Dual Functional Roles of Dentin Matrix Protein 1
IMPLICATIONS IN BIOMINERALIZATION AND GENE TRANSCRIPTION BY
ACTIVATION OF INTRACELLULAR Ca2+ STORE*
Karthikeyan
Narayanan
,
Amsaveni
Ramachandran
,
Jianjun
Hao
,
Gen
He
,
Kyle Won
Park§,
Michael
Cho§, and
Anne
George
¶
From the
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
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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.
 |
INTRODUCTION |
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
-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.
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MATERIALS AND METHODS |
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
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-
-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
-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
-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.
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RESULTS |
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.
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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.
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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
-importin by GST-
-importin pull-down assay. Binding
assays clearly demonstrated that mutations at the NLS3 (N3) site
affected the interaction of DMP1 with
-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
-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.
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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
-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 -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
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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
-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
-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 -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).
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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.
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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- 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- -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.
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|
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·
-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 [
-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-
-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 [ -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 |
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-
-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
-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-
-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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