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• W2025592015 abstract "Dentin matrix protein 1 (DMP1) is a bone- and teeth-specific protein initially identified from mineralized dentin. Here we report that DMP1 is primarily localized in the nuclear compartment of undifferentiated osteoblasts. In the nucleus, DMP1 acts as a transcriptional component for activation of osteoblast-specific genes like osteocalcin. During the early phase of osteoblast maturation, Ca2+ surges into the nucleus from the cytoplasm, triggering the phosphorylation of DMP1 by a nuclear isoform of casein kinase II. This phosphorylated DMP1 is then exported out into the extracellular matrix, where it regulates nucleation of hydroxyapatite. Thus, DMP1 is a unique molecule that initiates osteoblast differentiation by transcription in the nucleus and orchestrates mineralized matrix formation extracellularly, at later stages of osteoblast maturation. The data presented here represent a paradigm shift in the understanding of DMP1 function. This information is crucial in understanding normal bone formation, remodeling, fracture healing, and skeletal tissue repair. Dentin matrix protein 1 (DMP1) is a bone- and teeth-specific protein initially identified from mineralized dentin. Here we report that DMP1 is primarily localized in the nuclear compartment of undifferentiated osteoblasts. In the nucleus, DMP1 acts as a transcriptional component for activation of osteoblast-specific genes like osteocalcin. During the early phase of osteoblast maturation, Ca2+ surges into the nucleus from the cytoplasm, triggering the phosphorylation of DMP1 by a nuclear isoform of casein kinase II. This phosphorylated DMP1 is then exported out into the extracellular matrix, where it regulates nucleation of hydroxyapatite. Thus, DMP1 is a unique molecule that initiates osteoblast differentiation by transcription in the nucleus and orchestrates mineralized matrix formation extracellularly, at later stages of osteoblast maturation. The data presented here represent a paradigm shift in the understanding of DMP1 function. This information is crucial in understanding normal bone formation, remodeling, fracture healing, and skeletal tissue repair. dentin matrix protein 1 osteocalcin alkaline phosphatase fluorescein isothiocyanate phosphate-buffered saline bovine serum albumin nuclear localization signal nuclear export signal green fluorescent protein open reading frame glutathione S-transferase casein kinases I and II 5,6-dichloro-1-औ-d-ribofuranosylbenzimidazole Hanks' balanced salt solution charge-coupled device tetramethylrhodamine isothiocyanate extracellular matrix myocyte enhancer factor 2 histone deacetylase inositol 1,4,5-trisphosphate 1,2-bis(o-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid Mesenchymal stem cells have the potential to differentiate into several cell types that give rise to bone, cartilage, fat, and muscles. Proliferation and differentiation of mesenchymal cells to osteoblastic lineage is regulated by an intrinsic genetically defined program, which is well-controlled by various transcription factors, cytokines, morphogens, and secreted growth factors. There are two known transcription factors, namely Cbfa1 and osterix, that regulate osteoblast differentiation and skeletal formation during embryonic development (1Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3630) Google Scholar). Cbfa1-deficient mice have an osteopenic skeleton (2Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar) and are known to regulate the expression of bone sialoprotein, osteopontin, dentin matrix protein 1, osteocalcin, and collagen type I (3Ducy P. Starbuck M. Priemel M. Shen J. Pinero G. Geoffroy V. Amling M. Karsenty G. Genes Dev. 1999; 13: 1025-1036Crossref PubMed Scopus (707) Google Scholar). Recently osterix has been shown to act downstream of Cbfa1 and functions to regulate the differentiation of preosteoblasts into mature osteoblasts (4Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2784) Google Scholar). Differentiated osteoblasts synthesize a number of calcium-binding proteins like bone sialoprotein, osteopontin, and osteocalcin and secrete a complex extracellular matrix that has the capacity to nucleate hydroxyapatite crystal formation when adequate amounts of calcium and phosphate are supplied (reviewed in Ref. 5Karsenty G. Endocrinology. 