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DMP1 (dentin matrix protein 1) is an extracellular matrix protein highly expressed in bones. Studies of Dmp1 knockout (KO) mice led to the discovery of a rare autosomal recessive form of hypophosphatemic rickets (ARHR) caused by DMP1 mutations. However, there are limitations for using this mouse model to study ARHR, including a lack of Haversian canals and osteons (that occurs only in large mammalian bones), high levels of fibroblast growth factor 23 (FGF23), and PTH, in comparison with a moderate elevation of FGF23 and unchanged PTH in human ARHR patients. To better understand this rare disease, we deleted the DMP1 gene in rabbit using CRISPR/Cas9. This rabbit model recapitulated many features of human ARHR, such as the rachitic rosary (expansion of the anterior rib ends at the costochondral junctions), moderately increased FGF23, and normal PTH levels, as well as severe defects in bone mineralization. Unexpectedly, all DMP1 KO rabbits died by postnatal week 8. They developed a severe bone microarchitecture defect: a major increase in the central canal areas of osteons, concurrent with massive accumulation of osteoid throughout all bone matrix (a defect in mineralization), suggesting a new paradigm, where rickets is caused by a combination of a defect in bone microarchitecture and a failure in mineralization. Furthermore, a study of DMP1 KO bones found accelerated chondrogenesis, whereas ARHR has commonly been thought to be involved in reduced chondrogenesis. Our findings with newly developed DMP1 KO rabbits suggest a revised understanding of the mechanism underlying ARHR. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

Genetic mouse models are widely used for understanding human diseases but we know much less about the anatomical structure of the auditory ossicles in the mouse than we do about human ossicles. Furthermore, current studies have mainly focused on disease conditions such as osteomalacia and rickets in patients with hypophosphatemia rickets, although the reason that these patients develop late-onset hearing loss is unknown. In this study, we first analyzed Dmp1 lac Z knock-in auditory ossicles (in which the blue reporter is used to trace DMP1 expression in osteocytes) using X-gal staining and discovered a novel bony membrane surrounding the mouse malleus. This finding was further confirmed by 3-D micro-CT, X-ray, and alizarin red stained images. We speculate that this unique structure amplifies and facilitates sound wave transmissions in two ways: increasing the contact surface between the eardrum and malleus and accelerating the sound transmission due to its mineral content. Next, we documented a progressive deterioration in the Dmp1-null auditory ossicle structures using multiple imaging techniques. The auditory brainstem response test demonstrated a conductive hearing loss in the adult Dmp1-null mice. This finding may help to explain in part why patients with DMP1 mutations develop late-onset hearing loss, and supports the critical role of DMP1 in maintaining the integrity of the auditory ossicles and its bony membrane., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression., (Copyright © 2017 by the Endocrine Society.)

Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1- knockout- lacZ -knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1 -loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis by DMP1 via the axis of "FGF23-renal phosphorus reabsorption" is vital for maintaining a healthy joint., Competing Interests: The authors have declared that no competing interest exists.

Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8weeks: the prevention group (starting at age 4weeks) and the treatment group (starting at age 12weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (>15%) and the biomechanical properties (3-point bending, ~60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Human DMP1 mutations or Dmp1-null (KO) mice display hypophosphatemia rickets, suggesting a causative role of low phosphate (P) in development of osteomalacia. To address the direct contribution of P to the in vivo bone mineralization we analyzed the properties of femurs obtained from Dmp1 null mice and wild type (WT) mice under a normal or high phosphorous (HiP) diet using combined assays, including histological examination, micro computed tomography (μCT), X-ray absorption near edge structure (XANES) spectroscopy and Raman spectroscopy. Histology and XANES indicate that WT mice have phosphate coordinated with Ca in the form of hydroxyapatite and tricalcium phosphate, while the KO mice have poorly coordinated soluble phosphates in their structure in both the normal and HiP diets. Raman spectroscopy and XANES indicate a higher carbonate/phosphate ratio and a low mineral/matrix ratio in the osteoid clusters in the KO femurs, which was only partially improved by HiP diets. Thus, we conclude that the hypophosphatemia induced osteomalacia phenotype in Dmp1 KO mice is contributed by at least two factors: the low Pi level and the DMP1 local function in mineralization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

FAM20C is a kinase phosphorylating the small-integrin-binding ligand, N-linked glycoproteins (SIBLINGs), a group of extracellular matrix proteins that are essential for bone and dentin formation. Previously, we showed that Sox2-Cre;Fam20Cfl/fl mice had bone and dentin defects, along with hypophosphatemia and significant downregulation of dentin matrix protein 1 (DMP1). While the assumed phosphorylation failure of the SIBLINGs is likely associated with the defects in the Fam20C-deficient mice, it remains unclear if the downregulation of Dmp1 contributes to these phenotypes. In this study, we crossed 3.6 kb Col1-Dmp1 transgenic mice with 3.6 kb Col1-Cre;Fam20Cfl/fl mice to overexpress Dmp1 in the mineralized tissues of Fam20C conditional knockout (cKO) mice. X-ray, micro-computed tomography, serum biochemistry and histology analyses showed that expressing the Dmp1 transgene failed to rescue the bone and dentin defects, as well as the serum levels of FGF23 and phosphate in the Fam20C-cKO mice. These results indicated that the downregulation of Dmp1 may not directly associate with, or significantly contribute to the bone and dentin defects in the Fam20C-cKO mice.

Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with αvβ3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as (NLS)DMP1) or to the extracellular matrix as the WT type (referred to as (SP)DMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted (NLS)DMP1 transgene driven by a 3.6-kb rat Col 1α1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the (SP)DMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent studies have revealed multiple roles of osteocytes in bone metabolism. However, detailed analyses of the embedded osteocytes in bone structure are still limited because of the high mineral content around these cells. In this study, we developed an innovative technique, the "FITC-Imaris technique", which combines FITC ([2, 5]-Fluorescein isothiocyanate), confocal microscopy and Imaris software. With this method, we could not only visualize the 3-D morphology of embedded osteocytes, but more importantly, we were able to statistically quantitate the osteocyte structure in the cell surface area, total cell volume, and dendrite numbers. Furthermore, we made a side-by-side comparison of the new method with the acid-etched SEM imaging technique, a common imaging method for studies of osteocyte morphology with a much smaller cell depth (< 3 μm). Finally, we used the FITC-Imaris technique to show both the morphological and statistical differences in the osteocyte structure between the Dmp1-null mice (the osteomalacia model) and their age-matched control littermates. We expect that this newly developed technique will become a powerful tool to disclose more roles that osteocytes play in bone health and diseases.

Dentin sialophosphoprotein (DSPP) plays a vital role in dentinogenesis. Previously, we showed that, in addition to dentin, DSPP is also highly expressed in alveolar bone and cellular cementum, and plays a crucial role in maintaining periodontal integrity; Dspp-deficient mice demonstrate severe periodontal defects, including alveolar bone loss, decreased cementum deposition, abnormal osteocyte morphology in the alveolar bone, and apical migration of periodontal ligament. Dentin sialophosphoprotein in dentin and bone is cleaved into NH₂ -terminal and COOH-terminal fragments. Whilst our previous study showed that the proteolytic processing of DSPP is critical for dentinogenesis, it is unclear whether the post-translational cleavage of DSPP also plays an essential role in maintaining a healthy periodontium. In this study, we analyzed the periodontal tissues from transgenic mice expressing the uncleavable full-length DSPP in the Dspp knockout (Dspp-KO) background (named 'Dspp-KO/D452A-Tg mice'), in comparison with those from wild-type mice, Dspp-KO mice, and mice expressing the normal Dspp transgene in the Dspp-KO background (designated 'Dspp-KO/normal-Tg mice'). We found that transgenic expression of the normal DSPP fully rescued the periodontal defects of the Dspp-KO mice, whereas this was not the case in Dspp-KO/D452A-Tg mice. These results indicate that proteolytic processing of DSPP is essential to periodontal integrity., (© 2013 Eur J Oral Sci.)

Dentin sialophosphoprotein (DSPP) is a large precursor protein that is proteolytically processed into a NH2 -terminal fragment [composed of dentin sialoprotein (DSP) and a proteoglycan form (DSP-PG)] and a COOH-terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH2 -terminal fragment of DSPP (i.e., DSP and DSP-PG) in dentinogenesis remain unclear. This study focuses on the function of the NH2 -terminal fragment of DSPP in dentinogenesis. Here, transgenic (Tg) mouse lines expressing the NH2 -terminal fragment of DSPP driven by a 3.6-kb type I collagen promoter (Col 1a1) were generated and cross-bred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found that dentin from the Dspp KO/DSP Tg mice was much thinner, more poorly mineralized, and remarkably disorganized compared with dentin from the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than did the Dspp null mice indicates that the NH2 -terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentinogenesis., (© 2013 Eur J Oral Sci.)

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.

DSPP, which plays a crucial role in dentin formation, is processed into the NH(2)-terminal and COOH-terminal fragments. We believe that the proteolytic processing of DSPP is an essential activation step for its biological function in biomineralization. We tested this hypothesis by analyzing transgenic mice expressing the mutant D452A-DSPP in the Dspp-knock-out (Dspp-KO) background (referred to as "Dspp-KO/D452A-Tg" mice). We employed multipronged approaches to characterize the dentin of the Dspp-KO/D452A-Tg mice, in comparison with Dspp-KO mice and mice expressing the normal DSPP transgene in the Dspp-KO background (named Dspp-KO/normal-Tg mice). Our analyses showed that 90% of the D452A-DSPP in the dentin of Dspp-KO/D452A-Tg mice was not cleaved, indicating that D452A substitution effectively blocked the proteolytic processing of DSPP in vivo. While the expression of the normal DSPP fully rescued the dentin defects of the Dspp-KO mice, expressing the D452A-DSPP failed to do so. These results indicate that the proteolytic processing of DSPP is an activation step essential to its biological function in dentinogenesis.

