Dual Functional Roles of Dentin Matrix Protein 1

IMPLICATIONS IN BIOMINERALIZATION AND GENE TRANSCRIPTION BY ACTIVATION OF INTRACELLULAR Ca2+ STORE*

Karthikeyan NarayananDagger , Amsaveni RamachandranDagger , Jianjun HaoDagger , Gen HeDagger , Kyle Won Park§, Michael Cho§, and Anne GeorgeDagger

From the Dagger  Department of Oral Biology, University of Illinois, Chicago, Illinois 60612 and the § Department of Bioengineering, University of Illinois, Chicago, Illinois 60607

Received for publication, December 12, 2002, and in revised form, February 19, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (1). Cbfa1-deficient mice have an osteopenic skeleton (2) and are known to regulate the expression of bone sialoprotein, osteopontin, dentin matrix protein 1, osteocalcin, and collagen type I (3). Recently osterix has been shown to act downstream of Cbfa1 and functions to regulate the differentiation of preosteoblasts into mature osteoblasts (4). 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. 5). 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 beta -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. 52% 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 (6-9). 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 (10). 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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfections-- The mouse pre-osteoblastic cells, MC3T3-E1, were cultured with Dulbecco's modified Eagle's medium supplemented with 10% 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 100% activity.

FITC Labeling of DMP1-- 100 µg of recombinant DMP1 was adjusted to pH 9.5 with phosphate buffer (400 µl). 100 µl 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 µg/ml.

Antibody Purification, Immunostaining, and Immunoprecipitation-- Polyclonal DMP1 antibody was affinity-purified as described earlier (11) 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 5% BSA for 4 h. Upon washing with PBS containing 1% 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 (12).

Site-directed Mutagenesis-- 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. An in vitro site-directed mutagenesis system (GeneEditor, Promega Inc.) was used to achieve mutations. Mutated sites were verified by sequencing.

Plasmid Constructs-- 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 (10).

GST Pull-down Assays-- GST-importin constructs were obtained as a kind gift from Dr. Stephen Adams, Northwestern University, Chicago. Recombinant GST-importin alpha  bound to glutathione-Sepharose beads was used in GST pull-down assays. 100 µg 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.

In Vitro Phosphorylation of DMP1-- Recombinant DMP1 was in 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.

Casein Kinase II Assay-- 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-beta -D-ribofuranosylbenzimidazole, DRB). The reaction was allowed for 30 min at 30 °C. The proteins were resolved on a 10% 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 5% trichloroacetic acid followed by filter binding assay using PE81 filters.

Induction of Mineralization-- Mineralization was induced as described earlier (10). The microenvironment for maturation of osteoblasts and mineral nodule formation was created by treating the cells (80-90% confluent) with medium supplemented with 5% fetal bovine serum, 10 mM beta -glycerophosphate, and 100 µg/ml ascorbic acid. To mimic in vivo biomineralization microenvironment, the cells were treated with cyclopiazonic acid, at a concentration of 1 µM for 2 h and then imaged for DMP1 localization.

Calcium Imaging-- 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 µM 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 beta -glycerophosphate and 100 µg/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 µM DRB, whereas calcium was inhibited by 30 µM 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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DMP1 Is Localized in the Nucleus of Undifferentiated Osteoblasts-- Based on immunohistochemical data from mature bone tissue we had expected to localize the protein in the mineralized matrix (7). 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.


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Fig. 1.   a, immunocytochemical localization of DMP1. MC3T3-E1 cells grown on coverslips were fixed with paraformaldehyde and incubated with monoclonal anti-tubulin antibody (green) and affinity-purified anti-DMP1 antibody (red) for 4 h followed by incubation with appropriate secondary antibodies. After incubation the cells were washed and mounted on a glass slide. DNA was positively stained with Hoechst dye (blue). Cells were observed under a laser confocal microscope. Bar = 10 µm. In a: upper left, tubulin antibody; upper right, DMP1 antibody; lower left, nuclear staining by Hoechst dye; and lower right, composite of first three images. b, uptake of DMP1 by osteoblasts. Recombinant DMP1 was labeled with FITC as described under the "Materials and Methods." Free uncoupled FITC was removed by dialysis. MC3T3-E1 cells were grown on coverslips; labeled DMP1 was added exogenously to the cells at a concentration of 30 µg/ml. For the control experiments, BSA was labeled in a similar manner and added exogenously at the same concentration (data not shown). The cells were fixed at different time intervals and observed under laser confocal microscope. Panel A, migration of labeled DMP1 at different time points and its accumulation in the nucleus; panel B, immunostaining with monoclonal anti-tubulin antibody; and panel C, the composite of A and B. Note the accumulation of the protein around the nuclear envelope at 15 min after the addition of DMP1, and migration of the labeled protein into the nucleus was observed within 30 min. Bar = 20 µm.