2001; 142: 2731-2733Crossref PubMed Scopus (169) Google Scholar). Understanding the regulatory mechanisms that control differentiation of osteoblast phenotype during proliferation, maturation, and mineralization is necessary for understanding various skeletal disorders. MC3T3-E1 cells are a well-established preosteoblast cell line derived from mouse calvaria and maintain much of the tightly linked controls between proliferation and differentiation. These cells, when treated with औ-glycerophosphate and ascorbic acid, differentiate into mature osteoblast phenotype and produce a calcifiable matrix that recapitulates in vivo conditions. Mineralized nodule formation takes place at least 18–21 days after induction of mineralization. During the early stage (3–5 days) of induction the preosteoblastic cells undergo proliferation, and at later stage (8–12 days) the cells differentiate to mature osteoblast capable of synthesis and assembly of mineralized matrix with increased alkaline phosphatase activity and production of type I collagen. Dentin matrix protein 1 (DMP1)1 is a non-collagenous extracellular matrix protein identified from mineralized matrix of dentin and bone. DMP1 is highly anionic and rich in aspartic acid, glutamic acid, and serine residues. 527 of these serines can be potentially phosphorylated by casein kinase II. Based on its high negative charge, it has been postulated to play an important role in mineralized tissue formation, more specifically, by initiation of nucleation and modulation of mineral phase morphology (6George A. Sabsay B. Simonian P.A.L. Veis A. J. Biol. Chem. 1993; 268: 12624-12630Abstract Full Text PDF PubMed Google Scholar, 7George A. Gui J. Jenkins N.A. Gilbert D.J. Copeland N.G. Veis A. J. Histochem. Cytochem. 1994; 42: 1527-1531Crossref PubMed Scopus (80) Google Scholar, 8D'Souza R.N. Cavender A. Sunavala S.G. Alvarez J. Ohshima T. Kulkarni A.B. MacDougall M. J. Bone Miner. Res. 1997; 12: 2040-2049Crossref PubMed Scopus (298) Google Scholar, 9MacDougall M. Gu T.T. Luan X. Simmons D. Chen J. J. Bone Miner. Res. 1998; 13: 422-431Crossref PubMed Scopus (135) Google Scholar). Recent experiments demonstrated that overexpression of DMP1 in embryonic mesenchymal cells resulted in characteristic morphological changes accompanied by transcriptional up-regulation of OCN and AP. Blocking the translation of DMP1 by antisense expression inhibited the expression of OCN and AP genes. Furthermore, stable cell lines overexpressing antisense DMP1 failed to initiate mineralized nodule formation in cell culture systems (10Narayanan K. Srinivas R. Ramachandran A. Hao J. Quinn B. George A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4516-4521Crossref PubMed Scopus (215) Google Scholar). These experiments laid the foundation for speculation of a dual functional role for DMP1 during osteoblast differentiation. In this report we demonstrate that DMP1 resides in the nucleus, cytoplasm, and extracellular matrix of osteoblasts depending on their differentiation state and exhibits pleiotropic effects. Combined with experimental evidence, we suggest a bifunctional role for DMP1 during osteoblast differentiation and maturation. The mouse pre-osteoblastic cells, MC3T3-E1, were cultured with Dulbecco's modified Eagle's medium supplemented with 107 heat-inactivated fetal bovine serum (Celgro). Transient transfections with reporter plasmids were performed with Superfect (Qiagen) as per the manufacturer's protocol. Reporter transfections were carried out in triplicates and repeated at least three times to obtain a mean value. All the transfections contained an internal control vector pRLSV40, which contains a Renilla luciferase gene driven by SV40 promoter. Promoter activity at the control point was taken as 1007 activity. 100 ॖg of recombinant DMP1 was adjusted to pH 9.5 with phosphate buffer (400 ॖl). 