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J Biol Chem, Vol. 273, Issue 45, 29935-29941, November 6, 1998
From the Department of Basic Sciences, University of Texas-Houston
Dental Branch, Houston, Texas 77030
In osteoblastic ROS 17/2.8 cells,
1,25-(OH)2-vitamin D3 stimulates
transcription of the extracellular matrix phosphoprotein osteopontin
(OPN). We now show post-translational regulation of OPN production by
1,25-(OH)2D3. Prior to transcriptional
up-regulation of OPN, 1,25-(OH)2D3 induces a
shift in OPN isoelectric point (pI) from 4.6 to 5.1. Loading equal
amounts of OPN recovered from ROS 17/2.8 cells exposed to
1,25-(OH)2D3 or carrier for 3 h reveals that the pI shift represents reduced phosphorylation. Trypsin cleavage
patterns of OPN produced after 1,25-(OH)2D3
treatment indicates phosphorylation changes in the resulting peptides.
Using structural analogs to 1,25-(OH)2D3, we
found that analog AT (25-(OH)-16-ene-23-yne-D3), which
triggers Ca2+ influx but does not bind to the vitamin D
receptor, mimicked the OPN pI shift, whereas analog BT
(1,25-(OH)2-22-ene-24-cyclopropyl-D3), which
binds to the vitamin D receptor without triggering Ca2+
influx, did not. Likewise, inclusion of the Ca2+ channel
blocker nifedipine blocks the charge conversion of OPN. Isolation of
OPN from rat femurs and tibiae demonstrates the existence of two OPN
charge forms in vivo. We conclude that
1,25-(OH)2D3 regulates OPN not only at the
transcriptional level, but also modulates OPN phosphorylation state.
The latter involves a short term (<3 h) treatment and is associated
with membrane-initiated Ca2+ influx.
Osteopontin (OPN)1 is a
non-collagenous, glycosylated phosphoprotein originally found in bone
matrix (1, 2), but now known to be expressed in many tissues including
kidney, hypertrophic chondrocytes, placenta, T-lymphocytes,
macrophages, secretory epithelia and ganglia of the inner ear, and
smooth muscle of the vascular system (3, 4). OPN is also found in
biological fluids such as milk (5), urine (6), and plasma (7), and it
displays elevated expression in many transformed cells (8). OPN is
highly acidic with approximately 25% of the amino acid composition
aspartate and glutamate and significant numbers of phosphoserine and
phosphothreonine (1, 2). The amino acid sequence contains a conserved
Gly-Arg-Gly-Asp-Ser (GRGDS) sequence (2, 3). As a result, OPN binds
effectively to the Hormone regulation plays an important role in OPN production. Our
laboratory has examined the effect of the seco-steroid hormone 1,25-dihydroxyvitamin D3
(1,25-(OH)2D3) on OPN expression and secretion.
After uptake of 1,25-(OH)2D3 into a cell, the
hormone binds to the vitamin D receptor (VDR), translocates to the
nucleus, dimerizes preferentially with the retinoid X receptor, then
binds to the vitamin D response element (VDRE) located in the promoter region of 1,25-(OH)2D3-responsive genes (19).
In the case of OPN, this results in increased transcription as seen by
higher OPN mRNA steady state levels at 24-48 h (20) and,
eventually, higher secreted protein levels (21). The OPN gene in rat is regulated by the additive action of two VDREs (3), which respond to
both 1,25-(OH)2D3 and to bioactive analogs
of 1,25-(OH)2D3 that bind to nuclear
receptors (20).
1,25-(OH)2D3 regulation also occurs through
rapid plasma membrane-initiated responses, which have been well studied
in osteoblasts. We previously reported the
1,25-(OH)2D3 regulation of L-type
voltage-sensitive calcium channels (VSCC) in ROS 17/2.8 osteosarcoma
cells (22, 23). Using patch-clamp techniques, nanomolar concentrations of 1,25-(OH)2D3 were shown to increase open
times of VSCCs and shift the threshold of activation toward the resting
potential of the plasma membrane (22). Ca2+ influx assays
show this increase in VSCC open time leads to elevated Ca2+
influx into the cell (23). 1,25-(OH)2D3
activates other osteoblast signaling pathways that are independent of
transcription such as a rapid increase in phospholipase C activity
(24), activation of protein kinase C (25), and regulation of whole cell
chloride currents (26).
In this publication, we report the effect of
1,25-(OH)2D3 on osteoblast-like ROS 17/2.8
cells at a time period between the classic genomic response and the
rapid membrane-associated responses. Examination of OPN secreted by ROS
17/2.8 cells after a 3-h exposure to
1,25-(OH)2D3 reveals the production of a form
of OPN with a higher isoelectric point (pI) than the original.
Structural analogs of 1,25-(OH)2D3 and
Ca2+ channel blockers were used to determine if a nuclear
receptor-mediated response or a membrane-initiated response was
responsible for the OPN pI shift. Our results provide evidence for
short term regulation by 1,25-(OH)2D3 of
post-translational modification of OPN, a phenomenon that may modify
its functional properties in bone matrix.
