Inductive effect of SORT1 on odontoblastic differentiation of human dental pulp-derived stem cells.

This study investigated the expression of sortilin 1 (SORT1) in cultured human dental pulp-derived stem cells (hDPSCs) and its role in their odontoblastic differentiation. Permanent teeth were extracted from five patients, and the dental pulp was harvested for explant culture. Fluorescence-activated cell sorting was used to analyze the outgrowth of adherent cells and cells that had migrated from the tissue margin. SORT1 expression was detected in hDPSCs simultaneously expressing the mesenchymal stem cell markers CD44 and CD90. The odontoblastic differentiation potential of SORT1-positive hDPSCs was examined via staining for alkaline phosphatase (ALP), an early odontoblastic differentiation marker. ALP staining was more intense in SORT1-positive than in SORT1-negative hDPSCs. Consistently, the expression of mRNA encoding SORT1 and p75NTR, a binding partner of SORT1, increased in SORT1-positive hDPSCs during odontoblastic differentiation. In addition, pro-nerve growth factor (NGF), a ligand for SORT1-p75NTR co-receptor, promoted ALP expression in SORT1-positive hDPSCs, and the interaction between SORT1 and p75NTR was detected using a coimmunoprecipitation assay. The function of SORT1 in odontoblastic differentiation was examined via RNA interference using shRNA targeting SORT1. ALP staining intensity in SORT1/shRNA-transfected cells was markedly lower than in control/shRNA-transfected cells. SORT1 knockdown decreased JUN phosphorylation and recruitment of phosphorylated JUN to the ALP promoter. Collectively, these results indicate that SORT1 is involved in the odontoblastic differentiation of hDPSCs through the JUN N-terminal kinases (JNK)/JUN signaling pathway and that the binding of SORT1 and p75NTR plays an important role in this process.

Bone metastatic mammary tumor cell-derived extracellular vesicles inhibit osteoblast maturation via JNK signaling.

The metastases of breast cancer to bone often cause osteolytic lesions not only by stimulating osteoclasts to resorb the bone but also by inhibiting osteoblasts from bone formation. Although tumor cell-derived extracellular vesicles (EVs) promote osteoclast differentiation and bone resorption, their roles in osteoblast differentiation and functions have not been elucidated. In this study, we investigated the effects of breast cancer cell-derived EVs on osteoblast differentiation and functions in vitro. We found that upon osteogenic induction, 4T1 bone metastatic mouse mammary tumor cell-derived EVs (4T1-EVs) were inhibited matrix mineralization of ST2 mouse bone marrow stromal cells. Temporal expression analysis of osteoblast marker genes, including runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), collagen type I (Col1a1), bone sialoprotein (Bsp), and osteocalcin (Bglap) revealed that 4T1-EVs decreased their expression during the late stage of osteoblast differentiation. Elevated levels of c-Jun N-terminal kinase (JNK) phosphorylation, upon osteogenic induction, were diminished by 4T1-EVs, significantly. In contrast, the nullification of reduced JNK phosphorylation by anisomycin, a potent JNK activator, increased the expression levels of osteoblast differentiation markers. Overall, our data indicated that 4T1-EVs affect osteoblast maturation, at least partially, through the regulation of JNK activity, which provides novel insights into the pathological impact of osteolytic bone metastasis and the role of EVs in osteoblast differentiation.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, a P300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrate or propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

miR-92a-3p encapsulated in bone metastatic mammary tumor cell-derived extracellular vesicles modulates mature osteoclast longevity.

Aberrant osteoclast formation and activation are the hallmarks of osteolytic metastasis. Extracellular vesicles (EVs), released from bone metastatic tumor cells, play a pivotal role in the progression of osteolytic lesions. However, the mechanisms through which tumor cell-derived EVs regulate osteoclast differentiation and function have not been fully elucidated. In this study, we found that 4T1 bone metastatic mouse mammary tumor cell-derived EVs (4T1-EVs) are taken up by mouse bone marrow macrophages to facilitate osteoclastogenesis. Furthermore, treatment of mature osteoclasts with 4T1-EVs promoted bone resorption, which was accompanied by enhanced survival of mature osteoclasts through the negative regulation of caspase-3. By comparing the miRNA content in 4T1-EVs with that in 67NR nonmetastatic mouse mammary tumor cell-derived EVs (67NR-EVs), miR-92a-3p was identified as one of the most enriched miRNAs in 4T1-EVs, and its transfer into mature osteoclasts significantly reduced apoptosis. Bioinformatic and Western blot analyses revealed that miR-92a-3p directly targeted phosphatase and tensin homolog (PTEN) in mature osteoclasts, resulting in increased levels of phospho-Akt. Our findings provide novel insights into the EV-mediated regulation of osteoclast survival through the transfer of miR-92a-3p, which enhances mature osteoclast survival via the Akt survival signaling pathway, thus promoting bone resorption.