100 ॖl of FITC solution (0.1 mg/ml) was added and incubated at 4 °C for 2 h. The reaction mixture was passed through a G-25 column to remove excessive FITC. Furthermore, the labeled protein was dialyzed against PBS buffer at 4 °C for 18 h. Bovine serum albumin was labeled in a similar manner and was used as a control. For the uptake studies labeled proteins were added exogenously to the cells at a concentration of 30 ॖg/ml. Polyclonal DMP1 antibody was affinity-purified as described earlier (11Srinivasan R. Chen B. Gorski J.P. George A. Connect. Tissue Res. 1999; 40: 251-258Crossref PubMed Scopus (51) Google Scholar) using rDMP1 coupled to CNBr-activated Sepharose column. For immunostaining, cells grown on coverslips were fixed with paraformaldehyde. Fixed cells were incubated with DMP1 antibody in the presence of 57 BSA for 4 h. Upon washing with PBS containing 17 Triton X-100, the cells were incubated with appropriate secondary antibody (fluorescent labeled) for 2 h. The coverslips were then mounted and observed under laser confocal microscope (Zeiss, LSM 510). Monoclonal tubulin antibody was purchased from Sigma; nucleus staining dye, propidium iodide, and Hoechst dye were purchased from Molecular Probes. Immunoprecipitation was carried out as described earlier (12Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 421-470Google Scholar). Site-directed mutagenesis was carried out with the following primers to mutate the respective potential NLS sites. The lowercase letters represent the modified bases. NLS1: 724 bp, 5′-TCAAGCaGGAcATCCTTCAGAAGGTCCgGGGTCTCT-3′, 760 bp; NLS2: 1294 bp, 5′-TCTCAGGACAGTAGCgGATCCAcAGAAGAGAGC-3′, 1327 bp; NLS3: 1384 bp, 5′-GCTGACAATgGGAcACTAATAGcTGATGCT-3′, 1414 bp. Anin vitro site-directed mutagenesis system (GeneEditor,Promega Inc.) was used to achieve mutations. Mutated sites were verified by sequencing. Double-stranded oligonucleotides synthesized for the NLS1 (724–760 bp), NLS2 (1294–1327 bp), NLS3 (1384–1414 bp), and NES (13–48 bp) were ligated to the carboxyl-terminal end of the GFP protein at SmaI site with ORF to GFP. pEGFP (Clontech, Palo Alto, CA) was used in this study. An osteocalcin promoter driving the luciferase gene was a gift from Dr. Gerard Karsenty at Baylor College of Medicine, Houston, TX. DMP1 sense and antisense plasmids were constructed as described earlier (10Narayanan K. Srinivas R. Ramachandran A. Hao J. Quinn B. George A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4516-4521Crossref PubMed Scopus (215) Google Scholar). GST-importin constructs were obtained as a kind gift from Dr. Stephen Adams, Northwestern University, Chicago. Recombinant GST-importin α bound to glutathione-Sepharose beads was used in GST pull-down assays. 100 ॖg of the extracted protein was added to the column and washed with 0.1 m NaCl in PBS buffer. GST beads were boiled in SDS-sample buffer, and the bound proteins were detected by Western blotting using DMP1 antibody. Recombinant DMP1 wasin vitro phosphorylated by casein kinases I and II mixture as described by the manufacturer (Upstate Biotechnology Inc., Lake Placid, NY). Phosphorylation was confirmed by monoclonal phosphoserine antibody. An CKII assay was carried out using the casein kinase II assay kit (Upstate Biotechnology). Briefly, 20 ng of recombinant DMP1 was incubated with the nuclear extracts of MC3T3-E1 cells in the presence and absence of CKII-specific peptide (RRRDDDSDDD) or CKII-specific inhibitor (5,6-dichloro-1-औ-d-ribofuranosylbenzimidazole, DRB). The reaction was allowed for 30 min at 30 °C. The proteins were resolved on a 107 SDS-PAGE and dried for autoradiography. The intensity of phosphorylation was measured using Kodak Digital Science software. In other cases, after phosphorylation the proteins were precipitated with 57 trichloroacetic acid followed by filter binding assay using PE81 filters. Mineralization was induced as described earlier (10Narayanan K. Srinivas R. Ramachandran A. Hao J. Quinn B. George A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4516-4521Crossref PubMed Scopus (215) Google Scholar). The microenvironment for maturation of osteoblasts and mineral nodule formation was created by treating the cells (80–907 confluent) with medium supplemented with 57 fetal bovine serum, 10 mm औ-glycerophosphate, and 100 ॖg/ml ascorbic acid. To mimic in vivo biomineralization microenvironment, the cells were treated with cyclopiazonic acid, at a concentration of 1 ॖm for 2 h and then imaged for DMP1 localization. Cells were seeded onto glass coverslips at least 24 h prior to use. Attached cells were washed four times with Hanks' balanced salt solution (HBSS) without calcium chloride. The cells were then loaded with Fluo3 (Molecular Probes, Eugene, OR, 10 ॖm final concentration) for 45 min in the dark. They were then washed four times with HBSS, mounted onto a slide with elevated edges allowing to be flushed by the buffer (HBSS containing 