Cell Culture--
ROS 17/2.8 cells were grown in Dulbecco's
modified Eagle's medium/F12 medium (Atlanta Biologicals, Norcross, GA)
containing 10% fetal bovine serum (Life Technologies, Inc.). ROS
17/2.8 osteoblast-like osteosarcoma cells were chosen because of their
responsiveness to 1,25-(OH)2D3 and because of
their ability to synthesize and secrete high levels of OPN mRNA and
protein. Cells were seeded at low density (30,000 cells/ml) and allowed
to grow until they reached 60-70% confluence. For protein isolation,
cells were transferred to serum-free medium overnight, then treated
with 2.5 nM 1,25-(OH)2D3 (Biomol,
Plymouth Meeting, PA), 2.5 nM analog AT
(25-(OH)-16-ene-23-yne-D3), 25 nM analog BT
(1,25-(OH)2-22-ene-24-cyclopropyl-D3) (gifts of Dr. Anthony Norman), or carrier (ethanol), and cultured for the indicated times prior to harvest. Concentrations of the analogs used
were based on previous studies (23). 50 nM nifedipine
(Calbiochem, La Jolla, CA) was used to block L-type Ca2+ channels.
For radiolabeling, cells were transferred to serum-free Dulbecco's
modified Eagle's medium/F12 medium without
Na3PO4, or methionine, respectively for 18 h. Phosphate in the form of [32P]orthophosphate or
[32P]Na3PO4 or methionine in the
form of [35S]methionine (NEN Life Science Products) was
added to the medium 30 min prior to the addition of reagents (167 µCi/20 ml medium). Experiments were performed in serum-free medium to
prevent binding of 1,25-(OH)2D3 to the vitamin
D-binding protein. Previous studies have shown that cells remain viable
under these conditions for 48-72 h.
OPN Purification from ROS 17/2.8 Cell Medium by Barium Citrate
Precipitation--
Medium was collected from ROS 17/2.8 cells that had
been exposed to 1,25-(OH)2D3 with or without
Ca2+ channel blockers, analog AT, analog BT, or carrier
(ethanol) alone. OPN was isolated using the barium citrate procedure
(5). Briefly, 3.8% sodium citrate and 15% BaCl2 were
added to the medium at 1/10 of the total volume. After shaking for 10 min at 4 °C, the mixture was centrifuged for 10 min at 3800 rpm. The
supernatant was discarded, and the pellet was then washed for 10 min
with 15% BaCl2 at 4 °C. After centrifugation for 10 min
at 3800 rpm, the supernatant was again discarded, and the pellet was
washed with H2O for 10 min at 4 °C. After another round
of centrifugation, the supernatant was removed, and OPN was eluted from
the pellet by dissolution in 0.2 M sodium citrate, pH 6.8. Excess salts were removed with a desalting column (Pierce). Further
purification was accomplished by passing the sample through an affinity
column (described below).
Immunoaffinity Chromatography--
IgG was immobilized with
cyanogen bromide-activated Sepharose 4B (Sigma) and coupled as
described (27). Briefly, 1 g of cyanogen bromide-activated
Sepharose 4B with 5 mg of
Medium from ROS 17/2.8 cells was dialyzed through three buffer changes
in either 10 mM Na3PO4, pH 7.5, or
0.25 mM ammonium bicarbonate (if performed following barium
citrate precipitation). Samples were passed over the immunoaffinity
column and OPN was eluted from the column with washes of Tween 20, PBS,
0.5 M NaCl, 0.025% SDS in PBS, and PBS.
OPN Purification from Rat Long Bone--
Purification of OPN
from rat long bone was performed as described (1). Briefly, extraction
of proteins from rat femurs and tibiae was accomplished by
demineralization using 4 M guanidinium HCl in 50 mM Tris-HCl, pH 7.2, for 24 h at 4 °C followed by
decalcification in 4 M guanidinium HCl, 0.5 M
EDTA in 50 mM Tris-HCl, pH 7.2, for 48 h at 4 °C.
All extractions contained the following protease and alkaline
phosphatase inhibitors: 10 mM EDTA, 0.1 M
6-aminohexanoic acid, 5 mM benzamidine hydrochloride, 1 mM sodium iodoacetate, 10 mM
phenylmethylsulfonyl fluoride, 5 mg/liter pepstatin, 1 mg/liter soybean
trypsin inhibitor, and 5 mM levamisole. The extracts were pooled, centrifuged at 30,000 × g for 15 min, and
concentrated by ultrafiltration.
Concentrated samples were subjected to gel filtration on a 2.5 × 30-cm Sephacryl S-200 column and eluted with 4 M
guanidinium HCl in 50 mM Tris-HCl, pH 7.2. The high
molecular weight fraction was concentrated to one-sixth volume, passed
over a 2 × 30-cm column of Bio-Gel P30, and eluted with 6 M urea in 50 mM Tris-HCl, pH 7.2. The elution
was then run on a 2.5 × 20-cm DEAE-Sephacel column. The column
was eluted with a linear gradient formed from 750 ml each of 6 M urea in 50 mM Tris-HCl, pH 7.2, and 6 M urea in 50 mM Tris-HCl, pH 7.2 containing 0.7 M NaCl. The fractions eluting at 0.28 M NaCl
(designated peak D4a) and 0.36 M NaCl (designated peak D4b)
were rechromatographed on the 2.5 × 20-cm DEAE-Sephacel column,
but with a shallow NaCl gradient (0.14-0.4 M).