Novel Function of DMP1 as a Transcriptional Regulator in the Nucleus-- 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 (10). 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.


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Fig. 2.   A, Northern blot showing the expression of OCN and AP (alkaline phosphatase) in the MC3T3-E1 cells overexpressing sense and antisense DMP1. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. B, effect of DMP1 on the OCN promoter activity: OCN promoter driving the luciferase gene was co-transfected with an increasing concentration of DMP1 (pcDNA3.1-DMP1). Cells were lysed and assayed for luciferase. All transfections were carried out in triplicates and contained an internal control of Renilla luciferase driven by an SV40 promoter. Luciferase activity was normalized with the Renilla luciferase activity. OCN promoter activity in the control MC3T3 cell was taken as 100%. C, OCN promoter activity during osteoblast differentiation. MC3T3 cells were induced to undergo differentiation and transfected with OCN promoter driving the luciferase gene. Transfections were carried out on different days of differentiation as described in B. pcDNA3.1-DMP1 (15 µg) plasmid was co-transfected for the +DMP1 samples. Stable cell line overexpressing antisense DMP1 (10) was also induced to undergo differentiation (AS-DMP1). OCN promoter activity was analyzed as described above.

DMP1 Contains a Functional NLS Sequence at the Carboxyl End-- The regulated transport of proteins across the nuclear envelope has been recognized as a critical step in vast number of cellular processes (13, 14). 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 alpha -importin by GST-alpha -importin pull-down assay. Binding assays clearly demonstrated that mutations at the NLS3 (N3) site affected the interaction of DMP1 with alpha -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 alpha -importin leading to the import of DMP1 into the nucleus.


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Fig. 3.   Identification of putative NLS in DMP1. Analysis of the primary sequence of DMP1 led to the identification of three potential NLS sequences (NLS1, NLS2, and NLS3). To investigate the function of these domains, mutations were made on these sequences as described under "Materials and Methods." Mutated proteins were expressed recombinantly and labeled with FITC as described earlier. The labeled proteins (panel A) were added at a concentration of 30 µg/ml to the cells grown on coverslips. The cells were fixed with paraformaldehyde and counterstained with tubulin antibody (panel B). The nuclear compartment was stained with Hoechst dye (panel C). Panel D is the composite of A-C. NLS1 and NLS2 bar = 10 µm and NLS3 bar = 5 µm.

DMP1 Is Localized in the Extracellular Matrix during Biomineralization-- 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 beta -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).


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Fig. 4.   a, localization of DMP1 during mineralization. Cells were induced to undergo mineralization for 2 days with beta -glycerophosphate and ascorbic acid, and the localization of DMP1 was demonstrated by the DMP1 antibody (green), nuclear staining by Hoechst dye. Please note that mineralization stimulates nuclear export of DMP1, and it is now localized in the extracellular matrix. Bar = 20 µm. b, release of intracellular calcium stimulates nuclear export of DMP1. MC3T3-E1 cells were treated with cyclopiazonic acid (+) (1 µM) for 2 h, fixed, and immunostained with DMP1 antibody. Cells without cyclopiazonic acid served as the control (-). Panel A shows localization of DMP1, panel B shows nuclear staining with propidium iodide, and panel C is the composite of A and B. Note the export of DMP1 from the nucleus with the release of intracellular calcium. Bar = 10 µm. c, localization of phosphorylated DMP1 in MC3T3-E1 cells. Recombinant DMP1 was phosphorylated in vitro by casein kinase mixture (I and II) and used in comparison with the non-phosphorylated DMP1 for uptake studies. Phosphorylated and unphosphorylated DMP1 were FITC-labeled and added to the cells and fixed after 16 h. Panel A shows the localization of FITC-labeled DMP1 (phosphorylated (P) and unphosphorylated (NP)), panel B shows nuclear staining by propidium iodide, and panel C is the composite of A and B. Bar = 20 µm

Release of Intracellular Calcium Is Required for Export of DMP1 to the ECM-- 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 (15). Confocal imaging in the presence of cyclopiazonic acid (1 µM, "+") 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.

