Biomedical subjects

Keiichi Ohya

Publications and source records attributed to Keiichi Ohya.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of osteoclast differentiation and function by the CaMK-CREB pathway.

Calcium (Ca(2+)) signaling is essential for a variety of cellular responses and higher biological functions. Ca(2+)/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis) that is activated by receptor activator of NF-kappaB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases.

Animals↗

PubMed2006

Amelioration of bone loss in collagen-induced arthritis by neutralizing anti-RANKL monoclonal antibody.

Receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are pivotal regulators of osteoclast differentiation. RANK and RANKL also mediate T cell/dendritic cell (DC) interaction. Previous study has shown that RANK/RANKL interaction induces prolonged DC survival and antigen presentation. In the present study, we have newly established a hybridoma which produces neutralizing anti-RANKL monoclonal antibody (IK22-5). By treating collagen-induced arthritis (CIA) mice with IK22-5, we have investigated the role of RANKL in the pathogenesis of CIA. Although IK22-5 had no effect on immune responses or inflammation, it ameliorated bone loss at the site of inflammation. Histological analyses revealed that osteoclast formation was impaired at the site of joint inflammation in IK22-5-treated CIA mice. These results suggest the utility of anti-RANKL mAb for the prevention of osteoporosis associated with joint inflammation in RA.

Animals↗

PubMed2006

A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss.

Activating receptor activator of NF-kappaB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-alpha-induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF-induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-alpha promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.

Amino Acid Sequence↗

PubMed2006

Bovine deciduous dentine is more susceptible to osteoclastic resorption than permanent dentine: results of quantitative analyses.

Many clinical reports suggest that deciduous teeth exhibit a greater susceptibility to resorption than permanent ones. To examine the difference between deciduous and permanent dentine in their susceptibility to osteoclastic resorption, osteoclast-like cells (OCLs) were cultured on deciduous and permanent dentine slices. The number, area, depth, and volume of resorption pits were then measured, using image-analyzing systems. We measured the level of degraded collagen (cross-linked N-telopeptide of type I collagen; NTx) in culture medium using an enzyme-linked immunosorbent assay (ELISA). The levels of cathepsin K, matrix metallo proteinase (MMP)-9, and MMP-13 mRNAs in the cells attached to dentine were also analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). Deciduous dentine slices exhibited a significant (twofold) increase in resorbed area compared with the permanent slices. Three-dimensional analysis revealed that the volume of pits in deciduous dentine differed significantly (fourfold) compared to that in the permanent dentine. The depth of pits also followed the same trend. However, there was no significant difference in the number of pits or osteoclasts on the dentine slices. The NTx level in deciduous media was significantly more than that in permanent media. The mRNA levels also followed the same trend. These results suggest that deciduous dentine is more susceptible to resorption than permanent dentine and signals from the substrate play an important role in physiological resorption.

Animals↗

PubMed2006

Expression of MT1-MMP during deciduous tooth resorption in odontoclasts.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-bound matrix metalloproteinase capable of mediating pericellular proteolysis of extracellular matrix components. In osteoclasts, the localization of MT1-MMP has been reported at the tips of specialized membrane protrusions (podosomes and lamellipodia) so that osteoclasts might use MT1-MMP to perform focal proteolysis and move through the extracellular matrix to the bone surface. The objectives of this study were to investigate an association of MT1-MMP in physiological root resorption of the deciduous tooth by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis, and to identify MT1-MMP-producing cell during deciduous tooth resorption by in situ hybridization and immunohistochemistry. RT-PCR and Northern blot analysis revealed the exclusively high expression of MT1-MMP mRNA in bovine root-resorbing tissue, which lies between the root of the deciduous tooth and its permanent successor. Expression of MT1-MMP mRNA was seen in odontoclasts aligning in the surface layer of the root-resorbing tissue at sites of root resorption. Furthermore, immmunohistochemistry also confirmed the localization of MT1-MMP protein to the odontoclasts. The present identification of MT1-MMP in odontoclasts during deciduous tooth resorption might be relevant to the migration activity that these cells have to gain access to the root surface.

Animals↗

PubMed2006

Bone regeneration by modified gene-activated matrix: effectiveness in segmental tibial defects in rats.

Gene-activated matrix (GAM) is a matrix, such as collagen-containing plasmid vector, that encodes a protein to stimulate tissue regeneration. In the original GAM system, gene transfer efficiency was extremely low. We have recently reported that modifying GAM with calcium-phosphate precipitates (CaP) enhances the efficiency of gene transfer. The purpose of this study was to evaluate the effects of our modified GAM on tissue regeneration. We prepared critical size segmental bone defects in rat tibiae and transplanted GAM consisting of bovine atelocollagen and expression plasmid vector (bmp2), which encodes human BMP2, with or without CaP. The tibiae were later examined radiographically, histologically, and mechanically. Implantation of bmp2-CaP-collagen at 12 microg bmp2 bridged the bone defect at 4 weeks, and the strength of the bone was comparable to that of an intact tibia at 6 weeks. Implantation of bmp2-collagen at the same dose of bmp2 bridged the defect to a smaller extent. Neither collagen alone nor vacant vector-CaP-collagen bridged the defect. These results indicate that our modified GAM with CaP has the potential to be effective in tissue regeneration at lower plasmid DNA doses than used in previous studies.