Precipitation of Total Protein from ROS 17/2.8
Cells--
Extraction buffer (1% Chaps, 4 M guanidinium
HCl, PIC I and II (28), and 20 mM Tris-HCl, pH 7.0) was
added to the plates for 10 min. The extraction buffer fraction was
added to the medium fraction. Proteins were precipitated using 10%
trichloroacetic acid, 3% phosphotungstic acid for 2 h at 4 °C.
The precipitate was centrifuged, and the pellets were solubilized in
2% cholate and sonicated. The soluble fraction was collected and
dialyzed against 10 mM Tris, 0.1% cholate.
Trypsin Cleavage of OPN--
OPN isolated from ROS 17/2.8 cells
exposed to 1,25-(OH)2D3 or carrier (ethanol)
for times indicated in figures was lyophilized. The lyophilized protein
was resuspended at 37 °C for 3 h in 2.5 mg/ml trypsin in 0.125 M Tris-HCl, pH 6.8. After incubation, the peptide fragments
were lyophilized, placed in SDS sample buffer, and separated by
SDS-PAGE as described below.
SDS-Polyacrylamide Gel Electrophoresis--
SDS-PAGE was
performed using a modification of procedures previously described (29).
Acrylamide and bisacrylamide concentrations were altered for a 12.5%
resolving gel. Gels were stained with 20 mg of Stains All (Bio-Rad) in
10 ml of formamide, 50 ml of isopropyl alcohol, 1.0 ml of 3 M Tris-HCl, pH 8.8, 139 ml of H2O overnight in
a dark room.
Two-dimensional Gel Electrophoresis and Isoelectric Focusing Gel
Electrophoresis--
Proteins were suspended in a minimum volume of
lysis buffer containing 9.5 M urea, 2% Triton X-100, 2%
ampholines (1.6% pH 5-7, 0.4% pH 3-10 or 1.0% pH 2-4, 1.0% pH
3-10), and 5%
Isoelectric gel electrophoresis was performed using IsoGel agarose IEF
plates (FMC Bioproducts, Rockland, ME). Proteins were lyophilized and
resuspended in deionized H2O. Proteins were focused on pH
range 3-10 gels following the manufacturer's instructions. Gels were
then dried and exposed to autoradiographic film or transferred to
nitrocellulose using a press blot procedure as described by the manufacturer.
Immunoblot Analysis--
SDS-polyacrylamide gels were
transferred to nitrocellulose at 15 V in transfer buffer (0.025 M Tris, 0.2 M glycine, 20% methanol) overnight. Blots were dried and then blocked in PBS containing 3% BSA
and 0.15% Tween 20 at room temperature for 2 h. Blots were then
incubated in PBS containing 3% BSA and 0.15% Tween 20 with a 1:10,000
dilution of primary antibody (goat Effect of 1,25-(OH)2D3 on OPN pI--
We
examined the effect of 1,25-(OH)2D3 on OPN
production during the first 3 h of treatment, prior to nuclear
receptor-mediated transcriptional up-regulation. Proteins from the cell
fraction were isolated as described under "Experimental Procedures"
and visualized by two-dimensional gel electrophoresis. OPN from ROS 17/2.8 cells treated with vehicle was found to focus in two discrete spots at pI 4.6 and 5.1 (Fig.
1A). The identity of the spots
focusing at these locations as OPN was confirmed by Western blotting
(data not shown) using goat
For further verification of the pI shift, OPN was isolated from ROS
17/2.8 cell medium by immunoprecipitation. ROS 17/2.8 cells were
cultured as described above, with the exception that the time of
exposure to carrier or 1,25-(OH)2D3 was
increased to 18 h. This was done to permit the secretion of
measurable levels of OPN protein prior to immunoprecipitation.
Visualization of equal counts/min of immunoprecipitated OPN protein was
accomplished by two-dimensional gel electrophoresis and
autoradiography. Immunoprecipitated OPN from ROS 17/2.8 cells exposed
to vehicle alone focused at pI 4.6 (Fig. 1C). OPN
immunoprecipitated from ROS 17/2.8 cells treated with 2.5 nM 1,25-(OH)2D3 focused at pI 5.1 and, to a much lesser extent, pI 4.6 (Fig. 1D). Medium from
ROS 17/2.8 cells treated with vehicle or
1,25-(OH)2D3 and passaged over a pre-immune IgG
column produced no spots when analyzed by two-dimensional gel
electrophoresis (data not shown). These results show that OPN secreted
into the medium undergoes a charge shift similar to that of OPN in the
cell fraction (Fig. 1, A and B).
Effect of 1,25-(OH)2D3 on OPN
Phosphorylation Levels--
Because the
1,25-(OH)2D3-induced pI change was over a short
range (0.5 units), we hypothesized that this shift was the result of a
reduced phosphorylation of OPN. An altered level of phosphorylation was
a likely possibility because of the number of potential OPN phosphorylation sites. Radiolabeled medium was collected and proteins were precipitated using sodium citrate and BaCl2 as
described under "Experimental Procedures." OPN was isolated from
the group of precipitated proteins by immunoaffinity chromatography as
described above. Equal amounts of OPN (50 µg) from vehicle-treated
and 1,25-(OH)2D3-treated cells were visualized
after separation by 12.5% SDS-PAGE (Fig. 2A). OPN from cells treated
with 1,25-(OH)2D3 (lane
2) contained lower levels of radioactive phosphate
incorporation compared with OPN from control cells (lane
1). Measurement by densitometry and scintillation counting
showed a 70% decrease in phosphorylation of OPN from
1,25-(OH)2D3-treated cells compared with that
from control cells. Fig. 2B shows Western blot analysis of
OPN (50 µg) from vehicle-treated and
1,25-(OH)2D3-treated cells. We consistently observed increased immunostaining of the antibody to OPN from 1,25-(OH)2D3-treated cells, which may reflect
increased accessibility to antigenic sites on the core protein.
