Removal of cationic dye methylene blue by zero-valent iron: Effects of pH and dissolved oxygen on removal mechanisms.

Effects of pH and dissolved oxygen on mechanisms for decolorization and total organic carbon (TOC) removal of cationic dye methylene blue (MB) by zero-valent iron (ZVI) were systematically examined. Decolorization and TOC removal of MB by ZVI are attributed to the four potential mechanisms, i.e. reduction, degradation, precipitation and adsorption. The contributions of four mechanisms were quantified at pH 3.0, 6.0 and 10.0 in the oxic and anoxic systems. The maximum efficiencies of decolorization and TOC removal of MB were found at pH 6.0. The TOC removal efficiencies at pH 3.0 and 10.0 were 11.0 and 17.0%, respectively which were considerably lower as compared with 68.1% at pH 6.0. The adsorption, which was favorable at higher pH but was depressed by the passive layer formed on the ZVI surface at alkaline conditions, characterized the effects of pH on decolorization and TOC removal of MB. The efficiencies of decolorization and TOC removal at pH 6.0 under the anoxic condition were 73.0 and 59.0%, respectively, which were comparable to 79.9 and 55.5% obtained under the oxic condition. In the oxic and anoxic conditions, however, the contributions of removal mechanisms were quite different. Although the adsorption dominated the decolorization and TOC removal under the oxic condition, the contribution of precipitation was largely superior to that of adsorption under the anoxic condition.

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.

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.

Inhibitory Effects of a Tryptamine Derivative on Ultraviolet Radiation-Induced Apoptosis in MC3T3-E1 Mouse Osteoblasts.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

Inhibitory effects of a tryptamine derivative on ultraviolet radiation-induced apoptosis in MC3T3-E1 mouse osteoblasts.

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

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.

The effects of Sp7/Osterix gene silencing in the chondroprogenitor cell line, ATDC5.

Chondrocytes are known to express Sp7/Osterix (Osx) to varying degrees, but the role of Osx in chondrocytes is still unknown. In the current study, we investigated the role of the Osx gene using the clonal mouse embryonic cell line ATDC5, which retains the properties of the chondroprogenitor. ATDC5 cells express Osx; therefore, the effects of Osx gene silencing with shRNA lentiviral particles on chondrocyte marker gene expression and alkaline phosphatase (ALP) activity were investigated. At confluency, gene silencing down-regulated expression of the Sox trio (Sox5, 6, 9), Dlx5 and Alp mRNA, as well as ALP enzyme activity. Bone morphogenetic protein 2 (BMP2) is known to induce Osx gene expression in chondrocytes, and stimulation with BMP2 rescued Osx expression accompanied by up-regulation of Alp expression and ALP enzyme activity in a dose-dependent manner. To clarify the role of Osx in chondrocyte differentiation, cells induced to differentiate by 10µg/ml insulin for 21days were examined. Gene silencing inhibited the expression of the hypertrophic chondrocyte marker gene, type X collagen (Col X), and attenuated up-regulation of Dlx5 and Alp mRNA, as well as ALP enzyme activity. Taken together, these results suggest that Osx is involved in chondrogenic gene activation and is a positive regulator of the chondrocyte differentiation.

FGF8 regulates myogenesis and induces Runx2 expression and osteoblast differentiation in cultured cells.

In the current study, treatment of the rat osteogenic cell line ROB-C26 cells with fibroblast growth factor 8 (FGF8) stimulated alkaline phosphatase (ALP) activity, and also induced the expression of the Runx2 transcription factor, and increased the activity of a luciferase reporter gene containing the osteocalcin (OCN) promoter and six copies of the osteoblast specific cis-acting element 2 (OSE2) response element. In contrast, FGF8 treatment of the mouse myoblast cell line C2C12 inhibited the expression of desmin and the synthesis of myotubes. The expression of MyoD, Myogenin, Foxc2, and Hand1 was also decreased by FGF8. Transient expression of Foxc2 in C2C12 cells induced the expression of Hand1, and chromatin immunoprecipitation (ChIP) analysis indicated that Foxc2 binds to the promoter region of the Hand1 gene. These results indicated that Foxc2 is directly involved in the regulation of Hand1 expression. The results of the current study indicate that FGF8 regulates myoblast differentiation through the regulation of MyoD expression, and that this regulation is independent of Hand1 in cultured cells. Conversely, FGF8 supports bone development and cell differentiation though the induction of Runx2 expression.

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

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.

A tryptamine derivative, SST-VEDI-1, inhibits apoptosis and stimulates mineralization in osteoblasts.