Objective: Dentine sialophosphoprotein (DSPP) was initially thought to be unique for dentine formation during tooth development, whilst recent reports have shown a much broader expression pattern such as in bone, periodontium and inner ear. Our goal was to explore its expression and potential impact during early nasal cartilage formation in comparison with tooth development., Study Design: We investigated DSPP expression in the nasal cartilage by immunohistochemistry and in situ hybridisation. We also cloned a 719bp partial DSPP cDNA from nasal cartilage and analysed its homology to the published mouse DSPP cDNA sequence. In addition, quantitative RT-PCR was undertaken to compare the expression pattern of DSPP in nasal cartilage and tooth germs during embryonic development., Results: The expression of DSPP in mouse nasal chondrocytes was detected using in situ hybridisation and immunohistochemical staining. The quantitative RT-PCR data showed that expression levels of DSPP in nasal cartilage are similar to that of tooth: low at E18, and increased during development with the peak level at P3. Furthermore, DSPP levels in nasal cartilage are lower than tooth but higher than bone., Conclusion: DSPP is expressed in nasal cartilage, and a similar temporal expression pattern in cartilage and tooth indicates the potential importance of DSPP during development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Dentin sialophosphoprotein (DSPP) in the extracellular matrix of dentin is cleaved into dentin sialoprotein and dentin phosphoprotein, which originate from the NH(2)-terminal and COOH-terminal regions of DSPP, respectively. In the proteolytic processing of mouse DSPP, the peptide bond at Gly(451)-Asp(452) has been shown to be cleaved by bone morphogenetic protein 1 (BMP1)/Tolloid-like metalloproteinases. In this study, we generated transgenic mice expressing a mutant DSPP in which Asp(452) was substituted by Ala(452). Protein chemistry analyses of extracts from the long bone of these transgenic mice showed that the D452A substitution partially blocked DSPP processing in vivo. When the full-length form of mutant DSPP (designated "D452A-DSPP") isolated from the transgenic mice was treated with BMP1 in vitro, a portion of the D452A-DSPP was cleaved, suggesting the presence of secondary peptide bond(s) that can be broken by BMP1. To identify the potential secondary DSPP cleavage site(s), site-directed mutagenesis was performed to generate nine DNA constructs expressing DSPP-bearing substitutions at potential scission sites. These different types of mutant DSPP made in eukaryotic cell lines were treated with BMP1 and the digestion products were assessed by Western immunoblotting. All of the mutant DSPP molecular species were partially cleaved by BMP1, giving rise to a protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to that of normal dentin sialoprotein. Taken together, we concluded that in addition to the peptide bond Gly(451)-Asp(452), there must be a cryptic cleavage site or sites close to Asp(452) in the mouse DSPP that can be cleaved by BMP1.

Family with sequence similarity 20,-member C (FAM20C) is highly expressed in the mineralized tissues of mammals. Genetic studies showed that the loss-of-function mutations in FAM20C were associated with human lethal osteosclerotic bone dysplasia (Raine Syndrome), implying an inhibitory role of this molecule in bone formation. However, in vitro gain- and loss-of-function studies suggested that FAM20C promotes the differentiation and mineralization of mouse mesenchymal cells and odontoblasts. Recently, we generated Fam20c conditional knockout (cKO) mice in which Fam20c was globally inactivated (by crossbreeding with Sox2-Cre mice) or inactivated specifically in the mineralized tissues (by crossbreeding with 3.6 kb Col 1a1-Cre mice). Fam20c transgenic mice were also generated and crossbred with Fam20c cKO mice to introduce the transgene in the knockout background. In vitro gain- and loss-of-function were examined by adding recombinant FAM20C to MC3T3-E1 cells and by lentiviral shRNA-mediated knockdown of FAM20C in human and mouse osteogenic cell lines. Surprisingly, both the global and mineralized tissue-specific cKO mice developed hypophosphatemic rickets (but not osteosclerosis), along with a significant downregulation of osteoblast differentiation markers and a dramatic elevation of fibroblast growth factor 23 (FGF23) in the serum and bone. The mice expressing the Fam20c transgene in the wild-type background showed no abnormalities, while the expression of the Fam20c transgene fully rescued the skeletal defects in the cKO mice. Recombinant FAM20C promoted the differentiation and mineralization of MC3T3-E1 cells. Knockdown of FAM20C led to a remarkable downregulation of DMP1, along with a significant upregulation of FGF23 in both human and mouse osteogenic cell lines. These results indicate that FAM20C is a bone formation "promoter" but not an "inhibitor" in mouse osteogenesis. We conclude that FAM20C may regulate osteogenesis through its direct role in facilitating osteoblast differentiation and its systemic regulation of phosphate homeostasis via the mediation of FGF23., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Dmp1 (dentin matrix protein1) null mice (Dmp1(-/-)) display hypophosphatemic rickets with a sharp increase in fibroblast growth factor 23 (FGF23). Disruption of Klotho (the obligatory co-receptor of FGF23) results in hyperphosphatemia with ectopic calcifications formed in blood vessels and kidneys. To determine the role of DMP1 in both a hyperphosphatemic environment and within the ectopic calcifications, we created Dmp1/Klotho compound deficient (Dmp1(-/-)kl/kl) mice., Procedures: A combination of TUNEL, immunohistochemistry, TRAP, von Kossa, micro CT, bone histomorphometry, serum biochemistry and Scanning Electron Microscopy techniques were used to analyze the changes in blood vessels, kidney and bone for wild type control, Dmp1(-/-), Klotho deficient (kl/kl) and Dmp1(-/-)kl/kl animals., Findings: Interestingly, Dmp1(-/-)kl/kl mice show a dramatic improvement of rickets and an identical serum biochemical phenotype to kl/kl mice (extremely high FGF23, hyperphosphatemia and reduced parathyroid hormone (PTH) levels). Unexpectedly, Dmp1(-/-)kl/kl mice presented elevated levels of apoptosis in osteocytes, endothelial and vascular smooth muscle cells in small and large blood vessels, and within the kidney as well as dramatic increase in ectopic calcification in all these tissues, as compared to kl/kl., Conclusion: These findings suggest that DMP1 has an anti-apoptotic role in hyperphosphatemia. Discovering this novel protective role of DMP1 may have clinical relevance in protecting the cells from apoptosis in high-phosphate environments as observed in chronic kidney disease (CKD).

