Expression Of Dmp1 Research Articles

Conditional deletion ofPkd1in osteocytes disrupts skeletal mechanosensing in mice

We investigated whether polycystin-1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1-Cre with Pkd1(flox/m1Bei) mice, in which the m1Bei allele is nonfunctional. We assessed in wild-type and Pkd1-deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1-Cre;Pkd1(flox/+)) and null mice (Pkd1(Dmp1-cKO)) exhibited a ∼ 40 and ∼ 90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2-II, Osteocalcin, Dmp1, and Phex. Anabolic load-induced periosteal lamellar MAR (0.58 ± 0.14; Pkd1(Dmp1-cKO) vs. 1.68 ± 0.34 μm/d; control) and increases in Cox-2, c-Jun, Wnt10b, Axin2, and Runx2-II gene expression were significantly attenuated in Pkd1(Dmp1-cKO) mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1(null/null) and Pkd1(m1Bei/m1Bei)-derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild-type controls. These data indicate that polycystin-1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.

Sclerostin is a locally acting regulator of late-osteoblast/preosteocyte differentiation and regulates mineralization through a MEPE-ASARM-dependent mechanism

The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose- and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.

Inhibition of Proprotein Convertase SKI-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone (dec-RRLL-cmk), inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Because SKI-1 is required for activation of SREBP and CREB (cAMP-response element-binding protein)/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5-3.0-fold including Phex, Dmp1, COL1A1, COL11A1, and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1, and fibronectin was reduced by both, whereas COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1-activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19-kDa caspase-3. Conversely, overexpression of SKI-1-activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells and possibly osteocytes depends upon the non-apoptotic actions of SKI-1.

Identification of secretory odontoblasts using DMP1-GFP transgenic mice

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue- and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts.

Mice Lacking Bone Sialoprotein (BSP) Lose Bone after Ovariectomy and Display Skeletal Site-Specific Response to Intermittent PTH Treatment

Bone sialoprotein (BSP) belongs to the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, whose members play multiple and distinct roles in the development, turnover, and mineralization of bone and dentin. The functions of BSP in bone remodeling are not yet well established. We previously showed that BSP knockout (BSP(-/-)) mice exhibit a higher trabecular bone volume, concomitant with lower bone remodeling, than wild-type (BSP(+/+)) mice. To determine whether bone turnover can be stimulated in the absence of BSP, we subjected BSP(+/+) and BSP(-/-) mice to catabolic [ovariectomy (OVX)] or anabolic (intermittent PTH administration) hormonal challenges. BSP(-/-) mice progressively develop hypocalcemia and high serum PTH between 2 and 4 months of age. Fifteen and 30 d after OVX, microtomography analysis showed a significant decrease of trabecular bone volume in tibiae of both genotypes. Histomorphometric parameters of bone formation and resorption were significantly increased by OVX. PTH treatment resulted in an increase of trabecular thickness and both bone formation and resorption parameters at all skeletal sites in both genotypes and a decrease of trabecular bone volume in tibiae of BSP(+/+) but not BSP(-/-) mice. PTH increased cortical thickness and bone area in BSP(+/+) but not BSP(-/-) mice and stimulated the bone formation rate specifically in the endosteum of BSP(+/+) mice and the periosteum of BSP(-/-) mice. PTH enhanced the expression of RANKL, MEPE, and DMP1 in both genotypes but increased OPG and OPN expression only in BSP(-/-) mice. In conclusion, despite the low basal turnover, both catabolic and anabolic challenges increase bone formation and resorption in BSP(-/-) mice, suggesting that compensatory pathways are operative in the skeleton of BSP-deficient mice. Although up-regulation of one or several other SIBLINGs is a possible mechanism, further studies are needed to analyze the interplay and cross-regulation involved in compensating for the absence of BSP.

