Bone Generation Following Repeated Administration of Recombinant Bone Morphogenetic Protein 2.

BACKGROUND: The delivery of recombinant human bone morphogenetic protein 2 (rhBMP2) by using various carriers has been used to successfully induce bone formation in many animal models. However, the effect of multiple administration of rhBMP2 on bone formation and BMP2 antibody production has not been determined. Our aim was to examine the bone formation activity of rhBMP2 and serum levels of anti-BMP2 antibodies following the repeated administration of rhBMP2 in mice. METHODS: Absorbable collagen sponges or polyphosphazene hydrogels containing rhBMP2 were subcutaneously implanted or injected into one side on the back of six-week-old C57BL/6 mice. Three or 4 weeks later, the same amount of rhBMP2 was administered again with the same carrier into the subcutaneous regions on the other side of the back or into calvarial defects. The effects of a single administration of rhBMP2 on the osteoinductive ability in the ectopic model were compared with those of repeated administrations. In vivo ectopic or orthotopic bone formation was evaluated using microradiography and histological analyses. Serum concentrations of anti-rhBMP2 antibodies were measured by ELISAs. RESULTS: Re-administration of the same amount of rhBMP2 into the subcutaneous area showed a comparable production of ectopic bone as after the first administration. The bone forming ability of repeated rhBMP2 administrations was equal to that of single rhBMP2 administration. The administration of rhBMP2 into calvarial defects, following the first subcutaneous administration of rhBMP2 on the back, completely recovered the defect area with newly regenerated bone within 3 weeks. Repeated administration of rhBMP2 at 4-week intervals did not significantly alter the serum levels of anti-BMP2 antibodies and did not induce any inflammatory response. The serum obtained from rhBMP2-exposed mice had no effect on the ability of rhBMP2 to induce osteogenic gene expressions in MC3T3-E1. CONCLUSION: We suggest that the osteoinductive ability of rhBMP2 is not compromised by repeated administrations. Thus, rhBMP2 can be repeatedly used for bone regeneration at various sites within a short duration.

H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation.

Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.

Relaxin augments BMP-2-induced osteoblast differentiation and bone formation.

Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-ß activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.

Effect of transition metal dopants on mechanical properties and biocompatibility of zirconia ceramics.

In this study, the effect of transition metal dopants, originally added as colouring agents, on the mechanical properties and biocompatibility of sintered zirconia was investigated. This study confirmed that transition metal dopants could have a slight detrimental effect on the mechanical properties of zirconia. The addition of metal dopants did not affect the adhesion and proliferation of gingival fibroblasts.

Biocompatibility of mineral trioxide aggregate mixed with hydration accelerators.

INTRODUCTION: The aim of this study was to evaluate the biocompatibility of mineral trioxide aggregate mixed with selective hydration accelerators such as calcium chloride (CaCl2), citric acid (CA), and calcium lactate gluconate solution (CLG). METHODS: Inductively coupled plasma-atomic emission spectrometry analysis was used to measure calcium ions in the extracts of test materials. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay was performed using MG-63 cells to examine the cytotoxicity of the test materials. The surface of each sample and the growth pattern of the attached cells were observed using scanning electron microscopy (SEM). RESULTS: MTA mixed with 10 wt% CaCl2 and MTA mixed with 43.4 wt% CLG released a higher amount of calcium ions than the other groups. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay revealed that the cell viability of MTA mixed with 0.1 wt% CA was significantly higher than pure MTA on 7-day extract (P < .05). MTA mixed with 43.4 wt% CLG showed significantly higher cell viability than the other groups on 1-day extract (P < .05). MTA mixed with 10 wt% CaCl2 in all groups showed the lowest cell viability at all time points (P < .05). Under SEM, elongated and confluent cells were observed in all samples except in samples of MTA mixed with 10 wt% CaCl2. CONCLUSIONS: MTA mixed with 0.1 wt% CA showed good biocompatibility. MTA mixed with 43.4 wt% CLG showed favorable biocompatibility on 1 day. MTA mixed with 10 wt% CaCl2 in all groups showed the lowest cell viability at every time point and poor cell attachment under SEM.

Cytotoxicity of newly developed ortho MTA root-end filling materials.