Dentin sialophosphoprotein (DSPP) and its cleaved products, dentin phosphoprotein (DPP) and dentin sialoprotein (DSP), play important roles in biomineralization. Believed to be tooth specific, the authors' group revealed its expression in bone, and more recently, they and other groups also showed its expression in a few types of soft tissues. In this study, the authors systematically examined the expression of DSPP in a variety of non-mineralized tissues using reverse-transcription polymerase chain reaction (RT-PCR), real-time PCR, Western immunoblotting, and immunohistochemistry analyses in wild-type mice as well as β-galactosidase assays in the Dspp lacZ knock-in mice. These approaches showed the presence of DSPP in the salivary glands, cartilage, liver, kidney, and brain and its absence in the heart and spleen. Real-time PCR showed that the expression levels of DSPP mRNA in salivary glands, cartilage, liver, and kidney were higher than in the bone. Interestingly, DSPP was observed in the pericytes of blood vessels in the dental pulp, which are believed to be able to differentiate into odontoblasts. On the basis of these observations, the authors conclude that DSPP and/or its cleaved products may fulfill important functions in certain non-mineralized tissues in addition to its role in biomineralization.

Fibroblastic growth factor 23 (FGF23) is a circulating phosphaturic hormone. Inactivating mutations of the endopeptidase PHEX or the SIBLING protein DMP1 result in equivalent intrinsic bone mineralization defects and increased Fgf23 expression in osteocytes. The mechanisms whereby PHEX and DMP1 regulate Fgf23 expression are unknown. We examined the possibility that PHEX and DMP1 regulate Fgf23 through a common pathway by analyzing the phenotype of compound Phex and Dmp1 mutant mice (Hyp/Dmp1(-/-)). Compared to single-mutant littermates, compound-mutant Hyp/Dmp1(-/-) mice displayed nonadditive elevations of serum FGF23 (1912 ± 183, 1715 ± 178, and 1799 ± 181 pg/ml), hypophosphatemia (P(i): 6.0 ± 0.3, 5.8 ± 0.2, and 5.4 ± 0.1 mg/dl), and severity of rickets/osteomalacia (bone mineral density: -36, -36, and -30%). Microarray analysis of long bones identified gene expression profiles implicating common activation of the FGFR pathway in all the mutant groups. Furthermore, inhibiting FGFR signaling using SU5402 in Hyp- and Dmp1(-/-)-derived bone marrow stromal cells prevented the increase in Fgf23 mRNA expression (129- and 124-fold increase in Hyp and Dmp1(-/-) vs. 1.3-fold in Hyp+SU5402 and 2.5-fold in Dmp1(-/-)+SU5402, P<0.05). For all analyses, samples collected from nonmutant wild-type littermates served as controls. These findings indicate that PHEX and DMP1 control a common pathway regulating bone mineralization and FGF23 production, the latter involving activation of the FGFR signaling in osteocytes.

Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism., (Copyright © 2011 American Society for Bone and Mineral Research.)