10 mm औ-glycerophosphate and 100 ॖg/ml ascorbic acid) every 3 min. Images were taken using a cooled CCD camera (CoolSnapFX, Roper Scientific) mounted on a Nikon microscope. Metamorph software (Universal Imaging, PA) was used to obtain and analyze data. The intensity of Fluo3 and TRITC-DMP1 were calculated with appropriate background subtraction. For monitoring both the DMP1 and Ca2+, the cells were fed with TRITC-DMP1 for 3 h before loading with Fluo3. Inhibition of phosphorylation was carried out by 75 ॖm DRB, whereas calcium was inhibited by 30 ॖm BAPTA. BAPTA or DRB were added to cells for 30 min prior to imaging by confocal microscopy. The Fluo3 intensity was used to monitor the modulation in calcium level. Based on immunohistochemical data from mature bone tissue we had expected to localize the protein in the mineralized matrix (7George A. Gui J. Jenkins N.A. Gilbert D.J. Copeland N.G. Veis A. J. Histochem. Cytochem. 1994; 42: 1527-1531Crossref PubMed Scopus (80) Google Scholar). Contrary to expectations, immunostaining of MC3T3-E1 cells with an affinity-purified monospecific DMP1 antibody indicated DMP1 to be predominantly nuclear-localized. However, we also observed a small amount of fluorescence outside the nucleus (Fig.1a). In a second set of experiments, FITC-labeled recombinant DMP1, when added to the culture medium of MC3T3-E1 cells, migrated into the nucleus in a time-dependent manner. Labeled DMP1 was found evenly distributed in the cytoplasm within 10 min. After 15 min, DMP1 was found to be concentrated around the outer nuclear membrane, and optimum localization in the nucleus occurred within 30 min as demonstrated by confocal microscopy (Fig. 1B). Therefore, in proliferating preosteoblasts, DMP1 was predominantly nuclear. However, the mechanism by which DMP1 is taken up by osteoblasts is currently unknown. To address the role of DMP1 in the nucleus, we have previously shown that overexpression of DMP1 in MC3T3-E1 cells and C3H10T1/2 (embryonic mesenchymal) cells resulted in characteristic morphological changes accompanied by transcriptional up-regulation of osteocalcin and alkaline phosphatase (10Narayanan K. Srinivas R. Ramachandran A. Hao J. Quinn B. George A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4516-4521Crossref PubMed Scopus (215) Google Scholar). However, the antisense-mediated repression of DMP1 protein led to an inhibition of osteocalcin and alkaline phosphatase expression level (Fig.2A). Based on this experimental evidence we investigated if DMP1 could be operating directly as a transcriptional regulator for matrix genes involved in mineralization. To this end, we specifically investigated the effect of DMP1 on OCN (osteocalcin) promoter activity. Results in Fig.2B demonstrate that there is no significant increase in OCN promoter activity with increasing concentration of DMP1 plasmid. This result is not surprising because it is well established that OCN expression increases severalfold when preosteoblasts undergo differentiation. Overexpression of DMP1 during osteoblast differentiation did not have any significant effect on OCN promoter activity (Fig. 2C), however, MC3T3 cells overexpressing antisense DMP1 failed to show an increase in OCN promoter activity during differentiation (Fig. 2C). These data clearly demonstrates that DMP1 in conjunction with other osteoblast-specific transcription factors regulate the expression of osteocalcin gene. We speculate that DMP1 in the nucleus of preosteoblasts may initiate osteoblastic differentiation. The regulated transport of proteins across the nuclear envelope has been recognized as a critical step in vast number of cellular processes (13Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (386) Google Scholar, 14Misteli T. Science. 