To determine whether 1,25-(OH)2D3 treatment,
and consequent reduced phosphorylation, could alter sensitivity to
proteolytic cleavage, OPN from vehicle-treated and
1,25-(OH)2D3-treated cultures was subjected to
trypsin digestion (Fig. 3). ROS 17/2.8
cells were treated as in Fig. 2. OPN was cleaved by trypsin for 3 h at 37 °C. Equal counts/min of OPN were loaded onto each lane of a
15.0% SDS-polyacrylamide gel. Limit peptides large enough to remain on
the gel were found at 28, 25.9, 22.7, 20, and 17.4 kDa for both OPN
from control cells (lane 3) and cells exposed to 1,25-(OH)2D3 (lane 4).
Phosphorylation differences were found on all peptides visualized.
Trypsin digestion of OPN from
1,25-(OH)2D3-treated cultures produced
phosphorylated peptides at 22.7 and 17.4 kDa that are not found during
digestion of OPN from vehicle-treated cultures. In addition, the OPN
peptide from 1,25-(OH)2D3-treated cultures
migrating at 20 kDa produced a stronger band than the peptide from
control cultures (compare lane 4 to
lane 3). The largest peptide migrating at 28 kDa
appears to be resistant to further proteolysis in OPN from
vehicle-treated cultures (lane 3).
Effect of Structural Analogs to
1,25-(OH)2D3 on OPN pI--
To assess the
relative involvement of the VDR and Ca2+ influx in these
observed changes, we first utilized two structural analogs to
1,25-(OH)2D3 that vary in their ability to
stimulate pathways of 1,25-(OH)2D3 action on
ROS 17/2.8 cells (Fig. 4A).
ROS 17/2.8 cells were labeled with [32P]orthophosphate
and treated with 25 nM analog BT or 2.5 nM
analog AT in serum-free medium without Na3PO4.
OPN was isolated using the barium citrate precipitation followed by
immunoprecipitation as explained under "Experimental Procedures."
Equal counts/min of OPN were visualized using isoelectric gel
electrophoresis (Fig. 4B). In lane 1,
OPN from cells exposed to analog BT focused at pI 4.6. However, OPN
from cells exposed to analog AT duplicated the pI shift that occurred
with the addition of 1,25-(OH)2D3
(lane 2). Western blotting confirmed that the
focused spots were OPN (data not shown).
Nifedipine Inhibits the
1,25-(OH)2D3-induced Phosphorylation Change in
OPN--
Since analog AT was found to induce the OPN charge shift, we
hypothesized that Ca2+ influx through VSCCs was involved in
the pathway leading to a shift in the OPN charge state. To examine this
possibility, we inhibited the VSCCs with the L-type Ca2+
channel blocker nifedipine. ROS 17/2.8 cells were labeled with [32P]orthophosphate and treated with 2.5 nM
1,25-(OH)2D3 or carrier with or without 50 nM nifedipine in serum-free medium without Na3PO4. OPN was isolated by barium citrate
precipitation followed by immunoprecipitation as explained under
"Experimental Procedures." 20 µg per lane of OPN were visualized
by SDS-PAGE (Fig. 5). Consistent with
observations previously seen in Fig. 2, OPN from cells treated with
1,25-(OH)2D3 contained lower levels of
incorporated radioactive phosphate compared with OPN from control cells
(compare lanes 1 and 2). In
lane 3, cells treated with
1,25-(OH)2D3 and nifedipine showed levels of
radioactive phosphate incorporation comparable to control cells.
pI of OPN Isolated from Rat Long Bone--
To determine the
existence of similar multiple charge forms in vivo, OPN was
isolated from rat femurs and tibiae. Analysis of OPN from peaks D4a and
D4b of the DEAE-Sephacel column was accomplished by SDS-PAGE and by
isoelectric gel electrophoresis (Fig. 6).
OPN isolated from peak D4a migrated at 73.4 kDa (Fig. 6A,
lane D4a; Fig. 6B, lane
D4a). The streaking found in Fig. 6B
(lane D4a) is likely the result of
microheterogeneity associated with the aggregation of OPN or partial
degradation during the bone extraction procedure. A minor band of OPN
was found at 28.8 kDa, possibly the product of thrombin cleavage. OPN
from peak D4b migrated at 91.3 kDa (Fig. 6A, lane
D4b; Fig. 6B, lane D4b). After isoelectric focusing, the gel was transferred to nitrocellulose via a press blot procedure. Western analysis and detection by alkaline
phosphatase (Fig. 6C) was performed as described under "Experimental Procedures." OPN1 from peak D4b (lane
OPN1) was found to focus at pI 4.6, analogous to OPN from
control cells. Conversely, OPN2 from peak D4a (lane
OPN2) was found to focus at pI 5.1, similar to OPN from ROS
17/2.8 cells treated with 1,25-(OH)2D3 or
analog AT. Streaking can be found in both lanes OPN1 and OPN2 of Fig.