Dual effect of polyphosphate on mineralization of rat osteoblast ROS17/2.8 cells in a dose-dependent manner.

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate, is found at high concentrations in osteoblasts. We demonstrated the effects of various polyP concentrations on the mineralization of rat osteoblast ROS17/2.8 cells. Mineralization of ROS17/2.8 was induced by a high polyP concentration (1 mg/mL), which was accompanied by an upregulation of the bone sialoprotein and osteocalcin. In contrast, a low polyP concentration (1 × 10-2 mg/mL) reduced mineralization without affecting the osteogenic gene expression. Furthermore, gene expression profiling and forced expression analysis indicated that phosphodiesterase 11a could be a candidate involved in the dose-dependent effect of polyP on osteoblast mineralization.

Alkaline phosphatase determines polyphosphate-induced mineralization in a cell-type independent manner.

Polyphosphate [Poly(P)] has positive effects on osteoblast mineralization; however, the underlying mechanism remains unclear. In addition, it is unknown whether Poly(P) promotes mineralization in soft tissues. We investigated this by using various cells. Poly(P) concentrations of 1 and 0.5 mg/mL yielded high levels of mineralization in ROS17/2.8 osteoblast cells. Similarly, Poly(P) induced mineralization in cell types expressing alkaline phosphatase (ALP), namely, ATDC5 and MC3T3-E1, but not in CHO, C3H10T1/2, C2C12, and 3T3-L1 cells. Furthermore, forced expression of ALP caused Poly(P)-induced mineralization in CHO cells. These results suggest that ALP determines Poly(P)-induced mineralization in a cell-type independent manner.

Osteocytes up-regulate the terminal differentiation of pre-osteoblasts via gap junctions.

We examined cell-to-cell interaction between pre-osteoblasts and osteocytes using MC3T3-E1 and MLO-Y4, respectively. First, GFP expressing MC3T3-E1 (E1-GFP) cells were generated to isolate the cells from co-culture with MLO-Y4. No changes were observed in the expression of osteogenic transcription factors Runx2, Osterix, Dlx5 and Msx2, but expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in E1-GFP co-cultured with MLO-Y4 was 300-400-fold greater than that in mono-cultured E1-GFP. In addition, mineralized nodule formation was drastically increased in co-cultured E1-GFP cells compared to mono-cultured cells. Patch clamp assay showed the presence of gap junctions between E1-GFP and MLO-Y4. Furthermore, when the gap junction inhibitor carbenoxolone (CBX) was added to the culture, increased expression of ALP and BSP in E1-GFP co-cultured with MLO-Y4 was suppressed. These results suggest that gap junction detected between pre-osteoblasts and osteocytes plays an important role on the terminal differentiation of pre-osteoblasts.

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.

Reactive oxygen species generation required for autophagy induction during butyrate- or propionate-induced release of damage-associated molecular patterns from dying gingival epithelial Ca9-22 cells.

PURPOSE: Bacterial cells in mature dental plaque produce a high concentration of short-chain fatty acids (SCFAs) such as butyrate and propionate. SCFA-treatment on human gingival epithelial Ca9-22 cells induced cell death. However, the exact mechanism underlying cell death remains unclear. In this study, the relationship between reactive oxygen species (ROS) and autophagy induction during SCFA-induced cell death was examined. METHODS: Human gingival epithelial Ca9-22 cells were treated with butyrate or propionate to induce cell death and the number of dead cells were measured using SYTOX-green dye. A siRNA for ATG5 and N-acetylcysteine (NAC) were used for autophagy reduction and ROS-scavenging, respectively. Release of damage-associated molecular patterns (DAMPs) such as Sin3A-associated protein 130 (SAP130) and high-mobility group box 1 (HMGB1) were detected using western blot. RESULTS: Reducing autophagy significantly suppressed SCFA-induced Ca9-22 cell death. ROS generation was observed upon SCFA treatment, and scavenging ROS with NAC decreased cell death. NAC also reduced the SCFA-induced increase in microtubule-associated protein 1 light chain 3B (LC3B)-I and LC3B-II, and mitigated the release of DAMPs. CONCLUSION: The findings suggest that ROS generation is necessary for autophagy, which is required for SCFA-induced cell death and accompanying DAMP release.