SST-VEDI-1(VEDI-1) is a new synthetic compound that is synthesized from tryptamine, and has structural similarity to the SSH-BM family of compounds. However, the biological effects of VEDI-1 have yet to be well characterized. A recent report has demonstrated that SSH-BM-type compounds can stimulate osteoblast activity in cultured scales of goldfish. In this study, we examined the effects of VEDI-1 on osteoblastic differentiation as well as its effects on apoptosis, which is known to be closely related to osteoblastic differentiation. We found that VEDI-1 enhanced the formation of mineralized nodules in rat osteoblast cell lines, including ROS17/2.8 cells, and in mouse pre-osteoblast cell lines, including MC3T3-E1 cells, in a dose dependent manner, which was accompanied by increased expression of late osteoblast markers, bone sialoprotein (BSP) and osteocalcin (OC). Furthermore, VEDI-I inhibited apoptotic cell death and regulated the expression of proteins in the Bcl-2 family. These results suggest that VEDI-1 may facilitate late differentiation of osteoblasts and may have an inhibitory effect on apoptosis.

The effect of retinoic acid on a zinc finger transcription factor, AJ18, during differentiation of a rat clonal preosteoblastic cell line, ROB-C20, into osteoblasts.

OBJECTIVE: A zinc finger type transcription factor, AJ18, is thought to be a negative regulator of osteoblast differentiation, but its expression mechanism is not fully understood. Retinoic acid (RA) is a metabolite of vitamin A and involves the proliferation and differentiation in a variety of cells. To verify the effect of RA on osteoblast differentiation, AJ18 expression level was examined using a rat clonal preosteoblastic cell line, ROB-C20 (C20). DESIGN: Confluent C20 cells were treated with or without RA (10(-6)M) for several days. Northern, real time RT-PCR and Western blotting analyses were performed to examine AJ18 expression pattern in gene and protein levels. To identify the active promoter sequence of AJ18 gene, luciferase assay was designed. Furthermore, the effect of overexpressed AJ18 in C20 cells on alkaline phosphatase (ALP) mRNA expression and its activity was compared with that of RA-treated cells. RESULTS: RA increased the expression of AJ18 mRNA from 2 to 13 days as well as its protein production. However, no significant changes of Runx2 mRNA expression and undetectable osterix mRNA expression were observed in C20 cells treated with or without RA. Luciferase assay showed increases in promoter activities in some constructs of 5'-flanking region of AJ18 gene in RA-treated C20 cells. On the other hand, RA decreases enzymatic activity and mRNA expression level of ALP, but mRNA expression levels of bone sialoprotein and osteocalcin were not altered. Interestingly, reduced ALP activity and its mRNA expression level were detected in exogenous AJ18-overexpressing C20 cells. CONCLUSIONS: This study supports the hypothesis that RA may restrict to the differentiation of C20 cells into mature osteoblasts via inductive AJ18 expression with activation of multiple signal pathways.

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.

Changes in extracellular activin A:follistatin ratio during differentiation of a mesenchymal progenitor cell line, ROB-C26 into osteoblasts and adipocytes.

We investigated the effects of BMP-2 and dexamethasone (Dex) on follistatin (FS) and activin A expressions in a mesenchymal progenitor cell line, ROB-C26 (C26). C26 cells stimulated to differentiate into osteoblastic cells by blocking myogenic differentiation after BMP-2 treatment and into adipocytes with Dex treatment. Alkaline phosphatase (ALP) mRNA expression and its activity in the confluent C26 cells were dose- and time-dependently stimulated by BMP-2, but inhibited by Dex. The stimulatory effect on FS and activin A mRNA expressions by BMP-2 and Dex were dose-dependent. Cycloheximide pre-treatment indicated that FS and activin A expressions appear to be the direct target of BMP-2 and Dex signaling. BMP-2 time-dependently increased FS and activin A levels. Dex also increased FS level, but induced a time-dependent biphasic effect on activin A level, a decrease (2-6 h) followed by an increase (12-72 h). The data of the ratio of activin A concentration in the culture media to that of FS (activin A:FS ratio) measured by ELISA showed that BMP-2-induced osteoblastic differentiation involved an activin-dominant microenvironment, whereas Dex-induced adipocyte differentiation involved a FS-dominant microenvironment. Excess FS suppressed the stimulatory ALP activity of BMP-2, whereas activin A prevented not only Dex-induced inhibitory ALP activity, but also adipogenesis via suppression of the adipocyte transcriptional factor cascade. These results indicate that BMP-2-induced activin-dominant microenvironment may be critical for osteoblastic differentiation by restricting the antagonistic effects of FS on BMP activity, while Dex-induced FS-dominant microenvironment may be critical for adipocyte differentiation by restricting the inhibitory action of activin A on adipocyte differentiation.

Immunoelectron microscopic analysis of CD11c-positive dendritic cells in the periapical region of the periodontal ligament of rat molars.

To increase understanding of structural and phenotypic characteristics of dendritic cells (DCs) in the periapical region of the periodontal ligament (PDL) of rat molars, we performed immunoelectron microscopy for CD11c, a previously untested DC marker. Results demonstrated that CD11c clearly recognized DCs, although it also labeled certain macrophage subpopulation(s). In the normal PDL, resident DCs that extended several long cytoplasmic processes from their oval to slender cell body were identified. When the PDL was bacterially challenged by three days of unsealed pulp exposure, relatively small, irregularly shaped DCs with many thin and short cytoplasmic processes, most likely representing newly recruited DCs, were detected. The ratio of DCs in CD11c+ cells was significantly higher than that in ED1 (anti-macrophages and DCs)-positive and OX6 (anti-major histocompatibility complex class II molecules)-positive cells in normal and challenged PDL.