Patterns of MEPE, DMP1, Phex and Fgf23 expression in growth plate chondrocytes

Patterns of MEPE, DMP1, Phex and Fgf23 expression in growth plate chondrocytes

Odontoblast‐targeted Bcl‐2 overexpression impairs dentin formation

Apoptosis has been described extensively in tooth development, which is under tight control of multiple apoptosis regulators, including anti-apoptotic protein Bcl-2. However, it is totally unclear how Bcl-2 is related to odontogenesis, especially dentinogenesis. Using a transgenic mouse Col2.3Bcl-2 in which human Bcl-2 was overexpressed in odontoblasts, the effect of Bcl-2 on dentinogenesis was investigated. Overexpression of Bcl-2 was detected by immunohistochemistry and Western blot. Odontoblast apoptosis was evaluated by TUNEL and Western blot detection of cleaved caspase-3. Odontoblast differentiation was assessed by real-time PCR detection of dentin matrix expression. Dentin mineralization was evaluated by micro-CT in vivo, and alizarin red S staining and calcium content analysis in vitro. Bcl-2 was found to be overexpressed in odontoblasts and prevent their apoptosis. Odontoblast differentiation and mineralization was inhibited by Bcl-2, as evidenced by lower expressions of DMP-1, OC, and DSPP, and decreased odontoblast mineralization in vitro, as well as decreased dentin thickness and mineral density in vivo when compared to the wild-type animals. Inhibition of odontoblast differentiation by Bcl-2 occurs, at least partially, via a suppression of MEK-ERK1/2 signaling pathway. In conclusion, Bcl-2 overexpression prevents odontoblast apoptosis and impairs dentin formation, partially via an inhibition of odontoblast differentiation. This study revealed some novel functions of Bcl-2 in dentinogenesis in addition to its anti-apoptotic effect, which shed some light on the genetic complexity of tooth development.

New roles and mechanism of action of BMP4 in postnatal tooth cytodifferentiation

During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre and the BMP4 floxed and BMP4 knockout mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process leads to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10–12 months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, is also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine postnatal role of odontoblast derived BMP4 on the proper amelogenesis and formation of the enamel.

The Arf‐inducing transcription factor Dmp1 encodes a transcriptional activator of amphiregulin, thrombospondin‐1, JunB and Egr1

Dmp1 (Dmtf1) encodes a Myb-like transcription factor implicated in tumor suppression through direct activation of the Arf-p53 pathway. The human DMP1 gene is frequently deleted in non-small cell lung cancers, especially those that retain wild-type INK4a/ARF and/or p53. To identify novel genes that are regulated by Dmp1, transcriptional profiles of lung tissue from Dmp1-null and wild-type mice were generated using the GeneChip Microarray. Comparative analysis of gene expression changes between the two groups resulted in identification of numerous genes that may be regulated by Dmp1. Notably, amphiregulin (Areg), thrombospondin-1 (Tsp-1), JunB, Egr1, adrenomedullin (Adm), Bcl-3 and methyl-CpG binding domain protein 1 (Mbd1) were downregulated in the lungs from Dmp1-null mice while Gas1 and Ect2 genes were upregulated. These target genes were chosen for further analyses since they are involved in cell proliferation, transcription, angiogenesis/metastasis, apoptosis, or DNA methylation, and thus could account for the tumor suppressor phenotype of Dmp1. Dmp1 directly bound to the genomic loci of Areg, Tsp-1, JunB and Egr1. Significant upregulation or downregulation of the novel Dmp1 target genes was observed upon transient expression of Dmp1 in alveolar epithelial cells, an effect which was nullified by the inhibition of de novo mRNA synthesis. Interestingly, these genes and their protein products were significantly downregulated or upregulated in the lungs from Dmp1-heterozygous mice as well. Identification of novel Dmp1 target genes not only provides insights into the effects of Dmp1 on global gene expression, but also sheds light on the mechanism of haploid insufficiency of Dmp1 in tumor suppression.