2001; 291: 843-847Crossref PubMed Scopus (527) Google Scholar). For large proteins such as DMP1 (66 kDa), import into the nucleus would likely require the presence of nuclear localization signals (NLS) and its associated transport machinery. Inspection of the primary sequence of DMP1 led to the identification of three potential NLS sequences: NLS1 residues 242–252 (amino acid sequence SSRKSFRRSRVS), NLS2 residues 432- 442 (amino acid sequence SQDSSRSKEES), and NLS3 residues 472–481 (amino acid sequence ADNRKLIVDA). Mutations were introduced into the NLS1, 2, and 3 sequences as described under “Materials and Methods” to identify the functional NLS domain. Mutations in NLS1 and NLS2 did not hinder the transport of DMP1 into the nucleus (Fig.3). However, mutations in NLS3 resulted in intense cytoplasmic accumulation of the labeled protein (Fig. 3). Results from this mutation study clearly indicate that the NLS3 domain is functional and is required for nuclear import of DMP1. To further characterize the transport pathway, we investigated the interaction of DMP1 with α-importin by GST-α-importin pull-down assay. Binding assays clearly demonstrated that mutations at the NLS3 (N3) site affected the interaction of DMP1 with α-importin. However, mutations on NLS2 (N2) and NLS1 (N1) did not have any effect on importin binding (see Fig. 8B, panel IV). Thus, these results indicate the specific interaction of NLS3 domain with α-importin leading to the import of DMP1 into the nucleus.Figure 8A, characterization of the NLS and NES sequences in DMP1. The oligonucleotides corresponding to the putative NLS and NES domains were cloned into the 5′-end of the GFP plasmid with an ORF. MC3T3-E1 cells were transiently transfected with the GFP hybrids, and localization was monitored using confocal microscopy. Note the localization of NLS-pEGFP hybrid in the nucleus and the localization of the NES-pEGFP hybrid at the periphery of the cellular membrane and in the extracellular matrix. Bars = 20 (NLS-pEGFP), 10 (pEGFP and NES-pEGFP), and 3 ॖm (NES-pEGFP, enlarged view). InB: panel I, Western blot analysis of DMP1 expressed in different cellular compartments of MC3T3-E1 cells probed with DMP1 antibody; panel II, DMP1 proteins from different compartments were immunoprecipitated using DMP1 antibody and probed for phosphorylation with a monoclonal phosphoserine antibody (note the absence of phosphorylated DMP1 in the nucleus); panel III, protein extracts from different cellular compartments of MC3T3-E1 cells were loaded onto GST-α importin column to identify its interaction with DMP1. The binding of DMP1 was revealed by cross-blotting the proteins that bound to the beads using DMP1 antibody. For panels I, II, and III: T, C,N, and E represent total, cytosol, nuclear, and extracellular proteins. Panel IV, recombinant proteins were loaded onto a GST-α-importin column, washed, and eluted with SDS-PAGE sample buffer. Eluted proteins were analyzed for the presence of DMP1 using DMP1 antibody. For panel IV: C = control, N1 = NLS1-mutated DMP1, N2 = NLS2-mutated DMP1, and N3 = NLS3-mutated DMP1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunohistochemical studies have demonstrated the presence of DMP1 in the mineralized extracellular matrix of bone and dentin. This suggests that DMP1 must migrate from the nucleus rather than be maintained in a nuclear pool. To examine the export mechanism, MC3T3-E1 cells were treated for 2 days (early maturation stage) with ascorbic acid and औ-glycerophosphate, an organic phosphate, to stimulate differentiation, because Dulbecco's modified Eagle's medium does not have sufficient phosphate ion product to support normal mineral formation. Immunostaining of these mineralizing cultures with a DMP1 antibody demonstrated a striking relocation of DMP1: instead of being in the nucleus, DMP1 was now located in the cytoplasm and the plasma membrane (Fig.4a). These data corroborated well with published reports regarding the localization of DMP1 extracellularly, in the bone matrix. Also, in vivo DMP1 can be localized in the nucleus of preosteoblasts and in the mineralized matrix of a mature osteoblast (data not shown). One of the main events during osteoblast differentiation and maturation is the release of calcium from intracellular stores. We hypothesized that Ca2+ might serve as a signal for DMP1 export. Therefore, we investigated if the export of DMP1 from the nucleus during mineralized matrix formation might be in response to a stimulus from the calcium microenvironment. To directly analyze this question, we treated MC3T3-E1 cells with cyclopiazonic acid, a stimulant for the release of intracellular Ca2+ stores without altering the IP3 levels (15Demaurex N. Lew D.P. Krause K.H. J. Biol. Chem. 1992; 267: 2318-2324Abstract Full Text PDF PubMed Google Scholar). Confocal imaging in the presence of cyclopiazonic acid (1 ॖm, “+”) showed that the release of calcium from the endoplasmic reticulum, in fact, triggered the export of DMP1 from the