in cells committed to osteogenic differentiation following BMP‐2 treatment. The isoform2 OSX promoter region was found to be involved in the effects of Dex on cells during the early phase of differentiation. Furthermore, cells stably expressing OSX (isoform2) formed mineralized nodules even though they had been treated with Dex + BMP‐2 during the early phase of differentiation. It appears that Dex modulates osteogenesis and adipogenesis in mesenchymal stem cells by regulating OSX expression. J. Cell. Physiol. 226: 739–748, 2011.

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 × 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 × 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.

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.

Poly I:C-induced expression of intercellular adhesion molecule-1 in intestinal epithelial cells

Intercellular adhesion molecul‐1 (ICAM‐1) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules and plays perdominant roles in recruitment and trafficking of leucocytes to sites of inflammation. ICAM‐1 expression in intestinal epithelial cells (IECs) is enhanced by several stimuli, such as proinflammatory cytokines, bacterial infections or pathogen‐associated molecular patterns. One of these stimuli, double‐stranded RNA (dsRNA), is a by‐product of viral replication and can be recognized by its cognate receptor Toll‐like receptor 3 (TLR‐3). In spite of expression of both TLR‐3 and ICAM‐1 in IECs, correlation between TLR‐3‐signalling and ICAM‐1 expression has never been examined in IECs. In the present study, we investigated whether poly I:C, an analogue of dsRNA, can stimulate the expression of ICAM‐1 in IEC line, HT‐29. Poly I:C‐stimulation up‐regulated the expression of ICAM‐1 mRNA by real‐time polymerase chain reaction. Enhanced expression of ICAM‐1 was confirmed in protein level by immunofluoresense cell staining and enzyme‐linked immunosorbent assay by measuring the released soluble ICAM‐1 in culture supernatant. As the stimulation effect was reduced by pre‐treatment of the cells with anti‐TLR‐3 antibody, poly I:C‐binding signal was thought to be sensed by TLR‐3 on the surface of HT‐29. The results of luciferase assay and nuclear factor kappa‐b (NF‐kB) inhibitor treatment experiments indicated that the downstream signal was mainly transduced by transcription factor, NF‐kB. All these results demonstrated the connection between TLR‐3 signalling and ICAM‐1 expression in HT‐29 cells and indicated the importance of coordinated function of both innate and adaptive immunity against viral infections.

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.

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.

Oral bacterial extracts facilitate early osteogenic/dentinogenic differentiation in human dental pulp–derived cells

OBJECTIVES Bacterial metabolites demineralize dental hard tissues, and soluble factors lead to tertiary dentinogenesis in the area of the dentin-pulp complex. However, it is unclear whether the oral bacteria are directly involved in the differentiation of dental pulp cells. In this study, we evaluated the effect of oral bacterial extracts on cellular differentiation in human dental pulp-derived cells (hDPC). STUDY DESIGN The hDPC were obtained from third molar teeth, and the cells were subcultured. The sonicated extracts were obtained from Porphyromonas gingivalis (gram-negative) and Streptococcus mutans (gram-positive). The effect of bacterial extracts on cellular growth and differentiation in hDPC were tested. RESULTS Alkaline phosphatase activity and bone sialoprotein (BSP) gene expression were increased in hDPC exposed to low concentrations of both sonicated extracts, whereas the activity was decreased upon exposure to high concentrations of sonicated extracts from P. gingivalis. CONCLUSION This is the first evidence that oral bacteria have a positive effect on cellular differentiation in hPDC.

The P75 neurotrophin receptor regulates proliferation of the human MG63 osteoblast cell line.

The 75 kDa transmembrane protein, p75(NTR), is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75(NTR) in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75(NTR) in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line. MG63 cells and MC3T3-E1 cells expressing exogenous p75(NTR) protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75(NTR) induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75(NTR) stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63. To elucidate any different effects of p75(NTR) expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75(NTR). Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell proliferation. Inhibition of trks by K252a suppressed p75(NTR)-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in P75(NTR) from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75(NTR) signaling associated with trk receptors promotes both cell proliferation and osteoblast differentiation, but that p75(NTR)-mediated proliferation may be suppressed by signaling from the p75(NTR)/NgR complex.