Dentin matrix protein 1 (DMP1) is an acidic protein that plays critical roles in osteogenesis and dentinogenesis. Protein chemistry studies have demonstrated that DMP1 primarily exists as processed NH₂⁻ and COOH-terminal fragments in the extracellular matrix of bone and dentin. Our earlier work showed that the substitution of Asp²¹³ (a residue at a cleavage site) by Ala²¹³ blocks the processing of mouse DMP1 in vitro. Recently, we generated transgenic mice expressing this mutant DMP1 (designated 'D213A-DMP1'). By crossbreeding these transgenic mice with Dmp1-knockout (Dmp1-KO) mice, we obtained mice expressing the D213A-DMP1 transgene in the Dmp1-null background (named 'Dmp1-KO/D213A-Tg' mice). In this study, we analyzed the long bone, mandible, dentin, and cartilage of Dmp1-KO/D213A-Tg mice in comparison with wild-type, Dmp1-KO, and Dmp1-KO mice expressing the normal DMP1 transgene (Dmp1-KO/normal-Tg). Our results showed that D213A-DMP1 was barely cleaved in the dentin matrix of Dmp1-KO/D213A-Tg mice and the expression of D213A-DMP1 failed to rescue the developmental defects in Dmp1-null mice. Interestingly, enlarged growth plates and condylar cartilages were observed in Dmp1-KO/D213A-Tg mice, indicating a potential role of the full-length form of DMP1 in chondrogenesis., (Copyright © 2011 S. Karger AG, Basel.)

Mutations in FAM20C were recently identified as the cause of lethal osteosclerotic bone dysplasia, which highlighted the important role of this molecule in biomineralization. No systematic studies have been performed to evaluate the expression pattern of this relatively new molecule in the developmental processes of bone and tooth. In the present study, we analyzed in detail the expression profile of FAM20C during osteogenesis and odontogenesis using ISH and IHC approaches. The specimens analyzed were mouse tissues spanning embryonic day 13.5 (E13.5) to postnatal 8 weeks. The earliest presence of FAM20C was observed at E14.5. During osteogenesis, FAM20C mRNA was detected in the chondrocytes and osteoblasts of the long bone, whereas its protein was observed in the extracellular matrix (ECM) of bone and in the cytoplasm of the chondrocytes, osteoblasts, and osteocytes. During odontogenesis, FAM20C mRNA was detected in the ameloblasts, odontoblasts, cementoblasts, and periodontal ligament fibroblasts, whereas its protein was observed in the matrices of dentin, enamel, and alveolar bone and in the cytoplasm of the aforementioned cells. The temporospatial expression profile revealed in this study indicates that FAM20C is an ECM protein that may play an important role in controlling the mineralization of bone and tooth.

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH₂-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH₂-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis.

Dentin matrix protein 1 (DMP1), an acidic protein important to the formation of bone and dentin, primarily exists as the processed NH(2)-terminal and COOH-terminal fragments in the extracellular matrix of the two tissues. Previous in vitro studies showed that the substitution of residue Asp(213) by Ala(213) (D213A) at a cleavage site blocked the processing of mouse DMP1 in cells. In this study, we generated transgenic mice expressing mutant D213A-DMP1 (WT/D213A-Tg mice) to test the hypothesis that the proteolytic processing of DMP1 is an activation step essential to osteogenesis. By crossbreeding WT/D213A-Tg mice with Dmp1 knock-out (Dmp1-KO) mice, we obtained mice expressing D213A-DMP1 in a Dmp1-KO background; these mice will be referred to as "Dmp1-KO/D213A-Tg" mice. Biochemical, radiological, and morphological approaches were used to characterize the skeletal phenotypes of Dmp1-KO/D213A-Tg mice compared with wild-type mice, Dmp1-KO mice, and Dmp1-KO mice expressing the normal Dmp1 transgene. Protein chemistry analyses showed that DMP1 was barely cleaved in the bone of the Dmp1-KO/D213A-Tg mice, indicating that D213A substitution effectively blocked the proteolytic processing of DMP1 in vivo. While the expression of the normal Dmp1 transgene completely rescued the phenotypic skeletal changes of the Dmp1-KO mice, the expression of the mutant D213A-Dmp1 transgene failed to do so. These results indicate that the full-length form of DMP1 is an inactive precursor and its proteolytic processing is an activation step essential to the biological functions of this protein in osteogenesis.

DMP1 mutations in autosomal recessive hypophosphatemic rickets (ARHR) patients and mice lacking Dmp1 display an overlapping pathophysiology, such as hypophosphatemia. However, subtle differences exist between the mouse model and human ARHR patients. These differences could be due to a species specificity of human versus mouse, or it may be that the mutant DMP1 in humans maintains partial function of DMP1. In this study we report a deformed tooth phenotype in a human DMP1 deletion mutation case. Unexpectedly, the deletion of nucleotides 1484 to 1490 (c.1484&#95;1490delCTATCAC, delMut, resulting in replacement of the last 18 residues with 33 random amino acids) showed a severe dentin and enamel defect similar to a dentinogenesis imperfecta (DI) III-like phenotype. To address the molecular mechanism behind this phenotype, we generated delMut transgenic mice with the endogenous Dmp1 gene removed. These mutant mice did not recapture the abnormal phenotype observed in the human patient but displayed a mild rachitic tooth phenotype in comparison with that in the Dmp1-null mice, suggesting that the DI III-like phenotype may be due to an as-yet-undetermined acquired gene modifier. The mechanism studies showed that the mutant fragment maintains partial function of DMP1 such as stimulating MAP kinase signaling in vitro. Last, the in vitro and in vivo data support a role of odontoblasts in the control of fibroblast growth factor 23 (FGF-23) regulation during early postnatal development, although this regulation on Pi homeostasis is likely limited.