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.

Inductive effects of dexamethasone on the gene expression of Cbfa1, Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts.

Runx2/core binding factor alpha 1 (Cbfa1) and Osterix (Osx) are osteoblast-specific transcription factors essential for the development of a mature osteoblast phenotype and are thought to activate osteoblast marker genes in vivo to produce a bone-specific matrix. Dexamethasone (Dex) is known to be a potent stimulator of osteoblastic differentiation in vitro, however, the exact role is still unclear. To investigate the mechanisms of the stimulation of osteoblastic differentiation by Dex, we evaluated the effects of Dex on proliferation and mineralization as well as on mRNA expression of Cbfa1, Osx and osteoblast marker genes, osteocalcin (OC) and bone sialoprotein (BSP) mRNAs in differentiating foetal rat calvarial cells (FRCC), which were cultured for 35 days in the presence or absence of 10(-7) M Dex. Treatment of FRCC with Dex resulted in the stimulation of cell proliferation and increased the number of cells, which are able to produce bone-like nodules with a mineralized matrix when compared to untreated controls. Northern blot analysis revealed that, in the absence of Dex, Cbfa1 mRNA expressed at day 8, while Osx mRNA expressed at day 15. Subsequently expression of these mRNAs increased up to day 21, followed by constant expression during the culture period. The expression of OC and BSP mRNAs appeared to be synchronous with that of Osx mRNA and was detectable at day 15 with an increase thereafter. The presence of Dex resulted in an induction in Cbfa1 and Osx mRNA expression. The former appeared at day 5 and the latter appeared at day 11. Subsequently expression of Cbfa1 and Osx mRNAs increased up to day 15 with a decrease thereafter. Expression of OC and BSP mRNAs appeared to be coincident with that of Osx mRNA and was detectable at day 11 and reached a maximum at day 15 followed by constant expression. These observations indicate that induction of Cbfal and Osx mRNAs by Dex may be followed by activation of osteoblast marker genes such as OC and BSP mRNAs to produce a bone-specific matrix that subsequently becomes mineralized. Thus, it is likely that Dex may promote osteoblastic differentiation and mineralization of FRCC by inducing the expression of Cbfa1 and Osx genes in vitro.

Effects of bone morphogenetic protein-2 and transforming growth factor beta1 on gene expression of transcription factors, AJ18 and Runx2 in cultured osteoblastic cells.

Osteoblast differentiation is controlled by multiple transcription factors, Runx2, AJ18, Osterix, Dlx5 and Msx2. The mechanisms of regulation of AJ18 mRNA expression by the transforming growth factor beta (TGF-beta) superfamily remain poorly understood. However, it is known that BMP-2 induces differentiation of C26 cells into more mature osteoblastic cells. The present study, using Northern blot and real-time reverse transcription polymerase chain reaction analyses, investigated the effects of bone morphogenetic protein-2 (BMP-2) and TGF-beta1 on mRNA expression of AJ18 and Runx2 in a clonal osteoblast precursor cell line ROB-C26 (C26) cultured for 3, 6 or 9 days in the presence or absence of BMP-2. Although mRNA expression of Osterix and bone sialoprotein (BSP) was undetectable in the C26 culture, BMP-2 induced Osterix expression on days 3-9, but not BSP expression. BMP-2 also stimulated significantly Dlx5 expression on days 3-9, Msx2 and matrix Gla protein expressions on days 3 and 6, Runx2, alkaline phosphatase and osteocalcin expressions on days 6 and 9 in the culture. Furthermore, BMP-2 increased significantly Smad5 mRNA in the culture on day 3, indicating BMP-2 involvement in the regulation of Smad5 mRNA expression. In contrast, the inhibitory effects of BMP-2 on AJ18 mRNA expression were significant on days 3-9, indicating that a decrease in AJ18 mRNA expression is essential for the increased osteoblastic differentiation. Furthermore, TGF-beta1 (0, 0.1, 1.0 and 5.0 ng/ml) treatment of C26 cells cultured for 6 days in the presence or absence of BMP-2 for 24h stimulated mRNA levels of AJ18 and Runx2, maximal stimulation occurring principally at 1.0 ng/ml. These observations indicate that the expression of AJ18 and Runx2 mRNAs in C26 cells is under the control of BMP-2 and TGF-beta1, which exert different effects on AJ18 mRNA expression, but are potent stimulators of Runx2 mRNA expression during osteoblast differentiation.

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma.

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained (P < 0.05). Correlations were obtained between the CENP-F LI and tumor size (P < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G2 to M cells, compared to Ki-67.