6C, indicating the presence of multiple charge forms in addition to the two major bands or the presence of degradation products
in bone extracts.
Increasing evidence supports a role for
1,25-(OH)2D3 in cellular responses that either
do not include the VDR or are completely non-genomic. One of the most
studied responses is Ca2+ signaling initiated by
1,25-(OH)2D3. Patch-clamp and Ca2+
influx assays from our laboratory have shown that physiological concentrations of 1,25-(OH)2D3 increase VSCC
open time in ROS 17/2.8 cells, thereby increasing Ca2+
influx into the cells (22, 23). 1,25-(OH)2D3
can also stimulate Ca2+ influx through a similar mechanism
in skeletal muscle cells (30). Although the changes in phosphorylation
level of OPN from ROS 17/2.8 cells treated with
1,25-(OH)2D3 appear to be post-translational, involvement of the VDR was possible. Even though up-regulation of OPN
at the transcriptional level by 1,25-(OH)2D3
through the VDR is unlikely in this time frame, previous data have
shown up-regulation of other transcripts by
1,25-(OH)2D3 in less than 3 h (25,
31-33). Analog BT has been shown to bind to the VDR as well or better than 1,25-(OH)2D3 and can up-regulate OPN
mRNA steady state levels in ROS 17/2.8 cells comparable to
1,25-(OH)2D3 (20, 23). Analog AT increases
Ca2+ influx through VSCCs in ROS 17/2.8 cells similarly to
1,25-(OH)2D3 (22, 23). Analog BT, however, does
not increase VSCC activity, and analog AT does not bind to the VDR or
increase OPN mRNA steady state levels. Other analogs are also able
to stimulate separate pathways, with structural confirmation and side
chain additions or subtractions affecting the ability of the analog to
bind to VDR or stimulate Ca2+ influx (33-36).
To the growing list of non-genomic or membrane-initiated responses to
1,25-(OH)2D3 (37-45), we now report that
addition of 1,25-(OH)2D3 to ROS 17/2.8
osteoblast-like cells results in the production of a less
phosphorylated form of the secreted extracellular matrix protein OPN.
Ca2+ influx through VSCCs is necessary for production of
the less phosphorylated form of OPN as inhibition of VSCCs by the
Ca2+ channel blocker nifedipine prevents its production.
Although changes in phosphorylation state stimulated by
1,25-(OH)2D3 have been found in cytosolic and
plasma membrane fractions (46, 47), this is the first report of a
change in post-translational processing initiated by
1,25-(OH)2D3.
OPN has been found in multiple charge forms, previously. Nemir et
al. (48) reported the existence of a phosphorylated form and a
non-phosphorylated form of OPN produced by normal rat kidney cells.
Chang and Prince (49) showed that 1,25-(OH)2D3
could stimulate the synthesis of a non-phosphorylated form in mouse JB6
epidermal cells, which normally produce low amounts of a phosphorylated form. The stimulation, however, appears to be genomic, as protein levels increase after a 24-h exposure to
1,25-(OH)2D3.
Data from the in vitro experiments indicate the existence of
multiple charge forms of OPN secreted by osteoblast-like cells, but
does not answer the question of the existence of these charge forms in
bone extracellular matrix. Looking for equivalent charge forms in
vivo, we examined OPN purified from rat tibiae and femurs employing a well-developed isolation procedure (1). On the final
purification column (DEAE-Sephacel), OPN can be isolated from two peaks
(designated D4a and D4b). Since OPN from these peaks elutes at two
different concentrations of NaCl, we hypothesized that these peaks
might correspond to OPN with two distinct isoelectric points. The
functional significance of these charge forms must be determined.
With the addition of this new phosphorylation shift in OPN from ROS
17/2.8 cells stimulated by 1,25-(OH)2D3 and in
OPN from rat long bone, we have the following model representing
1,25-(OH)2D3 action on osteoblasts (Fig.
7). Osteoblasts, when not exposed to
1,25-(OH)2D3, produce a higher phosphorylated
OPN, designated OPN1. This form is found in rat long bone from peak
D4b. 1,25-(OH)2D3 produces, after 3 h, a
shift to a lower phosphorylation form of OPN, designated OPN2. This
form is found in rat long bone from peak D4a. Since the shift in OPN
phosphorylation state can be mimicked by analog AT or inhibited by
Ca2+ channel blockers in ROS 17/2.8 cells, this pathway
apparently does not involve the VDR, and appears to be a result of a
Ca2+-influx-dependent signaling response.