Assessing the combined impact of fatty liver-induced TGF-ß1 and LPS-activated macrophages in fibrosis through a novel 3D serial section methodology.

Non-alcoholic steatohepatitis (NASH), caused by fat buildup, can lead to liver inflammation and damage. Elucidation of the spatial distribution of fibrotic tissue in the fatty liver in NASH can be immensely useful to understand its pathogenesis. Thus, we developed a novel serial section-3D (SS3D) technique that combines high-resolution image acquisition with 3D construction software, which enabled highly detailed analysis of the mouse liver and extraction and quantification of stained tissues. Moreover, we studied the underexplored mechanism of fibrosis progression in the fatty liver in NASH by subjecting the mice to a high-fat diet (HFD), followed by lipopolysaccharide (LPS) administration. The HFD/LPS (+) group showed extensive fibrosis compared with control; additionally, the area of these fibrotic regions in the HFD/LPS (+) group was almost double that of control using our SS3D technique. LPS administration led to an increase in Tnfα and Il1ß mRNA expression and the number of macrophages in the liver. On the other hand, transforming growth factor-ß1 (Tgfß1) mRNA increased in HFD group compared to that of control group without LPS-administration. In addition, COL1A1 levels increased in hepatic stellate cell (HSC)-like XL-2 cells when treated with recombinant TGF-ß1, which attenuated with recombinant latency-associated protein (rLAP). This attenuation was rescued with LPS-activated macrophages. Therefore, we demonstrated that fatty liver produced "latent-form" of TGF-ß1, which activated by macrophages via inflammatory cytokines such as TNFα and IL1ß, resulting in activation of HSCs leading to the production of COL1A1. Moreover, we established the effectiveness of our SS3D technique in creating 3D images of fibrotic tissue, which can be used to study other diseases as well.

Possible roles of short-chain fatty acids produced by oral bacteria in the development of alveolar osteitis.

PURPOSE: Alveolar osteitis (dry sockets) is a painful condition characterized by a limited immune response. It is typically caused by the removal of blood clots from extracted tooth sockets, which leads to the fermentation of trapped food remnants by oral bacteria in the cavities, producing high concentrations of short-chain fatty acids (SCFAs). This study examined the effects of SCFAs on immunity and bone metabolism. METHODS: Mouse macrophage Raw264.7 cells were treated with oral bacteria supernatants or SCFA mixtures, and inducible nitric oxide synthase (iNOS) levels were determined by western blot. The same cells were treated with SCFA mixtures in the presence of receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoclast-like cells were counted. MC3T3-E1 cells were treated with SCFA mixtures and stained with alizarin red S. RESULTS: Raw264.7 cells treated with oral bacterial culture supernatants of Porphyromonas gingivalis and Fusobacterium nucleatum inhibited lipopolysaccharide (LPS)-induced iNOS production, likely due to SCFA content. SCFA mixtures mimicking these supernatants inhibited the number of RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive cells and MC3T3-E1 cell mineralization. CONCLUSION: These data suggest that SCFAs produced by P. gingivalis and F. nucleatum may reduce the inflammatory response and mildly induce mineralization of the alveolar walls. These results may contribute to the understanding of alveolar osteitis.

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/ß-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/ß-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 ß-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear ß-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 ß-catenin expression.

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.

Spatiotemporal expression patterns of R-spondins and their receptors, Lgrs, in the developing mouse telencephalon.