Animals↗

PubMed2006

A tumor necrosis factor-alpha antagonist inhibits inflammatory bone resorption induced by Porphyromonas gingivalis infection in mice.

BACKGROUND: A tumor-necrosis factor-alpha (TNF-alpha) antagonist, the WP9QY peptide, was designed based on the crystal structure of the TNF-beta/TNF-receptor complex in order to overcome the disadvantages of macromolecules such as antibodies or soluble receptors by reducing the molecular size of TNF-alpha antagonists. It efficiently antagonizes the effect of TNF-alpha binding to the TNF receptor (I). OBJECTIVES: The aim of the present study was to assess the effects of the WP9QY peptide on inflammatory bone resorption and osteoclast formation in the periodontal pathogen-infection model. MATERIAL AND METHODS: Live Porphyromonas gingivalis ATCC 33277 was injected once daily for 6 days into the subcutaneous tissue overlying the calvariae in mice. At the same time, the WP9QY peptide (1 mg/kg, 2 mg/kg or 4 mg/kg per day) was administrated via osmotic minipumps for 7 days. Histological observations and the radiological assessments of the calvariae as well as bone mineral density measurements were performed. RESULTS: The WP9QY peptide significantly prevented the P. gingivalis-induced reduction in the bone mineral density at the calvariae. The histomorphometric assessments revealed the inhibitiory effects of the WP9QY peptide on the P. gingivalis-induced increase in the number of the inflammatory cells and in the area of sagittal suture at the calvariae. Furthermore, there was also an inhibitory effect on the P. gingivalis-induced increase in the number of osteoclasts per unit bone surface at the calvariae. CONCLUSION: These results suggest that the strategy for the design to reduce the molecular size of the TNF-alpha antagonists would be beneficial for the treatment of local inflammatory bone loss induced by periodontal-pathogen infection.

Analysis of Variance↗

PubMed2006

Effects of exposure to 4-META/MMA-TBB resin on pulp cell viability.

Adhesive restorative systems have expanded the range of possibilities for direct pulp-capping technique, with evidences of clinical success in vital pulp therapy. However, quite few studies have described the direct responses of pulp cells following the application of resinous materials to pulp exposure. To address this issue, effects of exposure to an adhesive resin, 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin on cellular activity were investigated in an established rat dental pulp cell line (RPC-C2A). RPC-C2A cells were cultured on normal plastic plates or the disks prepared from 4-META/MMA-TBB resin (Super Bond C&B) in a-MEM containing 10% FBS. After 3, 7 and 14 days, DNA content and alkaline phosphatase (ALP) activity were measured. Total RNA in each group was extracted and RT-PCR analysis was performed. Moreover, the live cell ratio was also evaluated by cytotoxicity assay after treatment with various concentrations of 4-META/MMA-TBB. At day 3, 7 and 14, amount of DNA and ALP activity of the cells on normal plastic plates and the one on the 4-META/MMA-TBB were comparable. Cells of both groups expressed mRNA of type I collagen (Coll), ALP, osteopontin (OPN), osteocalcin (OC), and bone morphogenetic protein (BMP-2). Furthermore, 4-META/MMA-TBB (10(-1)% or less) did not influence dead cell ratio in the confluent state. According to the results of these in vitro studies, exposure to this resinous material would not induce cytotoxic response in the pulp cells.

Alkaline Phosphatase↗

PubMed2005

Regional distinctions in cortical bone mineral density measured by pQCT can predict alterations in material property at the tibial diaphysis of the Cynomolgus monkey.

