Jelica Gluhak-Heinrich

Mechanical Stimulation of Bone in Vivo Reduces Osteocyte Expression of Sost/Sclerostin

Sclerostin, the protein product of the Sost gene, is a potent inhibitor of bone formation. Among bone cells, sclerostin is found nearly exclusively in the osteocytes, the cell type that historically has been implicated in sensing and initiating mechanical signaling. The recent discovery of the antagonistic effects of sclerostin on Lrp5 receptor signaling, a crucial mediator of skeletal mechanotransduction, provides a potential mechanism for the osteocytes to control mechanotransduction, by adjusting their sclerostin (Wnt inhibitory) signal output to modulate Wnt signaling in the effector cell population. We investigated the mechanoregulation of Sost and sclerostin under enhanced (ulnar loading) and reduced (hindlimb unloading) loading conditions. Sost transcripts and sclerostin protein levels were dramatically reduced by ulnar loading. Portions of the ulnar cortex receiving a greater strain stimulus were associated with a greater reduction in Sost staining intensity and sclerostin-positive osteocytes (revealed via in situ hybridization and immunohistochemistry, respectively) than were lower strain portions of the tissue. Hindlimb unloading yielded a significant increase in Sost expression in the tibia. Modulation of sclerostin levels appears to be a finely tuned mechanism by which osteocytes coordinate regional and local osteogenesis in response to increased mechanical stimulation, perhaps via releasing the local inhibition of Wnt/Lrp5 signaling.

Runx2, Osx, and Dspp in Tooth Development

The transcription factors Runx2 and Osx are necessary for osteoblast and odontoblast differentiation, while Dspp is important for odontoblast differentiation. The relationship among Runx2, Osx, and Dspp during tooth and craniofacial bone development remains unknown. In this study, we hypothesized that the roles of Runx2 and Osx in the regulation of osteoblast and odontoblast lineages may be independent of one another. The results showed that Runx2 expression overlapped with Osx in dental and osteogenic mesenchyme from E12 to E16. At the later stages, from E18 to PN14, Runx2 and Osx expressions remained intense in alveolar bone osteoblasts. However, Runx2 expression was down-regulated, whereas Osx expression was clearly seen in odontoblasts. At later stages, Dspp transcription was weakly present in osteoblasts, but strong in odontoblasts where Osx was highly expressed. In mouse odontoblast-like cells, Osx overexpression increased Dspp transcription. Analysis of these data suggests differential biological functions of Runx2, Osx, and Dspp during odontogenesis and osteogenesis.

Mechanical Loading Stimulates Dentin Matrix Protein 1 (DMP1) Expression in Osteocytes In Vivo

Dentin matrix protein 1 (DMP1) was originally postulated to be dentin specific. Further analysis showed that DMP1 is also expressed in mature cartilage and bone. In bone tissue, DMP1 is expressed predominantly in late osteoblasts and osteocytes. DMP1 belongs to the SIBLING (Small Integrin Binding Ligand N‐linked Glycoprotein) family of cellular matrix proteins that also includes osteopontin, bone sialoprotein, dentin sialophosphoprotein, and others. In this study, we examined the effect of mechanical loading on expression of DMP1 mRNA and DMP1 protein in alveolar bone in the mouse tooth movement model by in situ hybridization and immunocytochemistry. The expression of DMP1 mRNA was determined quantitatively in mechanically loaded and control sites of dento‐alveolar tissue at several time points from 6 h to 7 days after loading. The tooth movement model allows simultaneous evaluation of bone resorption and bone formation sites. Expression of DMP1 mRNA in osteocytes increased 2‐fold as early as 6 h after treatment in both the bone formation and bone resorption sites. After 4 days, DMP1 expression in osteocytes increased to a maximum of 3.7‐fold in the bone formation sites and 3.5‐fold in the resorption sites. Osteoblasts responded in the opposite manner and showed a transient 45% decrease of DMP1 mRNA in bone formation sites and a constant decrease of DMP1 mRNA during the entire course of treatment in the bone resorption sites, with a peak inhibition of 67% at day 2. By immunocytochemistry using a C‐terminal region peptide antibody to DMP1, we found that there was a transient decrease in immunoreactivity at 3 days after treatment on both the formation side and the resorption side compared with the matched contralateral control tissue. However by 7 days of loading, there was a dramatic increase in DMP1 protein immunoreactivity on both the formation side and the resorption side. These results represent changes in epitope availability using this antibody or true changes in protein levels. The observations imply that the DMP1 protein is undergoing dynamic changes in either synthesis or other protein/matrix interaction after mechanical loading of alveolar bone. The findings indicate that DMP1 is involved in the responses of osteocytes and osteoblasts to mechanical loading of bone. These results support the hypothesis that osteocytes alter their matrix microenvironment in response to mechanical loading.

Glucocorticoid-induced autophagy in osteocytes.

Glucocorticoid (GC) therapy is the most frequent cause of secondary osteoporosis. In this study we have demonstrated that GC treatment induced the development of autophagy, preserving osteocyte viability. GC treatment resulted in an increase in autophagy markers and the accumulation of autophagosome vacuoles in vitro and in vivo promoted the onset of the osteocyte autophagy, as determined by expression of autophagy markers in an animal model of GC‐induced osteoporosis. An autophagy inhibitor reversed the protective effects of GCs. The effects of GCs on osteocytes were in contrast to tumor necrosis factor α (TNF‐α), which induced apoptosis but not autophagy. Together this study reveals a novel mechanism for the effect of GC on osteocytes, shedding new insight into mechanisms responsible for bone loss in patients receiving GC therapy.