Previous work in ROS 17/2.8 cells has provided evidence for a 44-kDa
form and a 55-kDa form of OPN (50). The 44-kDa form was found to be
highly phosphorylated, while the 55-kDa form was found to be less
phosphorylated. In ROS 17/2.8 cells, our data indicate the existence of
two different charge states. The 4.6 pI form apparently is equivalent
to this 44-kDa form, and the 5.1 pI form of OPN is equivalent to the
55-kDa form. Contrary to their conclusions, however, is our finding
that both forms can be isolated and do exist in rat long bone. OPN from
rat long bone migrates differently than these 44- and 55-kDa forms. We find that the more acidic form, the more phosphorylated form, migrates
slower on SDS-PAGE (Fig. 6B), compared with faster migration of the highly phosphorylated, more acidic 44-kDa OPN. These results are
similar to the findings of Nemir et al. (48), where the phosphorylated OPN migrated slower than the non-phosphorylated form. In
fact, the discrepancy in migration on SDS-PAGE between OPN from peaks
D4a and D4b might be attributed to decreased binding of SDS to OPN from
peak D4b. This decreased binding to SDS could result in slower
migration on SDS-PAGE.
Currently, our laboratory is investigating the phosphorylation sites
found on the two OPN isoforms isolated from rat long bone (D4a and
D4b). Analysis of phosphorylation from bovine OPN revealed the
existence of 28 sites of phosphorylation (51). Phosphorylation motifs
matched the recognition sequence utilized by CKI and CKII coinciding
with the evidence that a CKII-like activity phosphorylates OPN in the
Golgi (52). In rat long bone, OPN from peak D4a has been analyzed for
post-translational modifications (53). Phosphorylations were found on
equivalent sites as those in bovine OPN, but some of the
phosphorylations were partial (not found in every analysis). Partial
phosphorylation of residues might play a role in the unusual streaking
found when OPN is detected by Western blot procedures (Fig. 6).
Although staining reveals the presence of distinct bands (Fig.
6A), Western blot analysis of OPN from rat long bone reveals
not only the distinct bands, but also a broad range of forms (Fig.
6B). This is especially evident in the isoelectric gel (Fig.
6C), where OPN from D4a and D4b focused at major bands of pI
5.1 and 4.6, respectively, but was also detected in a broad band at
higher isoelectric points. Notice that the D4a fraction does not appear
to contain any of the lower pI fraction of OPN. Another interesting
result with antibody detection is that the antibody to OPN utilized in
this study apparently binds to the lower phosphorylated form of OPN more efficiently than to the higher phosphorylated form. This can be
seen in OPN isolated from ROS 17/2.8 cells (Fig. 2) and in OPN from rat
long bone (Fig. 6). A possible explanation is that this antibody was
developed using OPN purified from peak D4a, and, therefore, might be
more specific for the lower phosphorylated form.
OPN contains a GRGDS sequence that allows the protein bind to cells
containing the In conclusion, we have isolated two charge forms of the phosphoprotein
OPN from ROS 17/2.8 cells exposed to vehicle or
1,25-(OH)2D3 that have uniquely different
isoelectric points. This pI shift appears to be the result of reduced
phosphorylation in cells treated with
1,25-(OH)2D3. The loss in phosphorylation does
not occur through a VDR-mediated genomic event, but is associated with
Ca2+ influx. Finally, we report that the two charge
isoforms of differing pI can be isolated from rat long bone.
We thank Gail Wright for her contribution to
the early stages of this work, Jan Brunn for his column chromatography
work, and Dr. Anthony Norman for his donation of analogs to
1,25-(OH)2D3. We also thank Dr. Catherine
Bègue-Kirn and Dr. Daniel Carson for their assistance in editing
this manuscript.
*
This work was supported by National Institutes of Health
Grant AR39273 (to W. T. B. and M. C. F.-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.
The abbreviations used are:
OPN, osteopontin; 1, 25-(OH)2D3, 1,25-dihydroxyvitamin
D3; VDR, vitamin D receptor; VDRE, vitamin D response
element; VSCC, voltage-sensitive calcium channel; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; BSA, bovine serum albumin; CK, casein
kinase; AT, 25-(OH)-16-ene-23-yne-D3; BT, 1,25-(OH)2-22-ene-24-cyclopropyl-D3; Bicine, N,N-bis(2-hydroxyethyl)glycine; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
Modulation of Osteopontin Post-translational State by
1,25-(OH)2-Vitamin D3
DEPENDENCE ON Ca2+ INFLUX*
,
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
V
3 integrin (9), as
well as to the V5 and
V1 integrins (10). OPN also contains a
thrombin cleavage site located near the middle of the molecule. Both
peptides produced by thrombin cleavage are capable of supporting
integrin-mediated cell attachment (11). Potential roles for OPN
function through the V3 integrin were proposed (12). OPN can promote attachment of various cell types and can
initiate signal transduction through integrin-associated kinases. Other
proposed roles for OPN include chemotaxis (13, 14), inhibition of
nitric oxide synthase expression (15), activation of
pp60c-src (16), hydroxyapatite binding (17), and
Ca2+ binding (18).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-rat OPN IgG (purified using ImmunoPure
(A/G) IgG purification kit as per manufacturer's instructions
(Pierce)) in 0.1 M Bicine-HCl, pH 8.5, 0.5 M
NaCl was agitated continuously for 2 h at room temperature (coupling buffer). Remaining coupling sites were blocked by incubation in 1 M Tris-HCl, pH 8.0, with constant agitation for 2 h at room temperature. The gel was then washed extensively with
coupling buffer and then with 0.1 M citric acid to remove
nonspecifically bound protein.