Calcium Dynamics and DMP1 Export during Biomineralization-- 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 beta -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 (16). 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.


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Fig. 5.   Calcium dynamics during mineralization. Cells were seeded onto coverslips at least 18 h before use. TRITC-labeled DMP1 was fed to the cells for 3 h. 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 beta -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. The intensity of Fluo3 and TRITC-DMP1 were calculated with appropriate background subtraction. A, presence of TRITC-DMP1 in the cells; B, the calcium indicator Fluo3; C, the composite of A and B. Bar = 5 µm. Three different time frames are shown (0, 60, and 150 min, respectively). Please note the movement of calcium (green) and the export of DMP1 (red) from the nucleus.


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Fig. 6.   Mineralization 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 beta -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 µm. Three different time frames were shown (A, B, and C correspond to 0, 90, and 120 min, respectively).

Export of DMP1 from the Nucleus to the Cytoplasm Is Impaired by BAPTA-AM, a Calcium Chelator-- 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+.


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Fig. 7.   Role of calcium on the export of DMP1. Cells were seeded and processed as described in Fig. 5, except that the cells were treated with BAPTA-AM (30 µM) for 30 min before taking the images. 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. The intensity of Fluo3 and TRITC-DMP1 were calculated with appropriate background subtraction. Panel A represents the presence of TRITC-DMP1 in the cells. Panel B represents the calcium indicator Fluo3. Panel C represents the composite of A and B. Bar = 5 µm. Three different time frames were shown (0, 60, and 150 min, respectively). Please note the inability of calcium to be transported into the nucleus and the retention of DMP1 within the nucleus.

Transport of DMP1 Is Regulated by Functional NES and NLS-- 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.


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Fig. 8.   A, 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 µm (NES-pEGFP, enlarged view). In B: 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-alpha 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-alpha -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.

Phosphorylation of DMP1 Is Necessary for Nucleocytoplasmic Transport-- 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·alpha -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 was in 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 Is Phosphorylated by CKII in the Nucleus Prior to Its Export into ECM-- DMP1 has several consensus sites for phosphorylation by CKII. To identify the casein kinase responsible for in vivo phosphorylation of DMP1, proteins were extracted from the nucleus and cytosol and analyzed for their phosphorylating activity in the presence of [alpha -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-beta -ribofuranosylbenzimidazole, 75 µM), 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 µM) (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). Together, the specificity of these reactions clearly demonstrates the presence of casein kinase II-like enzyme in the nuclear extracts of MC3T3-E1 cells, and their phosphorylating activity on DMP1 can be augmented by the addition of calcium. Next we addressed the question of whether blocking DMP1 phosphorylation can inhibit its export. For this the cells were treated with DRB and then monitored for calcium and DMP1 dynamics using Fluo3, a Ca2+ indicator. Confocal results clearly indicate the movement of calcium into the nucleus, at the same time DMP1 was retained in the nuclear compartment (Fig. 10). Interestingly, these results demonstrate a direct correlation between inhibition of DMP1 phosphorylation and its retention within the nuclear compartment.


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Fig. 9.   A, phosphorylation of DMP1 by the nuclear extract and the role of calcium during this process. Nuclear extracts from MC3T3-E1 were incubated with recombinant DMP1 to monitor the phosphorylation of DMP1. Lane C represents the control; lane DRB represents phosphorylation of DMP1 in the presence of CKII-specific inhibitor, DRB (75 µM). Phosphorylation was also investigated in the presence of increasing concentrations of calcium. 1, 2, and 5 mM CaCl2 solutions were used in this experiment. Phosphorylation was also studied in the presence of BAPTA. Experiments were conducted in the presence of 5 mM CaCl2 along with a 30 µM concentration of BAPTA. The proteins were resolved on a 10% SDS-PAGE and dried for autoradiography. The intensity of phosphorylation was measured using Kodak Digital Science software and presented as histograms. B, CKII activity during the induced mineralization. Mineralization was induced as described earlier, and nuclear extracts were made at different time intervals during the mineralization process. CKII activity was measured by the incorporation of labeled phosphate from [gamma -32P]ATP. The reaction was stopped by the addition of 5% trichloroacetic acid, and a filter binding assay was carried out using PE81. The x axis represents the time in hours.