With age, there is a progressive loss of body balance function. Yet, the potential influence of osteoporosis on body balance is largely unknown. Dentin matrix protein 1 (DMP1) is highly expressed in bone and required for phosphate homeostasis and mineralization. Dmp1 null mice display striking defects in bone structure. In this study we reported circling behavior and hyper reaction to touching in Dmp1 null mice. Our histology, tartrate resistant acid phosphatase (TRAP) staining and µCT data showed dramatic changes, such as an expansion of poorly mineralized matrices, in the Dmp1 null porous bony structure in the vestibular apparatus. The targeted re-expression of DMP1 in the Dmp1 null bone fully rescued not only the bone phenotype, but also circling behavior and hyper reaction. Furthermore, X-gal stain and DMP1 immunohistochemistry assay showed that DMP1 was not expressed in neuron cells or balance related cells in the inner ear, suggesting that a defect in the bony labyrinth of the internal ear is indirectly responsible for the circling behavior and/or hyper reaction to touching. Finally, discovery of DMP1 lacZ signal in pericyte-like cells may suggest a new function of DMP1 in angiogenesis.

Dmp1-null mice and patients with mutations in dentin matrix protein 1 (DMP1) resulting in autosomal recessive hypophosphatemic rickets display similar skeletal defects. As mutations were observed in the last 18 amino acids of DMP1 in 1 subset of patients and as fragments of intact DMP1, a 37-kDa N-terminal and a 57-kDa C-terminal fragment, have been purified from bone and dentin, we hypothesized that the cleaved 57-kDa C-terminal fragment is the essential functional domain of DMP1. To test this hypothesis, different forms of recombinant DMP1 were expressed in 293EBNA, CHO and 2T3 cells. The results showed that DMP1 was processed into a 37-kDa N-terminal and a 57-kDa C-terminal fragment in vitro in all cell lines examined. DMP1 processing in CHO cells was blocked by a furin protease inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone, in a dose-dependent manner. Coexpression of PHEX, a potential upstream protease, had no apparent effect on DMP1 cleavage in 293EBNA cells, suggesting that PHEX may not be required for DMP1 processing. To test the in vivo role of the C-terminal fragment, transgenic mice overexpressing full-length DMP1 or the 57-kDa fragment controlled by the 3.6-kb Col1 promoter were generated. Overexpression of these transgenes had no effect on the wild-type skeleton, but on the Dmp1-null background showed expression in the osteoblast layer and throughout the bone matrix leading to the rescue of the null bone phenotype. This suggests that the 57-kDa C-terminal fragment may be able to recapitulate the function of intact DMP1 in vivo., (Copyright 2008 S. Karger AG, Basel.)

Autosomal recessive hypophosphatemic rickets (ARHR), which is characterized by renal phosphate wasting, aberrant regulation of 1alpha-hydroxylase activity, and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). ARHR resembles autosomal dominant hypophosphatemic rickets (ADHR) and X-linked hypophosphatemia (XLH), hereditary disorders respectively caused by cleavage-resistant mutations of the phosphaturic factor FGF23 and inactivating mutations of PHEX that lead to increased production of FGF23 by osteocytes in bone. Circulating levels of FGF23 are increased in ARHR and its Dmp1-null mouse homologue. To determine the causal role of FGF23 in ARHR, we transferred Fgf23 deficient/enhanced green fluorescent protein (eGFP) reporter mice onto Dmp1-null mice to create mice lacking both Fgf23 and Dmp1. Dmp1(-/-) mice displayed decreased serum phosphate concentrations, inappropriately normal 1,25(OH)(2)D levels, severe rickets, and a diffuse form of osteomalacia in association with elevated Fgf23 serum levels and expression in osteocytes. In contrast, Fgf23(-/-) mice had undetectable serum Fgf23 and elevated serum phosphate and 1,25(OH)(2)D levels along with severe growth retardation and focal form of osteomalacia. In combined Dmp1(-/-)/Fgf23(-/-), circulating Fgf23 levels were also undetectable, and the serum levels of phosphate and 1,25(OH)(2)D levels were identical to Fgf23(-/-) mice. Rickets and diffuse osteomalacia in Dmp1-null mice were transformed to severe growth retardation and focal osteomalacia characteristic of Fgf23-null mice. These data suggest that the regulation of extracellular matrix mineralization by DMP1 is coupled to renal phosphate handling and vitamin D metabolism through a DMP1-dependent regulation of FGF23 production by osteocytes.

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages., ((c) 2008 Wiley-Liss, Inc.)