nucleus to the extracellular matrix (Fig. 4b). This result is in good agreement with the in vivo localization of DMP1 in the extracellular matrix of bone and dentin. To investigate the functional role of Ca2+ in the export process of DMP1, MC3T3-E1 cells in mineralizing cultures were loaded first with TRITC-labeled DMP1 followed by the fluorescent calcium-sensitive dye Fluo3. Exposing the cells to a simulated mineralization medium containing औ-glycerophosphate (the concentration of inorganic phosphate used promoted biomineralization and not ectopic mineral deposition) initiated osteoblast differentiation. Strikingly, this process evoked a biphasic Ca2+ response. Live cell microscopic analysis revealed an initial rapid release of calcium from intracellular stores followed by a massive influx of this pool of Ca2+ into the nucleus. However, after 3 h, the elevated nuclear calcium levels declined and returned to the basal level. Interestingly, this influx of calcium into the nucleus triggered the export of DMP1 from the nucleus to the extracellular matrix (Fig. 5). This translocation of Ca2+ into the nucleus, under mineralization conditions, is probably specific for cells involved in synthesizing a mineralized matrix, because control fibroblastic NIH3T3 cells failed to respond in a similar manner (Fig.6). During bone formation, extracellular phosphate levels are raised significantly and induce changes in gene expression (16Beck Jr., G.R. Zerler B. Moran E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8352-8357Crossref PubMed Scopus (424) Google Scholar). We speculate that this elevated extracellular phosphate levels triggers the release of calcium from intracellular stores of osteoblasts resulting in the export of DMP1 from the nucleus of differentiating osteoblasts.Figure 6Mineralization condition effect on NIH3T3 cells. NIH3T3 cells were seeded onto the coverslips at least 18 h before use. Cells were washed with HBSS (calcium-free) and loaded with Fluo3 for 45 min. Cells were mounted onto a slide with elevated edges giving access for the buffer to flow through. Cells were replenished with buffer (HBSS containing औ-glycerophosphate and ascorbic acid) every 3 min. Cells were monitored at regular intervals of 10 min. Imaging was done using a CCD camera mounted on a Nikon microscope. Metamorph software was used to obtain and analyze the data.Bar = 30 ॖm. Three different time frames were shown (A, B, and C correspond to 0, 90, and 120 min, respectively).View Large Image Figure ViewerDownload Hi-res image Download (PPT) An important question is whether this Ca2+ influx into the nucleus plays a specific role in the export of DMP1 from the nucleus. Mineralization stimulus was found to evoke a steady increase in the nuclear calcium levels for over 90 min, after which it subsequently declined to the initial level. This influx and efflux of calcium from the nucleus coincided with the export of DMP1 from the nucleus. However, addition of BAPTA-AM, a well-known chelator for calcium, led to the accumulation of the TRITC-DMP1 in the nucleus. Furthermore, the influx of calcium observed under normal mineralizing conditions did not take place in the presence of BAPTA-AM (Fig. 7). These results suggest that, in differentiating osteoblasts, DMP1 export into the extracellular matrix is directly or indirectly related to the local release of Ca2+. To investigate the signal sequence responsible for controlling the export of DMP1 into the extracellular matrix in coordination with calcium release, the sequence of DMP1 was examined for a nuclear export signal (NES). Sequence analysis indicated the presence of a classic leucine-rich hydrophobic export signal present at the amino-terminal end (5–16 amino acids) of the polypeptide and is conserved in all species identified thus far. The functionality of both the NES and NLS domains in DMP1 was further confirmed by ligating the NES domain (LLTFLWGLSCAL) and the NLS3 sequence, individually to the carboxyl terminus of the GFP construct with an ORF. Transient transfections and confocal images demonstrated that NLS3-GFP hybrid protein accumulated in the nuclear compartment. On the other hand, the NES-GFP hybrid was found to accumulate at the cellular boundary and in the ECM (Fig.8A). These results confirmed that both NLS3 and NES peptide sequences are functional and are necessary for the rapid import and