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Fig. 10.   Role of CKII on DMP1 export. Cells were seeded onto the coverslips at least 18 h before use. TRITC-labeled DMP1 was fed to the cells for 3 h. Cells were washed with HBSS and loaded with Fluo3 for 45 min. Cells were loaded with DRB (75 µM) for 30 min before taking images. Cells were replenished with buffer every 3 min. Imaging was done using a CCD camera mounted on a Nikon microscope. Metamorph software was used to obtain and analyze data. The intensities of Fluo3 and TRITC-DMP1 were calculated with appropriate background subtraction. Panel A represents the presence of TRITC-DMP1 in the cells. Panel B represents the calcium indicator Fluo3. Panel C represents the composite of A and B. Bar = 20 µm. Three different time frames were shown (0, 60, and 150 min, respectively).

We further explored the role of Ca2+ on the mechanism of DMP1 phosphorylation. DMP1 has a high affinity for binding to calcium ions (17). Addition of calcium chloride to nuclear extracts increased the phosphorylating activity of CKII in a dose-dependent manner. This increase could be suppressed by the addition of BAPTA-AM (Fig. 9A). Reviewing the results obtained from calcium dynamics in the presence of BAPTA, DRB and phosphorylation studies clearly indicate the necessity for the presence of Ca2+ and phosphorylation by CKII to achieve the export of DMP1 from the nucleus to ECM during mineralization.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

For the first time, the results presented in this study shed new light on the mechanism by which nuclear localization of DMP1 might play an important role in the regulation of specific genes that control osteoblast differentiation. Experimental evidence presented here and elsewhere indicates a dual biological function for DMP1 both as a transcriptional signal during early differentiation of osteoblasts and as an initiator of mineralization during the terminal differentiation of osteoblasts (17). Different spatial and temporal profiles of DMP1 lead to different pleiotropic effects. The nuclear import of DMP1 is dependent on the NLS3 domain present at its carboxyl end. Positive interactions of GST-alpha -importin with the NLS3 domain of DMP1 further indicate the regulated, unidirectional import of DMP1 into the nucleus. This phenomenon exhibited by osteoblasts is cell type-specific, because fibroblasts and epithelial cells failed to respond in a similar manner (data not shown). However, the mechanism of DMP1 uptake in cells remains unclear.

The export of DMP1 from the nucleus during maturation of osteoblasts was found to be in response to a stimulus from calcium ions. Calcium ions have been reported as ubiquitous second messengers. The modulated release of Ca2+ from internal stores such as the endoplasmic reticulum can be transduced into an intracellular response. This signaling mechanism might convey information across individual cells and between connected cells by targeting a variety of calcium-sensitive elements that are pathway-specific. In this study Ca2+ was found to serve as a signal for DMP1 export. It has been reported that, under mineralization conditions, cells release calcium from their intracellular stores (18). For the first time, we were able to show that in mature osteoblasts there is a calcium influx into the nucleus, induced by the mineralization stimulus. This influx of Ca2+ into the nucleus is probably specific for cells involved in mineralized tissue formation, because the same effect was not observed in fibroblastic NIH3T3 cells. Two well-defined pathways have been reported for the entry of calcium into the nucleus. Malviya and co-workers (19) reported the existence of IP3 receptors on the inner nuclear membrane, and ryanodine receptors in the isolated liver nuclei (20) have also been found to be involved in calcium transport. More recently, Zadi and co-workers (21) clearly demonstrated the presence of IP3 and ryanodine receptors on the nuclear membrane of osteoblast cell line (MC3T3-E1), which were responsible for the transport of calcium into the nucleus. We speculate that the accumulated cytoplasmic Ca2+ gets transported to the nucleus either through the IP3/ryanodine receptor on the nuclear membrane or by an unknown carrier protein. However, to date there are no reports indicating the role of nuclear Ca2+dynamics with the induction of biomineralization.

Casein kinase II is a messenger-independent Ser/Thr kinase found predominantly in the nucleus of most cells (22). We speculate that phosphorylation of DMP1 by casein kinase II, present in the nuclear extracts of osteoblasts, might induce a conformational change in the protein and prevent its re-entry back into the nucleus from the cytoplasm. Confocal microscopy analysis demonstrated that phosphorylated DMP1 failed to migrate into the nucleus, whereas unphosphorylated rDMP1 became localized in the nucleus within 15 min. Thus DMP1 must be phosphorylated to be exported out into the ECM. This result corroborates well with a functional role for phosphorylated DMP1 in the ECM. Based on charge density, phosphorylated DMP1 is highly acidic and has the capacity to bind Ca2+ ions. Extracellularly, DMP1 has been hypothesized to play a regulatory role in the nucleation of hydroxyapatite within the collagenous matrix of bone and dentin (6). Thus, both Ca2+ signaling and phosphorylation of DMP1 by CKII are essential regulatory components required for the export of DMP1 during biomineralization.