We examined whether regional differences in cortical bone mineral density (Ct.BMD) measured by peripheral quantitative computed tomography is related to the heterogeneity of bone tissue and whether regional Ct.BMD is a better indicator of changes in bone material properties. Bilateral tibiae were obtained from 17 female adult Cynomolgus monkeys (Macaca fascicularis; mean age 16.8 years). After determining that Ct.BMD was similar between the right and left tibiae, the left tibiae were used for bone histomorphometry and the right for a three-point bending test. The Ct.BMD in the posterior quadrant was significantly higher than that in the anterior quadrant. In the bone histomorphometric analysis, all parameters (i.e., average osteonal area, average osteonal bone area, osteon population density, percent osteonal area [%On.Ar], percent osteonal bone area [%On.B.Ar], percent osteonal area of initial remodeling [%Il.On.Ar], percent osteonal area of secondary remodeling [%Sd.On.Ar], porosity, and percent osteoid area in the posterior region) were significantly lower than those in the anterior region. The results indicated that in the same cross-section, bone tissue structure was heterogeneous. Both total- and posterior-Ct.BMD were positively correlated with breaking stress and negatively correlated with toughness, whereas anterior-Ct.BMD was positively correlated with elastic modulus. Backward stepwise multiple regression analyses indicated that posterior-Ct.BMD and total-Ct.BMD were the best variables for predicting breaking stress and toughness, respectively, when age is taken into account. The %On.Ar, %On.B.Ar, and %Il.On.Ar in the posterior region were negatively correlated with elastic modulus. The %On.Ar, %On.B.Ar, and %Sd.On.Ar in the posterior region were positively correlated with toughness. These findings indicated that regional Ct.BMD measurement is useful to assess changes in the material properties of bone associated with the degree of mineralization. In particular, anterior-, posterior-, and total-Ct.BMD can be used separately to predict changes in the material properties of the tibial diaphysis.

Absorptiometry, Photon↗

PubMed2005

Establishment of rat model of drug-induced gingival overgrowth induced by continuous administration of phenytoin.

It is well-known that the anticonvulsant drug, phenytoin (PHT), induces gingival overgrowth as a side effect. The mechanism of PHT-induced gingival overgrowth, however, is not well understood. One reason for this is the lack of an adequate animal model for the PHT-induced gingival overgrowth. The purpose of this study was to establish a rat model of the drug-induced gingival overgrowth. Fourteen-day-old rats were randomly divided into 3 groups (5 rats/group). The control rats received only the vehicle. The rats in the experimental group were injected with 50 mg/kg per day (group L) and 100 mg/kg per day (group H) of PHT. They received a subcutaneous injection of vehicle or PHT twice a day for 42 days. A charge-coupled device (CCD) laser displacement sensor was used for measurement of the severity of gingival overgrowth of the mandibles. There was no significant difference in the growth of rats between the PHT-injected and the control groups. The CCD laser displacement sensor can measure minute changes in the gingival overgrowth in rats, and a significant extension of the buccal gingiva was observed in groups L and H. Using the CCD sensor, it is possible to quantify the change in the gingiva under precise control of the PHT dose.

Animals↗

PubMed2005

A new technique with calcium phosphate precipitate enhances efficiency of in vivo plasmid DNA gene transfer.

In vivo gene transfer with plasmid vector has been applied experimentally and clinically; however, the low level of gene transfer efficiency with plasmid vector is a problem. We speculated that the combination of calcium phosphate precipitate (CaP) and plasmid vector could solve this problem because CaP stabilizes plasmid DNA. In the present study, we used a plasmid exression vector encoding enhanced green fluorescent protein and combined the vector with CaP. Then, this combination was mixed with bovine type I atelocollagen. After incubating this mixture in phosphate-buffered saline, the amount of the plasmid DNA in the supernatant was low when the plasmid DNA was combined with CaP. Furthermore, the plasmid DNA, which was combined with CaP, was stable in DNase digestion in vitro. The plasmid vector with or without CaP, together with the atelocollagen, was transplanted subcutaneously or injected in the bone marrow of the femurs of rats. Then, the fluorescence was observed under a confocal laser scanning microscope and the fluorescence intensity in the tissue homogenates was measured. In these animal experiments, the fluorescence was extensive when the plasmid DNA was combined with CaP. These results indicate that our formula, collagen/CaP/DNA, appeared efficient for in vivo gene transfer.

Animals↗

PubMed2005

Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2.

Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and beta-glycerophosphate, under 0.8%-15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1 h and 6 h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48 h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain.

Alkaline Phosphatase↗

PubMed2005

Subcutaneous injections of a TNF-alpha antagonistic peptide inhibit both inflammation and bone resorption in collagen-induced murine arthritis.

The cyclic peptide WP9QY (YCWSQYLCY), which was designed to mimic the most critical tumor necrosis factor (TNF) alpha recognition loop on type1 TNF receptor, antagonizes the effects of TNFalpha. In this study, we investigated the effects of WP9QY peptide on collagen-induced arthritis (CIA) mice to evaluate its effects on inflammatory bone destruction. DBA/1J mice were injected intradermally at the base of the tail with bovine type II collagen, emulsified in complete Freund's adjuvant on day 0 and 21. The three sets of WP9QY peptide injections (24 mg/kg x 8 times per day) were performed before the onset of paw swelling. Mice were sacrificed at day 38 and thereafter, the arthritis scores as well as radiographical and histological outcomes were assessed. WP9QY peptide inhibited CIA-induced increase in the arthritis score. Furthermore, histomorphometric analysis of the tibial epiphysis region revealed that WP9QY peptide inhibited the increase of synovial pannus infiltration and the decrease of bone volume, which were induced by the CIA. The WP9QY treatment prevented the inflammation as well as bone destruction of the joints in the CIA mice, suggesting that the administration of WP9QY peptide might be useful for developing a drug to prevent inflammatory bone destruction.