The osteocyte: the underestimated conductor of the bone orchestra

The osteocyte is often considered to be a cell that is hardly active, yet it turns out to have a central role in bone remodeling. It functions as a mechanoreceptor that translates mechanical stimuli into biochemical signals. The expression of PGE2 by loaded osteocytes and the consequent activation of the cAMP/PKA pathway, together with stabilization of β-catenin, may permit cross talk with the canonical Wnt/β-catenin pathway, the activation of which is increased by the load-dependent reduction of the expression of DKK1 and of sclerostin (Sost, the secretion product of old osteocytes). These signal pathways, as well as TGF-β and BMP expression, stimulate the differentiation and activity of osteoblasts, reduce their apoptosis, and enhance bone formation. The expressions of MEPE, FGF-23 and DMP-1 further contribute to the osteocytic control of bone metabolism. Periosteocytic osteolysis, if definitively demonstrated, would constitute another fundamental function of osteocytes. A thorough understanding of the osteocyte’s activities could open up new strategies in the therapy of metabolic bone diseases.

Molecular characterization of young and mature odontoblasts

The odontoblast is the secretory cell responsible for primary, secondary and tertiary reactionary dentinogenesis. We provide evidence that the changes in secretory activity of odontoblasts reflect differential transcriptional control and that common regulatory processes may exist between dentine and bone. Introduction Based on the hypothesis that differential dentine secretion (primary and secondary dentinogenesis) is associated with changes in the transcriptional control within the cell, we have investigated the transcriptome of odontoblasts at young and mature stages and subsequently used this information to identify key regulatory intracellular pathways involved in this process. Materials and methods We used microarray analysis to compare the transcriptome of early stage (primary dentinogenesis) and late stage (secondary dentinogenesis) odontoblasts from 30 month old bovine teeth. Secondarily, we used post-array sqRT-PCR to confirm the differential expression of 23 genes in both populations of odontoblasts. Finally, immunohistochemistry was performed on bovine and murine tissues with antibodies to DMP1 and anti-phospho p38 proteins. Results DMP-1 and osteocalcin gene expression were up-regulated in the mature odontoblasts, whereas collagen I, DSPP, TGF-β1 and TGF-β1R gene expression were down-regulated. Microarray analysis highlighted 574 differentially regulated genes (fold change > 2 — p < 0.05). This study supports further existing similarities between pulp cells and bone cells. Using post-array Sq-RT-PCR we characterized transcript levels of genes involved in the p38 MAP kinase pathway (PTPRR, NTRKK2, MAPK13, MAP2K6, MKK3). Differential p38 gene activation was confirmed by immunohistochemistry for p38 protein in murine teeth. Finally, immunohistochemistry for DMP1 indicated that odontoblasts involved in primary and secondary dentinogenesis may coexist in the same tooth. Conclusion As established in bone cells, the transcriptome of the odontoblast was shown here to evolve with their stage and functional maturity. Identification of the involved signalling pathways, as highlighted for p38, will enable the deciphering of physiology and pathology of mineralised tissue formation.

The differentiation-inducing effect of conditioned media obtained from dental pulp cells

Conditioned media (CM) from human dental pulp cells (HDPC) was investigated for its effects on the proliferation and differentiation of HDPC and MG63 cells. CM prepared from the primary culture of HDPC was used for the culture of HDPC and MG63. Cell growth, alkaline phosphatase (ALPase) activity, mRNA expression of differentiation-related genes, and mineralization of the HDPC and MG63 cells in the media containing CM were assessed. HDPC CM increased the cell growth of HDPC but decreased MG63 cell growth. ALPase activity and the mineralization of both HDPC and MG63 were enhanced by HDPC CM. The CM also up-regulated the expressions of DSPP, DMP-1, and OCN mRNA in HDPC. Pretreatment of HDPC CM with a neutralizing antibody against TGF-beta completely eliminated the effect of CM on ALPase activity in HDPC. HDPC was able to secrete odontogenic differentiation-inducing factors, in which TGF-beta seems to a key element of the CM effects.