export of DMP1. DMP1 is a phosphoprotein with a high negative charge. Phosphate groups confer a very high capacity to DMP1 for binding calcium ions, which is important for its potential function in mineralization. If fully phosphorylated, DMP1 would bear a net charge of −175 per molecule of 473 residues. To examine whether DMP1 is differentially phosphorylated in vivo, we immunoprecipitated DMP1 from the nucleus, cytosol, and extracellular matrix of MC3T3-E1 cells and cross-blotted it with an anti-phosphoserine antibody. It was observed, that DMP1 from the cytosol and extracellular matrix were phosphorylated, however, the nuclear DMP1 was unphosphorylated (Fig. 8B, panel II). To investigate the role of phosphorylation in the nucleocytoplasmic transport of DMP1, we investigated the binding of DMP1 to importin, a soluble transport factor. GST pull-down assay performed using the GST·α-importin complex showed that the DMP1 from the nuclear compartment was able to bind to importin, while no detectable binding was observed for DMP1 isolated from the cytosol and extracellular compartments (Fig. 8B, panel III). Results from these two studies, namely phosphorylation and importin binding assay, indicate that, upon phosphorylation, DMP1 might undergo a conformational change enabling it to expose the NES domain leading to its export into the ECM. In a different approach, recombinant DMP1 wasin vitro phosphorylated by CKII enzyme and added exogenously to the cells. Confocal microscopy demonstrated that there was no uptake of phosphorylated DMP1 by MC3T3-E1 cells. On the contrary, unphosphorylated recombinant DMP1 localized in the nucleus within 15 min (Fig. 4c). Thus phosphorylation of DMP1 is necessary for nucleocytoplasmic transport. DMP1 has several consensus sites for phosphorylation by CKII. To identify the casein kinase responsible for in vivophosphorylation of DMP1, proteins were extracted from the nucleus and cytosol and analyzed for their phosphorylating activity in the presence of [α-32P]ATP as the phosphoryl donor. Initial results demonstrate that the kinase in the nuclear extract had a greater phosphorylating potential when compared with the components from the cytosol fraction (data not shown). The specificity of this phosphorylating activity was confirmed by DRB (5,6-dichloro-1-औ-ribofuranosylbenzimidazole, 75 ॖm), a well-characterized specific inhibitor for CKII and by competition with excess CKII-specific peptide. Interestingly, addition of exogenous calcium in the form of calcium chloride (1–5 mm) to the reaction mixture combined with the nuclear extracts increased the phosphorylating activity of CKII. Furthermore, this increase can be suppressed by the addition of BAPTA-AM (30 ॖm) (Fig.9A). Moreover, CKII activity during the mineralization process was shown to increase at least 2- to 3-fold within the 24 h after mineralization induction (Fig.9B). Tog" @default.
• W2025592015 created "2016-06-24" @default.
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• W2025592015 date "2003-05-01" @default.
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• W2025592015 title "Dual Functional Roles of Dentin Matrix Protein 1" @default.
• W2025592015 cites W1500109515 @default.
• W2025592015 cites W1589002191 @default.
• W2025592015 cites W1665071864 @default.
• W2025592015 cites W1900565974 @default.
• W2025592015 cites W1951130496 @default.
• W2025592015 cites W1968329569 @default.
• W2025592015 cites W1971163872 @default.
• W2025592015 cites W1975210552 @default.
• W2025592015 cites W1994511343 @default.
• W2025592015 cites W1994596418 @default.
• W2025592015 cites W1995911293 @default.
• W2025592015 cites W2002254327 @default.
• W2025592015 cites W2018084034 @default.
• W2025592015 cites W2025046506 @default.
• W2025592015 cites W2044406741 @default.
• W2025592015 cites W2055683188 @default.
• W2025592015 cites W2057031134 @default.
• W2025592015 cites W2058278101 @default.
• W2025592015 cites W2061189653 @default.
• W2025592015 cites W2076614498 @default.
• W2025592015 cites W2084776852 @default.
• W2025592015 cites W2116998164 @default.
• W2025592015 cites W2122367477 @default.
• W2025592015 cites W2135300227 @default.
• W2025592015 cites W2170802720 @default.
• W2025592015 cites W2170890561 @default.
• W2025592015 doi "https://doi.org/10.1074/jbc.m212700200" @default.
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