Animals↗

PubMed2005

[RANKL/RANK signaling-inhibitor].

Osteoclast differentiation requires the interaction of RANK (receptor activator of NF kappaB) and RANK ligand, conserved member of the TNF (tumor necrosis factor) receptor and TNF families, respectively. We tested the ability of a peptide mimic (WP9QY) of a critical contact site on the TNF receptor to inhibit RANK ligand induced osteoclastogenesis and discuss whether it could work as both TNF-alpha and RANKL antagonists, and the possibility of the drug development using the techniques in the structural biology.

Animals↗

PubMed2005

Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.

Alkaline Phosphatase↗

PubMed2004

Inhibition of RANKL-induced osteoclastogenesis by (-)-DHMEQ, a novel NF-kappaB inhibitor, through downregulation of NFATc1.

UNLABELLED: (-)-DHMEQ, a newly designed NF-kappaB inhibitor, inhibited RANKL-induced osteoclast differentiation in mouse BMMs through downregulation of the induction of NFATc1, an essential transcription factor of osteoclastogenesis. INTRODUCTION: Bone destruction is often observed in advanced case of rheumatoid arthritis and neoplastic diseases, including multiple myeloma. Effective and nontoxic chemotherapeutic agents are expected for the suppression of these bone destructions. RANKL induces activation of NF-kappaB and osteoclastogenesis in bone marrow-derived monocyte/macrophage precursor cells (BMMs). Targeted disruption or pharmacological suppression of NF-kappaB result in impaired osteoclastogenesis, but how NF-kappaB is involved in the regulation of osteoclastogenesis is not known. MATERIALS AND METHODS: The effect of (-)-dehydroxymethylepoxyquinomicin [(-)-DHMEQ] on osteoclast differentiation was studied using a culture system of mouse BMMs stimulated with RANKL and macrophage colony-stimulating factor. The mechanism of the inhibition was studied by biochemical analysis such as immunoblotting and retroviral transfer experiments. RESULTS: (-)-DHMEQ strongly inhibited RANKL-induced NF-kappaB activation in BMMs and inhibited RANKL-induced formation of TRACP(+) multinucleated cells. Interestingly, (-)-DHMEQ specifically inhibited the RANKL-induced expression of NFATc1 but not the expressions of TRAF6 or c-fos. Inhibition of osteoclast differentiation by (-)-DHMEQ was rescued by overexpression of NFATc1, suggesting that the inhibition is not caused by a toxic effect. Moreover, pit formation assays showed that (-)-DHMEQ also inhibited the bone-resorbing activity of mature osteoclasts. CONCLUSION: The inhibition of NF-kappaB suppresses osteoclastogenesis by downregulation of NFATc1, suggesting that NFATc1 expression is regulated by NF-kappaB in RANKL-induced osteoclastogenesis. Our results also indicate the possibility of (-)-DHMEQ becoming a new therapeutic strategy against bone erosion.

Animals↗

PubMed2004

Selective inhibition of NF-kappa B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo.

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-kappa B (NF-kappa B) has a crucial role in osteoclast differentiation, and blocking NF-kappa B is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the I kappa B-kinase complex, a crucial component of signal transduction pathways to NF-kappa B. The peptide inhibited RANKL-stimulated NF-kappa B activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-alpha and interleukin-1 beta, abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-kappa B activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption.

Animals↗

PubMed2004

Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues.

Dentin matrix protein 1 (DMP1) is an Arg-Gly-Asp-containing acidic phosphoprotein that was originally identified from a rat incisor cDNA library and was thought to be a dentin-specific protein. DMP1 was later shown to express in a number of hard tissue-forming cells, including osteoblasts, osteocytes, ameloblasts, and cementoblasts, and was considered to play important roles in mineralization. Further, DMP1 gene expression was also detected in fetal bovine brain and in newborn mouse brain. These findings indicate the possibility of DMP1 expression in other soft tissues. In the present study, to clarify the significance of DMP1 expression in nonmineralized tissues, we made a specific antibody to mouse DMP1 peptides and demonstrated that DMP1 protein was localized in mouse brain, pancreas, and kidney by immunohistochemistry. Further DMP1 mRNA was detected in nonmineralized mouse tissues including liver, muscle, brain, pancreas, and kidney by RT-PCR. Based on the evidence that the localization and the expression of DMP1 are not restricted to mineralized tissues, we assume that DMP1 may have functions other than the regulation of mineralization.

Amino Acid Sequence↗