In vitro odontoblast‐like cell differentiation of cranial neural crest cells induced by fibroblast growth factor 8 and dentin non‐collagen proteins

During tooth development, cranial neural crest (CNC) cells represent a population of pluripotent stem cells that give rise to various dental tissues. This study aimed to investigate whether CNC cells could differentiate into odontoblast-like cells by in vitro induction. CNC cells were isolated from explants of cranial neural tubes and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM)/F12 medium which contained fibroblast growth factor 8 (FGF8) and dentin non-collagen proteins (DNCP). The initiation of controlled differentiation was determined using histological assays, and the expression of specific gene phenotypes was detected using immunocytochemical staining and reverse transcription--polymerase chain reaction (RT--PCR). The first branchial arch phenotype of the CNC cells demonstrated negative Hoxa2 expression and positive vimentin expression in the presence of 100 ng/ml FGF8. Following DNCP induction, the CNC cells became bipolar, demonstrated high alkaline phosphatase (ALP) activity, and formed mineralized nodules. In addition, the expression of DSPP, DMP1, and collagen type I confirmed the odontoblast phenotype. The results indicate that the tissue-specific cellular differentiation (odontoblast-like cells) of early-stage embryonic-derived cells (such as CNC cells) can be induced by adult extracellular matrix proteins (such as DNCP). CNC cells may be used as a valuable cell model for research on dental tissue differentiation and regeneration.

Oxygen tension is an important mediator of the transformation of osteoblasts to osteocytes

Osteocytes are derived from osteoblasts, but reside in the mineralized bone matrix under hypoxic conditions. Osteocyte-like cells show higher expression of ORP150, which is induced by hypoxia, than osteoblast-like cells. Accordingly, we hypothesized that the oxygen tension may regulate the transformation of osteoblasts to osteocytes. MC3T3-E1 cells and calvariae from 4-day-old mice were cultured under normoxic (20% O(2)) or hypoxic (5% O(2)) conditions. To investigate osteoblastic differentiation and tranformation to osteocytes, alizarin red staining was done and the expression of various factors was assessed. Hypoxic culture promoted the increased synthesis of mineralized matrix by MC3T3-E1 cells. Alkaline phosphatase activity was initially increased during hypoxic culture, but decreased during osteogenesis. Osteocalcin production was also increased by hypoxic culture, but decreased after mineralization. Furthermore, expression of Dmp1, Mepe, Fgf23, and Cx43, which are osteocyte-specific or osteocyte-predominant proteins, by MC3T3-E1 cells was greater under hypoxic than under normoxic conditions. In mouse calvarial cultures, the number of cells in the bone matrix and cells expressing Dmp1 and Mepe were increased by hypoxia. In MC3T3-E1 cell cultures, ORP150 expression was only detected in the mineralized nodules under normoxic conditions, while its expression was diffuse under hypoxic conditions, suggesting that the nodules were hypoxic zones even in normoxic cultures. These findings suggest that a low oxygen tension promotes osteoblastic differentiation and subsequent transformation to osteocytes.

An experimental study on porcine fibroblasts transfected by dental matrix protein-1 gene

To evaluate the expression of dental matrix protein-l (DMP1) in porcine oral mucosa fibroblasts (POMF) transfected by DMP1 and the influences of the transfection. The full length of porcine DMP1 cDNA was linked into an eukaryotic expression vector pEGFP-C1. POMF and mesenchymal stem cells (MSC) were transfected with the pEGFP-DMP1. The expression of DMP1, dental sialoprotein (DSP), amelin and ameloblastin (Ambn) gene of transfected POMF and MSC were detected by RT-PCR. The expression of DMP1 and DSP protein was examined by immunocytochemical staining. The formation ratio of mineralized nodules of transfected cells was compared with un-transfected ones after mineralized induction. The formation of mineralized nodules of three-dimensional pellet transfected cells was compared with un-transfected ones after hematoxylin and eosin staining. The constructed pEGFP-DMP1 could produce 4.7 kb and 1.5 kb fragments. DMP1 gene, DSP gene and Ambn gene were expressed by POMF after transfection. Immunohistochemical staining and the quantitative analysis of protein showed that DMP1 and DSP protein was positive in transfected POMF and MSC. The formation ratio of mineralized nodules of transfected POMF and MSC was higher than that of un-transfected ones (P < 0.05). The expression of porcine DMP1 in POME after gene transfection can induce the expression of tooth-development-associated gene Ambn and DSP and enhance the formation of mineralized nodules.