INTRODUCTION: Various materials have been advocated for use as root-end filling materials. The purpose of the present in vitro study was to compare the cytotoxicity of 4 root-end filling materials: glass ionomer cement (GIC; Fuji II, GC Corp, Tokyo, Japan), reinforced zinc oxide-eugenol cement (IRM; Dentsply Tulsa Dental, Tulsa, OK), and 2 types of mineral trioxide aggregate. METHODS: This study used MG-63 cells derived from a human osteosarcoma. To quantitatively evaluate the cytotoxicity of test materials, the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used. The cells were exposed to the extracts and incubated. Cell viability was recorded by measuring the optical density of each test well in reference to controls. Each specimen was examined by scanning electron microscopy for the observation of cell morphology. RESULTS: The XTT assay showed that the cell viability of ProRoot MTA (Dentsply Tulsa Dental) was higher than that of GIC and Ortho MTA (BioMTA, Seoul, Republic of Korea) at all time points. IRM showed significantly lower cell viability than the other groups. The scanning electron microscopic analysis revealed that elongated, dense, and almost confluent cells were observed in the cultures of GIC, Ortho MTA, and ProRoot MTA specimens. In contrast, cells on the surface of IRM were rounded in shape, and the numbers and the density of the cells were smaller than that in the other groups. CONCLUSIONS: ProRoot MTA and GIC showed good biocompatibility in this study. However, Ortho MTA showed lower biocompatibility compared with ProRoot MTA and GIC.

H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iß.

Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iß, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iß to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iß strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iß is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene.

Negative regulation of JAK2 by H3K9 methyltransferase G9a in leukemia.

Histone methylation at specific lysine residues is a crucial regulatory process in transcriptional regulation. Using chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis, we found that the H3K9-me2 target gene JAK2 was an important factor during differentiation of the HL-60 promyelocytic leukemia cell line by all-trans-retinoic acid (ATRA) treatment. Here, we report that the H3K9 methyltransferase G9a negatively regulated JAK2 transcription in histone methyltransferase activity and in a YY1-dependent manner during ATRA-mediated leukemia cell differentiation. We found that G9a knockdown repressed ATRA-mediated HL-60 cell differentiation. We demonstrated that G9a interacts with YY1 and is recruited to the JAK2 promoter along with corepressors, including histone deacetylase, that induced H3K9-me2. Repression of JAK2 transcription by G9a decreased H3Y41 phosphorylation and promoted inhibition of the recently identified JAK2-H3Y41P-HP1α pathway-mediated leukemogenesis.

KDM3B is the H3K9 demethylase involved in transcriptional activation of lmo2 in leukemia.

Histone lysine methylation and demethylation are considered critical steps in transcriptional regulation. In this report, we performed chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis to examine the genome-wide occupancy of H3K9-me2 during all-trans-retinoic acid (ATRA)-induced differentiation of HL-60 promyelocytic leukemia cells. Using this approach, we found that KDM3B, which contains a JmjC domain, was downregulated during differentiation through the recruitment of a corepressor complex. Furthermore, KDM3B displayed histone H3K9-me1/2 demethylase activity and induced leukemogenic oncogene lmo2 expression via a synergistic interaction with CBP. Here, we found that KDM3B repressed leukemia cell differentiation and was upregulated in blood cells from acute lymphoblastic leukemia (ALL)-type leukemia patients. The combined results of this study provide evidence that the H3K9-me1/2 demethylase KDM3B might play a role in leukemogenesis via activation of lmo2 through interdependent actions with the histone acetyltransferase (HAT) complex containing CBP.

Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

Histone methyltransferase SETD3 regulates muscle differentiation.

Histone lysine methylation, as one of the most important factors in transcriptional regulation, is associated with a various physiological conditions. Using a bioinformatics search, we identified and subsequently cloned mouse SET domain containing 3 (SETD3) with SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) and Rubis-subs-bind domains. SETD3 is a novel histone H3K4 and H3K36 methyltransferase with transcriptional activation activity. SETD3 is expressed abundantly in muscular tissues and, when overexpressed, activates transcription of muscle-related genes, myogenin, muscle creatine kinase (MCK), and myogenic factor 6 (Myf6), thereby inducing muscle cell differentiation. Conversely, knockdown of SETD3 by shRNA significantly retards muscle cell differentiation. In this study, SETD3 was recruited to the myogenin gene promoter along with MyoD where it activated transcription. Together, these data indicate that SETD3 is a H3K4/K36 methyltransferase and plays an important role in the transcriptional regulation of muscle cell differentiation.

AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells.

This study examined the role of AMPK activation in osteoblast differentiation and the underlining mechanism. An AMPK activator (AICAR or metformin) stimulated osteoblast differentiation with increases in ALP and OC protein production as well as the induction of AMPK phosphorylation in MC3T3E1 cells. In addition, metformin induced the phosphorylation of Smad1/5/8 and expression of Dlx5 and Runx2, whereas compound C or dominant negative AMPK inhibited these effects. Transient transfection studies also showed that metformin increased the BRE-Luc and Runx2-Luc activities, which were inhibited by DN-AMPK or compound C. Down-regulation of Dlx5 expression by siRNA suppressed metformin-induced Runx2 expression. These results suggest that the activation of AMPK stimulates osteoblast differentiation via the regulation of Smad1/5/8-Dlx5-Runx2 signaling pathway.