Mutations in human cartilage oligomeric matrix protein (COMP) have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia; however, the functions of both wild-type and mutant COMP in the skeletogenesis remain unknown. In an effort to define the biological functions of COMP, a functional genetic screen based on the yeast two-hybrid system was performed. This led to the identification of granulin-epithelin precursor (GEP), an autocrine growth factor, as a COMP-associated partner. COMP directly binds to GEP both in vitro and in vivo, as revealed by in vitro pull down and co-immunoprecipitation assays. GEP selectively interacts with the epidermal growth factor repeat domain of COMP but not with the other three functional domains of COMP. The granulin A repeat unit of GEP is required and sufficient for association with COMP. COMP co-localizes with GEP predominantly in the pericellular matrix of transfected rat chondrosarcoma cell and primary human chondrocytes. Staining of musculoskeletal tissues of day 19 mouse embryo with antibodies to GEP is restricted to chondrocytes in the lower proliferative and upper hypertrophic zones. Overexpression of GEP stimulates the proliferation of chondrocytes, and this stimulation is enhanced by COMP. In addition, COMP appears to be required for GEP-mediated chondrocyte proliferation, since chondrocyte proliferation induced by GEP is dramatically inhibited by an anti-COMP antibody. These findings provide the first evidence linking the association of COMP and GEP and identifying a previously unrecognized growth factor (i.e. GEP) in cartilage.

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.

The osteocyte, a terminally differentiated cell comprising 90%-95% of all bone cells, may have multiple functions, including acting as a mechanosensor in bone (re)modeling. Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes and, when deleted in mice, results in a hypomineralized bone phenotype. We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (P(i)) homeostasis. Both Dmp1-null mice and individuals with a newly identified disorder, autosomal recessive hypophosphatemic rickets, manifest rickets and osteomalacia with isolated renal phosphate-wasting associated with elevated fibroblast growth factor 23 (FGF23) levels and normocalciuria. Mutational analyses showed that autosomal recessive hypophosphatemic rickets family carried a mutation affecting the DMP1 start codon, and a second family carried a 7-bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using Dmp1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. Our findings suggest a bone-renal axis that is central to guiding proper mineral metabolism.

Unlabelled: The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype., Introduction: Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls., Materials and Methods: Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (N(total) = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by muCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined., Results: The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on muCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and muCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values., Conclusions: The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca x P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.

Dentin matrix protein 1 (DMP1) is highly expressed in osteocytes and is mechanically responsive. To study osteocyte-specific and mechanically regulated DMP1 gene expression, the transcriptional activity of three cis-regulatory regions was first examined in an osteoblast differentiation model in vitro using a green fluorescent protein (GFP) reporter. Expression of the -9624 to +1996 bp (10 kb) and -7892 to +4439 bp (8 kb) DMP1 cis-regulatory regions dramatically increased in areas of mineralized matrix, in dendritic, osteocyte-like cells. Mineralizing cultures expressing the 8-kb construct show dramatic GFP increases in response to loading in cells with a dendritic morphology. Transgenic mice expressing the 8-kb DMP1-GFP and -2433 to +4439 bp (2.5 kb) DMP1-LacZ were generated. Osteocyte-specific expression was found with the 8 kb but not with the 2.5 kb in postnatal animals. However, the 2.5 kb could support expression in rapidly forming osteoblasts and pre-osteocytes in the embryo. Primary calvarial osteoblast cultures demonstrated that the 2.5 kb supports weak expression in a subset of osteoblasts and pre-osteocytes, but not in mature osteocytes. However, the 8 kb supports robust expression in primary bone marrow cultures. Therefore the region -7892 to -2433 bp, termed a 5.5-kb "Osteocyte Enhancer Module," appears to be required for osteocyte specificity. Ulnae of mice with the 8-kb DMP1-GFP were subjected to mechanical loading where GFP expression increased selectively and locally in osteocytes, distal to the mid-shaft and near the surface of the bone. Thus, the 8-kb region of the DMP1 gene is a target for mechanotransduction in osteocytes, and its cis-regulatory activity may be correlated to local strain in bone.

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development., (2005 S. Karger AG, Basel)

The non-covalent bond between aggrecan and hyaluronan is critical for maintaining the normal structure and function of the extracellular matrix in articular cartilage. The failure of this bond can cause the loss of aggrecan and destruction of the extracellular matrix of articular cartilage. In this study, the rupture force of the single bond between hyaluronan and hyaluronan binding protein - the complex of the hyaluronan binding region of aggrecan and link protein - was directly measured with a nanomechanical testing system as 40+/-11 pN. The results were compared to a theoretical prediction based on a smart version of the Monte Carlo simulation.