Development of the mammalian telencephalon, which is the most complex region of the central nervous system, is precisely orchestrated by many signaling molecules. Wnt signaling derived from the cortical hem, a signaling center, is crucial for telencephalic development including cortical patterning and the induction of hippocampal development. Secreted protein R-spondin (Rspo) 1-4 and their receptors, leucine-rich repeat-containing G-protein-coupled receptor (Lgr) 4-6, act as activators of Wnt signaling. Although Rspo expression in the hem during the early stages of cortical development has been reported, comparative expression analysis of Rspos and Lgr4-6 has not been performed. In this study, we examined the detailed spatiotemporal expression patterns of Rspo1-4 and Lgr4-6 in the embryonic and postnatal telencephalon to elucidate their functions. In the embryonic day (E) 10.5-14.5 telencephalon, Rspo1-3 were prominently expressed in the cortical hem. Among their receptors, Lgr4 was observed in the ventral telencephalon, and Lgr6 was highly expressed throughout the telencephalon at the same stages. This suggests that Rspo1-3 and Lgr4 initially regulate telencephalic development in restricted regions, whereas Lgr6 functions broadly. From the late embryonic stage, the expression areas of Rspo1-3 and Lgr4-6 dramatically expanded; their expression was found in the neocortex and limbic system, such as the hippocampus, amygdala, and striatum. Increased Rspo and Lgr expression from the late embryonic stages suggests broad roles of Rspo signaling in telencephalic development. Furthermore, the Lgr+ regions were located far from the Rspo+ regions, especially in the E10.5-14.5 ventral telencephalon, suggesting that Lgrs act via a Rspo-independent pathway.

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.

Inhibition of Wnt/ß-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/ß-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 ß-catenin protein expression by Dex occured concomitantly with the increased expression of the PPARγ protein. Dex decreased phosphorylation of Ser9-GSK3ß and expression of active ß-catenin protein. To examine the effects of Dex on Wnt/ß-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 GSK3ß 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/ß-catenin activity by Dex are mediated by GSK3ß activity. Furthermore, ß-catenin knockdown not only suppressed Dex-induced ALP-positive osteoblasts differentiation but also promoted Dex-induced adipocytes differentiation. These results suggest that inhibition of ß-catenin expression by Dex promotes the differentiation of mesenchymal progenitor cells into adipocytes.

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.

Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis.

SSH-BM-I, a tryptamine derivative, stimulates mineralization in terminal osteoblast differentiation but inhibits osteogenesis of pre-committed progenitor cells.

SSH-BM-I was synthesized from tryptamine by using a newly developed synthetic method, and it has structural similarity to bromomelatonin. Recently, it had been reported that SSH-BM-I increases osteoblasts in scales of gold fish. However, the effect of SSH-BM-I on osteoblast differentiation in mammalian cells has not yet been examined. Therefore, this study examined the effect of SSH-BM-I on osteoblast differentiation in mesenchymal progenitor-like cells and mature osteoblast-like cells. SSH-BM-I enhanced terminal osteoblast differentiation, as indicated by mineralization, which was accompanied by upregulation of the osteogenic marker genes bone sialoprotein (BSP) and osteocalcin (OC). However, in mesenchymal progenitor ROB-C26 cultures, no mineralized nodules were observed regardless of SSH-BM-I treatment, although BMP-2 was able to induce nodule formation in these cells. Furthermore, BMP-2-induced nodule formation was suppressed by SSH-BM-I treatment in ROB-C26 cultures. We further investigated the impact of the timing and duration of SSH-BM-I treatment on osteoblast differentiation. The effect of SSH-BM-I treatment on osteoblast differentiation of ROB-C26 in the presence of BMP-2 switches from negative to positive sometime between day 6 and 9, because SSH-BM-I treatment enhanced the formation of mineralized nodules when it was started on day 9, but suppressed nodule formation when it was started at day 6 or earlier. These results suggest that the stimulatory effects of SSH-BM-I on the formation of mineralized nodules depend on the degree of cell differentiation.

Bacitracin upregulates mbrAB transcription via mbrCD to confer bacitracin resistance in Streptococcus mutans.

Streptococcus mutans is a bacterial cause of dental caries that is resistant to bacitracin. The aim of this study was to elucidate the mbrABCD-related bacitracin resistance mechanism of S. mutans. Transcriptome data demonstrated that the expression levels of 33 genes were induced more than twofold by bacitracin. Fourteen genes were selected from the upregulated genes, and defective mutants of these genes were constructed for measurement of their sensitivity to bacitracin. Among the mutants, only the mbrA- or mbrB-deficient mutants exhibited 100- to 121-fold greater sensitivity to bacitracin when compared with the wild-type strain. Moreover, knockout of the mbrC and mbrD genes abolished the bacitracin-induced mbrAB upregulation. These results suggest that both mbrC and mbrD are required for mbrAB upregulation that confers the bacitracin-resistant phenotype on S. mutans.