Odontogenic Potential of Bone Marrow Mesenchymal Stem Cells

This study aimed to investigate the odontogenic potential of bone marrow mesenchymal stem cells (BM-MSCs) for seeding in tooth regeneration. In this study, BM-MSCs were co-cultured with oral epithelial cells derived from rat embryos. Expression of the odontogenic genes Pax9, DMP1, and DSPP was detected by the reverse-transcription polymerase chain reaction (RT-PCR) technique. To further characterize the odontogenic potential of BM-MSCs, the gold standard in vivo transplantation system was used. The results revealed that Pax9, DMP1, and DSPP expression was detected by RT-PCR only after co-culture of BM-MSCs and oral epithelial cells derived from embryos age E11.5. Histological analyses of the BM-MSCs/epithelial cell mass demonstrated the presence of tooth-like structures. The series of experiments both in vitro and in vivo demonstrated that BM-MSCs can differentiate into functional odontoblast-like cells. This implies that BM-MSCs may become a novel source of cells for seeding in tooth regeneration research.

Dmp1 and tumor suppression

Dmp1 (cyclin D binding myb-like protein 1; also called Dmtf1) is a transcription factor that was isolated in a yeast two-hybrid screen through its binding property to cyclin D2. Although it was initially predicted to be involved in the cyclin D-Rb pathway, overexpression of Dmp1 in primary cells induces cell cycle arrest in an Arf, p53-dependent fashion. Dmp1 is a unique Arf regulator, the promoter of which is activated by oncogenic Ras-Raf signaling. Dmp1 expression is repressed by physiological mitogenic stimuli as well as by overexpressed E2F proteins; thus, it is a novel marker of cells that have exited from the cell cycle. Spontaneous and oncogene-induced tumor formation is accelerated in both Dmp1(+/-) and Dmp1(-/-) mice; the Dmp1(+/-) tumors often retain and express the wild-type allele; thus, Dmp1 is haplo-insufficient for tumor suppression. Tumors from Dmp1(+/-) and Dmp1(-/-) mice often retain wild-type Arf and p53, suggesting that Dmp1 is a physiological regulator of the Arf-p53 pathway. The human DMP1 (hDMP1) gene is located on chromosome 7q21, the locus of which is often deleted in myeloid leukemia and also in some types of solid tumors. Post-translational modification of Dmp1 and its role in human malignancy remain to be investigated.