The novel finding that DMP1 is localized in the nucleus and acts as a transcriptional regulator for OCN expression is appealing, because it is consistent with the observation that OCN might be one of the downstream genes known, to be regulated by DMP1. Based on our overexpression studies, we also speculate that DMP1 may directly activate transcriptional pathways leading to expression of alkaline phosphatase in osteoblasts (10). Thus our findings suggest a bifunctional role for DMP1 during biomineralization and support a model in which Ca2+ is actively supplied to the mineralized matrix. The model (Fig. 11) predicts that DMP1, synthesized by preosteoblasts, is transported into the nucleus by binding to soluble transport factors such as alpha -importin. In the nucleus DMP1 is responsible for the transcription of matrix genes involved in mineralized tissue formation. In polarized osteoblasts, the nucleocytoplasmic transport of DMP1 is mediated by calcium ions. Additionally, the phosphates present at the time of calcified tissue formation can trigger the release calcium from intracellular stores. The released calcium gets transported to the nucleus either by an unknown carrier protein or through the IP3/ryanodine receptors, present on the nuclear membrane of osteoblasts. In the nucleus, calcium binds to DMP1 and undergoes structural modifications and phosphorylation is facilitated by activating casein kinase II. The overall conformational change leads to the exposure of the NES sequence and the export of the DMP1·Ca2+ complex to the extracellular matrix where phosphorylated DMP1 with high anionic characteristics initiates the nucleation of hydroxyapatite and orchestrates calcified tissue formation. Movement of transcription factors between the nucleus and the cytoplasm has been shown to control various cellular activities (23). Such tight regulation has important functional consequences for cell metabolism and differentiation. Recently, the mechanism involved in the repression of myogenesis differentiation in the presence of histone deacetylase has been reported. Briefly, the differentiation of myoblast depends on the availability of free myocyte enhancer factor 2 (MEF2). Histone deacetylase (HDAC) represses myogenesis by forming a complex with MEF2. Calcium/calmodulin-dependent protein kinase induces the myoblast differentiation by phosphorylating HDAC, thereby disrupting the complex with MEF2, and the phosphorylated HDAC gets exported to the cytoplasm (25). Phosphorylation has also been reported to play a major role in the transport of factors into the nucleus and in export to the cytoplasm (24-28).


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Fig. 11.   A hypothetical model. The export of DMP1 from the nucleus to the extracellular matrix in a differentiating osteoblast is illustrated. In polarized osteoblasts, the nucleocytoplasmic transport of DMP1 is mediated by calcium ions. The phosphates present at the time of calcified tissue formation can trigger the intracellular calcium stores to release calcium. The released calcium gets transported to the nucleus either by an unknown carrier protein or through the IP3/ryanodine receptors on the nuclear membrane. In the nucleus, calcium binds to DMP1, which undergoes structural modification (I). Casein kinase II then phosphorylates DMP1, leading to a conformational change that exposes the NES sequence (II). This triggers the export of the DMP1·Ca2+ complex to the extracellular matrix where the phosphorylated, highly anionic DMP1 initiates the nucleation of hydroxyapatite formation.


    ACKNOWLEDGEMENTS

We thank Dr. Trisha Gura and Dr. Primal de Lanerolle for critical editing and helpful comments on the manuscript. We acknowledge Dr. Mei Lin at the confocal microscope center for her assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DE-11657 (to A. G.), DE-13836 (to A. G.), and GM60741 (to M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Oral Biology, University of Illinois, 801 S. Paulina St., Chicago, IL 60612. Tel.: 312-413-0738; Fax: 312-996-6044; E-mail: anneg@uic.edu.

Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M212700200

    ABBREVIATIONS

The abbreviations used are: DMP1, dentin matrix protein 1; OCN, osteocalcin; AP, alkaline phosphatase; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NLS, nuclear localization signal; NES, nuclear export signal; GFP, green fluorescent protein; ORF, open reading frame; GST, glutathione S-transferase; CKI/II, casein kinases I and II; DRB, 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole; HBSS, Hanks' balanced salt solution; CCD, charge-coupled device; TRITC, tetramethylrhodamine isothiocyanate; ECM, extracellular matrix; MEF2, myocyte enhancer factor 2; HDAC, histone deacetylase; IP3, inositol 1,4,5-trisphosphate; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid.

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