-mercaptoethanol. Isoelectric focusing was performed
in tube gels overnight at 200 V or for 3.5 h at 750 V. Gels were
removed from the tubes and soaked for 20 min in sample buffer (0.06 M Tris-HCl, pH 6.8, 2% SDS, 5% -mercaptoethanol, and
10% glycerol), then overlaid onto 8.75% or 12.5% polyacrylamide
gels. The system was electrophoresed according to previously described
procedures for SDS-PAGE (29). Two-dimensional gels were stained with
Coomassie G, then dried and exposed to autoradiographic film.
-rat OPN) at room temperature for
1 h. Blots were then washed five times for 10 min each in PBS
containing 1.5% BSA and 0.075% Tween 20. Next, blots were incubated
in PBS containing 3% BSA and 0.15% Tween 20 with a 1:100,000 dilution
of peroxidase-conjugated donkey -goat IgG secondary antibody
(Jackson Immunoresearch, West Grove, PA) or a 1:50,000 dilution of
alkaline phosphatase-conjugated swine -goat IgG secondary antibody
(Boehringer Mannheim). Blots were again washed 5 times for 10 min each
in PBS containing 1.5% BSA and 0.075% Tween 20. For alkaline
phosphatase detection, blots were incubated in carbonate buffer with
detection substrate (0.35 mM nitro blue tetrazolium, 0.35 mM 5-bromo-4-choloro-3-indolyl phosphate, 0.1 M
NaH2CO3, 1.0 mM MgCl2,
pH 9.8) until appearance of coloration. Peroxidase was detected using a
chemiluminescent procedure. Blots were incubated in 50%
Luminol/enhancer solution, 50% stable peroxide solution (Pierce) for 5 min and exposed to autoradiographic film for 1 min.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
-rat OPN. The ratio of protein found at pI 5.1 compared with pI 4.6 was 0.26:1 assessed by densitometry. In
Fig. 1B, OPN from ROS 17/2.8 cells exposed to 2.5 nM 1,25-(OH)2D3 for 3 h
focuses almost completely at pI 5.1, with only a small fraction (12:1
ratio) at pI 4.6. Western blotting confirmed the focused spots as OPN
(data not shown). These experiments were repeated with cells labeled
with [35S]methionine to visualize total protein from the
cell fraction. Most spots were unchanged in location, but the spot
corresponding to OPN shifted to a more basic pI after 3 h
1,25-(OH)2D3 treatment (data not shown). The
identity of the spot which disappears altogether (small
arrow in Fig. 1, A and B) is presently
unknown.
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Fig. 1.
Two-dimensional analysis of OPN from vehicle
and 1,25-(OH)2D3-treated cells.
A, total protein from ROS 17/2.8 cells labeled with
[32P]Na3PO4 and treated with
vehicle (ethanol) for 3 h wasprecipitated as described under "Experimental Procedures."
Visualization of the precipitated proteins was accomplished using
two-dimensional gel electrophoresis. Molecular weight is indicated on
the left; isoelectric point is indicated on the
bottom. Large arrows indicate
two-dimensional focusing of OPN at pI 4.6 and 5.1 in the first
dimension and 66 kDa in the second dimension. Small
arrow indicates the presence of unknown phosphorylated
protein. pI was determined by comparison to standards and by
measurement of first dimension gel slices at set intervals.
B, total protein from ROS 17/2.8 cells labeled with
[32P]Na3PO4 and treated with 2.5 nM 1,25-(OH)2D3 for 3 h was
precipitated and visualized as in A. Large
arrows indicate two-dimensional focusing of OPN at pI 4.6 and pI 5.1, both at 66 kDa in the second dimension. Small
arrow indicates absence of phosphorylated protein found in
A. C, OPN was immunoprecipitated from medium
isolated from ROS 17/2.8 cells labeled with
[32P]Na3PO4 and treated with
vehicle (ethanol) for 18 h. Immunoprecipitation was performed as
described under "Experimental Procedures." Visualization of equal
counts/min of immunoprecipated OPN was performed as in A.
Arrow indicates two-dimensional focusing of
immunoprecipitated OPN at pI 4.6 and 66 kDa. D, OPN was
immunoprecipitated from medium isolated from ROS 17/2.8 cells labeled
with [32P]Na3PO4 and treated with
2.5 nM 1,25-(OH)2D3 for 18 h.
Visualization of equal counts/min of immunoprecipitated OPN was
performed as in A. Arrows indicate
two-dimensional focusing of immunoprecipitated OPN at pI 5.1 and 4.6 in
the first dimension and 66 kDa in the second dimension.
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Fig. 2.
Change in OPN pI associated with reduced
phosphorylation. A, OPN was isolated from ROS 17/2.8
cells labeled with [32P]orthophosphate and treated with
vehicle (ethanol) or 2.5 nM
1,25-(OH)2D3 for 3 h using the barium
citrate precipitation described under "Experimental Procedures."
Analysis was performed on 12.5% SDS-PAGE. Molecular weight standards
are indicated on the left. Lane 1, 50 µg of OPN from ROS 17/2.8 cells exposed to vehicle alone.
Lane 2, 50 µg of OPN from ROS 17/2.8 cells
exposed to 2.5 nM 1,25-(OH)2D3.
B, Western blot analysis of OPN isolated by the barium
citrate precipitation from ROS 17/.28 cells treated with vehicle or 2.5 nM 1,25-(OH)2D3 for 3 h.