Targeted expression of csCSF-1 in op/ op mice ameliorates tooth defects

Objective The aim of this study was to characterize the tooth phenotype of CSF-1-deficient op/ op mice and determine whether expression of csCSF-1 in these mice has a role in primary tooth matrix formation. Design Ameloblasts and odontoblasts, isolated from wt/ wt frozen sections using laser capture microdissection, were analysed for csCSF-1, sCSF-1 and CSF-1R mRNA by RT-PCR. Mandibles, excised from 8 days op/ op and wt/ wt littermates, were examined for tooth morphology as well as amelogenin and DMP1 expression using in situ hybridisation. op/ opCS transgenic mice, expressing csCSF-1 in teeth and bone using the osteocalcin promoter, were generated. Skeletal X-rays and histomorphometry were performed; teeth were analysed for morphology and matrix proteins. Results Normal dental cells in vivo express both CSF-1 isoforms and CSF-1R. Compared to wt/ wt, op/ op teeth prior to eruption showed altered dental cell morphology and dramatic reduction in DMP1 transcripts. op/ opCS mice showed marked resolution of osteopetrosis, tooth eruption and teeth that resembled amelogenesis imperfecta-like phenotype. At 3 weeks, op/ op teeth showed severe enamel and dentin defects and barely detectable amelogenin and DMP1. In op/ opCS mice, DMP1 in odontoblasts increased to near normal and dentin morphology was restored; amelogenin also increased. Enamel integrity improved in op/ opCS, although it was thinner than wt enamel. Conclusions Results demonstrate that ameloblasts and odontoblasts are a source and potential target of CSF-1 isoforms in vivo. Expression of csCSF-1 within the tooth microenvironment is essential for normal tooth morphogenesis and may provide a mechanism for coordinating the process of tooth eruption with endogenous matrix formation.

Expression of Dmp1 in specific differentiated, nonproliferating cells and its regulation by E2Fs

Dmp1 is a Myb-like transcription factor that transmits oncogenic Ras-Raf signaling to the Arf-p53 pathway and induces cell cycle arrest. Immunohistochemical staining was performed to identify the pattern of Dmp1 expression in normal murine tissues compared with the proliferation marker, Ki67. In thymus, the nuclei of mature T lymphocytes in the medulla were strongly positive for Dmp1, whereas Ki67 was detected only in the cortex. In intestine, Dmp1 was detected in the nuclei of superficial layers of the villi, whereas Ki67-positive cells were confined to the lower one-third of the crypt. Double staining for Dmp1 and Ki67 revealed that these two proteins were expressed in mutually exclusive fashion in nearly all the tissues examined. Subsets of E2Fs were specifically bound to the Dmp1 promoter upon mitogenic signaling and E2Fs 1-4 inhibited the Dmp1 promoter in a reporter assay. The Dmp1 promoter was repressed when the cells entered the S to G2/M phase of the cell cycle when both Dmp1 and Arf expressions were downregulated. The Dmp1 mRNA was not downregulated by serum in E2F-DB(+) cells, suggesting that the Dmp1 promoter repression is E2F-dependent. This explains why the Dmp1 and Ki67-positive cells are stained in mutually exclusive fashion in normal tissues.

Cellular phenotyping of the mouse skeleton using synchrotron based nano-computed tomography

Whole body vibration (WBV) is a promising tool for counteracting bone loss. Most WBV studies on animals have been performed at acceleration <1g and frequency between 30 and 90 Hz. Such WBV conditions trigger bone growth in osteopenia models, but not in healthy animals. In order to test the ability of WBV to promote osteogenesis in young animals, we exposed seven-week-old male mice to vibration at 90 Hz and 2g peak acceleration for 15 min/day, 5 days/week. We examined the effects on skeletal tissues with micro-computed tomography and histology. We also quantified bone vascularization and mechanosensitive osteocyte proteins, sclerostin and DMP1. Three weeks of WBV resulted in an increase of femur cortical thickness (+5%) and area (+6%), associated with a 25% decrease of sclerostin expression, and 35% increase of DMP1 expression in cortical osteocytes. Mass-structural parameters of trabecular bone were unaltered in femur or vertebra, while osteoclastic parameters and bone formation rate were increased at both sites. Three weeks of WBV resulted in higher blood vessel numbers (+23%) in the distal femoral metaphysis.After 9-week WBV, we have not observed the difference in structural cortical or trabecular parameters. However, the tissue mineral density of cortical bone was increased by 2.5%.Three or nine weeks of 2g/90 Hz WBV treatment did not affect longitudinal growth rate or body weight increase under our experimental conditions, indicating that these are safe to use.These results validate a potential of 2g/90 Hz WBV to stimulate trabecular bone cellular activity, accelerate cortical bone growth, and increase bone mineral density.