Objective: To investigate the effect of isolated hydrostatic pressure on proteoglycan metabolism in chondrocytes., Methods: Bovine articular chondrocytes cultured in agarose gels were subjected to 5 MPa hydrostatic pressure for 4 hours in either a static or a pulsatile (1 Hz) mode, and changes in glycosaminoglycan (GAG) synthesis, hydrodynamic size, and aggregation properties of proteoglycans and aggrecan messenger RNA (mRNA) levels were determined., Results: The application of 5 MPa static pressure caused a significant increase in GAG synthesis of 11% (P < 0.05). Column chromatography showed that this increase in GAG synthesis was associated with large proteoglycans. In addition, semiquantitative reverse transcriptase-polymerase chain reaction showed a 4-fold increase in levels of aggrecan mRNA (P < 0.01)., Conclusion: Hydrostatic pressure in isolation, which does not cause cell deformation, can affect proteoglycan metabolism in chondrocytes cultured in agarose gels, indicating an important role of hydrostatic pressure in the regulation of extracellular matrix turnover in articular cartilage.

Tooth is made of crown and root. It is widely believed that dentin formation in crown and root uses the same regulatory mechanism. However, identification of nuclear factor 1 C (NFIC)'s unique function in determining root but not crown dentin formation challenges the old thinking. In searching for the target molecules downstream of NFIC, we unexpectedly found a sharp reduction of osterix (OSX), the key transcription factor in skeleton formation, in the Nfic knockout (Nfic-KO) tooth root. We then demonstrated a dose-dependent increase of Osx in the odontoblast cell line due to a transient transfection of Nfic expression plasmid. Studies of global and conditional Osx-KO mice revealed no apparent changes in the crown dentin tubules and dentin matrix. However, the OSX conditional KO (cKO) mice (crossed to the 2.3-kb collagen type 1 [Col1]-Cre) displayed an increase in cell proliferation but great decreases in expressions of root dentin matrix proteins (dentin matrix protein 1 [DMP1] and dentin sialophosphoprotein [DSPP]), leading to an inhibition in odontoblast differentiation, and short, thin root dentin with few dentin tubules. Compared to the Nfic-KO tooth, which contains essentially no dentin tubules and remains in a "root-less" status at adult stages, the Osx-cKO root phenotype had partially improved at the late stage, indicating that other factors can compensate for OSX function. Thus, we conclude that OSX, one of the key downstream molecules of NFIC, plays a critical role in root, but not crown, formation.

This study investigates bony protrusions within a narrowed periodontal ligament space (PDL-space) of a human bone-PDL-tooth fibrous joint by mapping structural, biochemical, and mechanical heterogeneity. Higher resolution structural characterization was achieved via complementary atomic force microscopy (AFM), nano-transmission X-ray microscopy (nano-TXM), and microtomography (MicroXCT™). Structural heterogeneity was correlated to biochemical and elemental composition, illustrated via histochemistry and microprobe X-ray fluorescence analysis (μ-XRF), and mechanical heterogeneity evaluated by AFM-based nanoindentation. Results demonstrated that the narrowed PDL-space was due to invasion of bundle bone (BB) into PDL-space. Protruded BB had a wider range with higher elastic modulus values (2-8GPa) compared to lamellar bone (0.8-6GPa), and increased quantities of Ca, P and Zn as revealed by μ-XRF. Interestingly, the hygroscopic 10-30μm interface between protruded BB and lamellar bone exhibited higher X-ray attenuation similar to cement lines and lamellae within bone. Localization of the small leucine rich proteoglycan biglycan (BGN) responsible for mineralization was observed at the PDL-bone interface and around the osteocyte lacunae. Based on these results, it can be argued that the LB-BB interface was the original site of PDL attachment, and that the genesis of protruded BB identified as protrusions occurred as a result of shift in strain. We emphasize the importance of bony protrusions within the context of organ function and that additional study is warranted.

Transforming growth factor beta (TGFβ) signaling plays an important role during osteogenesis. However, most research in this area focuses on cortical and trabecular bone, whereas alveolar bone is largely overlooked. To address the role of TGFβR2 (the key receptor for TGFβ signaling) during postnatal alveolar bone development, we conditionally deleted Tgf β r2 in early mesenchymal progenitors by crossing Gli1-Cre ERT 2; Tgf β r2 flox / flox ; R26R tdTomato mice (named early cKO) or in osteoblasts by crossing 3.2kb Col1-Cre ERT 2 ; Tgf β r2 flox / flox ; R26R tdTomato mice (named late cKO). Both cKO lines were induced at postnatal day 5 (P5) and mice were harvested at P28. Compared to the control littermates, early cKO mice exhibited significant reduction in alveolar bone mass and bone mineral density, with drastic defects in the periodontal ligament (PDL); conversely, the late cKO mice displayed very minor changes in alveolar bone. Mechanism studies showed a significant reduction in PCNA+ PDL cell numbers and OSX+ alveolar bone cell numbers, as well as disorganized PDL fibers with a great reduction in periostin (the most abundant extracellular matrix protein) on both mRNA and protein levels. We also showed a drastic reduction in β-catenin in the early cKO PDL and a great increase in SOST (a potent inhibitor of Wnt signaling). Based on these findings, we conclude that TGFβ signaling plays critical roles during early alveolar bone formation via the promotion of PDL mesenchymal progenitor proliferation and differentiation mechanisms. [ABSTRACT FROM AUTHOR]