Lane 1, 50 µg OPN from ROS 17/2.8 cells exposed
to vehicle. Lane 2, 50 µg of OPN from ROS
17/2.8 cells exposed to 2.5 nM
1,25-(OH)2D3.
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Fig. 3.
Susceptibility to trypsin digestion.
Equal counts/min of 32P were loaded on to 15.0% SDS-PAGE
for each lane. OPN was tryptically digested as described under
"Experimental Procedures." OPN was isolated from ROS 17/2.8 cells
using the barium citrate precipitation. Lane 1,
OPN from ROS 17/2.8 cells exposed to vehicle (ethanol) for 3 h.
Lane 2, OPN from ROS 17/2.8 cells exposed to 2.5 nM 1,25-(OH)2D3 for 3 h.
Lane 3, OPN from ROS 17/2.8 cells exposed to vehicle for
3 h and tryptically digested for 3 h at 37 °C.
Lane 4, OPN from ROS 17/2.8 cells exposed to 2.5 nM 1,25-(OH)2D3 for 3 h and
tryptically digested for 3 h at 37 °C.
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Fig. 4.
Effect of structural analogs of
1,25-(OH)2D3 on OPN pI shift.
A, structure of 1,25-(OH)2D3, analog
AT, and analog BT. B, OPN was analyzed on 3-10 isoelectric
focusing gels as described under "Experimental Procedures."
Lane 1, OPN precipitated by the barium citrate
procedure from ROS 17/2.8 cells exposed to 25 nM analog BT
for 3 h. pI was determined as in Fig. 1A.
Arrow indicates isoelectric focusing at pI 4.6. Lane 2, OPN precipitated by the barium citrate
procedure from ROS 17/2.8 cells exposed to 2.5 nM analog AT
for 3 h. Arrow indicates isoelectric focusing at pI
5.1.
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Fig. 5.
Effect of the Ca2+ channel
blocker nifedipine on the OPN phosphate incorporation. OPN was
isolated from ROS 17/2.8 cell medium and visualized by SDS-PAGE as
described under "Experimental Procedures." Cells were labeled with
[32P]orthophosphate and treated with vehicle (ethanol) or
2.5 nM 1,25-(OH)2D3 with or without
50 nM nifedipine. Molecular weight standards are indicated
on the left. Lane 1, 20 µg of OPN
from ROS 17/2.8 cells exposed to vehicle alone. Lane
2, 20 µg of OPN from ROS 17/2.8 cells exposed to 2.5 nM 1,25-(OH)2D3. Lane
3, 20 µg of OPN from ROS 17/2.8 cells exposed to 2.5 nM 1,25-(OH)2D3 and 50 nM nifedipine.
View larger version (39K):
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Fig. 6.
SDS-PAGE and isoelectric gel electrophoresis
of OPN isolated from rat long bone. OPN isolated from rat femurs
and tibiae as described under "Experimental Procedures" was
analyzed by SDS-PAGE and by 3-10 isoelectric focusing gel.
A, protein components of peaks D4a and D4b of the DEAE-Sephacel
column were visualized on SDS-PAGE via Stains All staining.
Lane STD, molecular weight standards.
Lane D4a, protein of peak D4a. Lane
D4b, protein of peak D4b. Asterisks indicate
migration of OPN. B, Western blot analysis of SDS-PAGE using
chemiluminescent detection. Lane D4a, OPN
purified from peak D4a. Lane D4b, OPN purified
from peak D4b. Asterisks indicate migration of OPN.
C, Western blot analysis by alkaline phosphatase detection
of 3-10 isoelectric focusing gel was performed on the product of peaks
D4a and D4b of the DEAE-Sephacel column. Lane
OPN1, OPN purified from peak D4b. pI was determined as in
Fig. 1A. Arrow indicates isoelectric focusing of
OPN at pI 4.6. Lane OPN2, OPN purified from peak
D4a. Arrow indicates pI of 5.1.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (11K):
[in a new window]
Fig. 7.
Model of 1,25-(OH)2D3
action on osteoblasts. Osteoblasts (ob) secrete OPN1
into newly synthesized osteoid unless signaled by
1,25-(OH)2D3 to deposit the lower
phosphorylated form, OPN2. OPN1 is designated as the 4.6 pI charge form
isolated from ROS 17/2.8 cells or the product of peak D4b during
purification of OPN from rat long bones. OPN2 is designated as the 5.1 pI charge form or the product of peak D4a during purification of OPN
from rat long bones.
V3 integrin. As previously
mentioned, OPN was found in both phosphorylated and non-phosphorylated
forms in normal rat kidney cells (48). Phosphorylated OPN showed cell surface association, apparently GRGDS-dependent, while the
non-phosphorylated form was not found on the cell surface. Instead, the
non-phosphorylated OPN was associated with fibronectin (via
co-precipitation). Another study of OPN phosphorylation demonstrated
that partially dephosphorylating OPN with tartrate-resistant acid
phosphatase created a form that could no longer bind to osteoclasts
(54). It may be that the OPN phosphorylation loss after
1,25-(OH)2D3 treatment results in decreased
binding to cells containing the V3 integrin.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Biological Sciences, 117 Wolf Hall,
University of Delaware, Newark, DE 19716.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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