Differential Regulation of Dentin Sialophosphoprotein Expression by Runx2 during Odontoblast Cytodifferentiation

Dentin sialophosphoprotein (DSPP) consists of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). The spatial-temporal expression of DSPP is largely restricted during differentiational stages of dental cells. DSPP plays a vital role in tooth development. It is known that an osteoblast-specific transcription factor, Runx2, is essential for osteoblast differentiation. However, effects of Runx2 on DSPP transcription remain unknown. Here, we studied different roles of Runx2 in controlling DSPP expression in mouse preodontoblast (MD10-F2) and odontoblast (MO6-G3) cells. Two Runx2 isoforms were expressed in preodontoblast and odontoblast cells, and in situ hybridization assay showed that DSPP expression increased, whereas Runx2 was down-regulated during odontoblast differentiation and maturation. Three potential Runx2 sites are present in promoters of mouse and rat DSPP genes. Runx2 binds to these sites as demonstrated by electrophoretic mobility shift assay and supershift experiments. Mutations of Runx2 sites in mouse DSPP promoter resulted in a decline of promoter activity in MD10-F2 cells compared with an increase of its activity in MO6-G3 cells. Multiple Runx2 sites were more active than a single site in regulating the DSPP promoter. Furthermore, forced overexpression of Runx2 isoforms induced increases of endogenous DSPP protein levels in MD10-F2 cells but reduced its expression in MO6-G3 cells consistent with the DSPP promoter analysis. Thus, our results suggest that differential positive and negative regulation of DSPP by Runx2 is dependent on use of cytodifferentiation of dental ectomesenchymal-derived cells that may contribute to the spatial-temporal expression of DSPP during tooth development.

Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides –97 and –72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.

Mechanical Loading Stimulates Differentiation of Periodontal Osteoblasts in a Mouse Osteoinduction Model: Effect on Type I Collagen and Alkaline Phosphatase Genes

Abstract The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer.

Effect of Mechanical Loading On Periodontal Cells

Mechanical loading is an important regulatory factor in alveolar bone homeostasis, and plays an essential role in maintaining the structure and mass of the alveolar processes throughout lifetime. A better understanding of the cellular and molecular responses of periodontal cells is a prerequisite for further improvements of therapeutic approaches in orthodontics, periodontal and alveolar bone repair and regeneration, implantology, and post-surgical wound healing. The purpose of this review is to provide an insight into some cell culture and animal models used for studying the effects of mechanical loading on periodontal cells, and into the recent developments and utilization of new in vivo animal models. There has been an increased awareness about the need for improvement and development of in vivo models to supplement the widely used cell culture models, and for biological validation of in vitro results, especially in the light of evidence that developmental models may not always reflect bone homeostasis in an adult organism. Due to the limitations of in vivo models, previous studies on mechanical regulation of alveolar bone osteoblasts and cementoblasts mostly focused on proliferative responses, rather than on the stimulation of cell differentiation. To address this problem, we have recently characterized and implemented a mouse osteoinductive tooth movement model for studying mechanically induced regulation of osteoblast- and cementoblast-associated genes. In this model, a defined and reproducible mechanical osteogenic loading is applied during a time course of up to two weeks. Regulation of gene expression in either wild-type or transgenic animals is assessed by a relative quantitative measurement of the level of target mRNAs directly within the subpopulations of periodontal cells. To date, results demonstrate a defined temporal pattern of cell-specific gene regulation in periodontal osteoblasts mechanically stimulated to differentiate and deposit bone matrix. The responses of osteoblast-associated genes to mechanical loading were 10- to 20-fold greater than the increase in the numbers of these cells, indicating that the induction of differentiation and an increase of cell function are the primary responses to osteogenic loading. The progression of the osteoblast phenotype in the intact mouse periodontium was several-fold faster compared with that in cultured cells, suggesting that the mechanical signal may be targeting osteoblast precursors in the state of readiness to respond to an environmental challenge, without the initial proliferative response. An early response of alkaline phosphatase and bone sialoprotein genes was detected after 24 hrs of treatment, followed by a concomitant stimulation of osteocalcin and collagen I between 24 and 48 hrs, and deposition of osteoid after 72 hrs. Although cementoblasts constitutively express biochemical markers similar to those of osteoblasts, distinct responses of osteocalcin, collagen I, and bone sialoprotein genes to mechanical loading were observed in the two cell phenotypes. This finding indicates that differential genetic responses to mechanical loading provide functional markers for distinction of the cementoblast and osteoblast phenotypes.

Temporal Pattern of Stimulation of Osteoblast-Associated Genes During Mechanically-Induced Osteogenesis In Vivo: Early Responses of Osteocalcin and Type I Collagen

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.

Bmp2 Is Required for Odontoblast Differentiation and Pulp Vasculogenesis

Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKOod) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKOod do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also describe an indirect effect of the Bmp2 gene in odontoblasts on formation of the vascular bed and associated pericytes in the pulp. This vascular niche and numbers of CD146+ pericytes are likely controlled by odontogenic and Bmp2-dependent VegfA production in odontoblasts. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.