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Expression and ultrastructural immunolocalization of a major 66 kDa phosphoprotein synthesized by chicken osteoblasts during mineralization in vitro.

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THE ANATOMICAL RECORD 228:93-103 (1990)
Expression and Ultrastructurat Immunolocalization
of a Major 66 kDa Phosphoprotein Synthesized
by Chicken Osteoblasts During Mineralization
In Vitro
L.C. GERSTENFELD, Y. GOTOH, M.D. McKEE, A. NANCI, W.J. LANDIS, AND
M.J. GLIMCHER
Department of Orthopedic Surgery, Laboratory for the Study of Skeletal Disorders and
Rehabilitation, Harvard Medical School, The Children’s Hospital, Boston, Massachusetts
(L.C.G., Y.G., M.D.M., W.J.L., M.J.G.); Departments of Anatomy and Stomatology,
University of Montreal, Montreal, QC Canada (A.N.)
ABSTRACT
Embryonic chicken osteoblasts cultured over a 30 day period were
used as a model system for studying the expression of bone phosphoproteins during
cellular differentiation and the possible role of these proteins in extracellular
matrix mineralization. Accumulation of total phosphoprotein in the cultures, as
determined by 0-phosphoserine (Ser-P) and 0-phosphothreonine (Thr-P) amino
acid analysis, revealed a > 10-fold increase over the 30 day period. Total phosphoprotein synthesis, a s assessed by (32P)-,(3H)-Ser-P, and (I4C)-Thr-P protein
labeling, showed the highest levels concurrent with initial mineral deposition
within the matrix. The major phosphoprotein present in chicken bones and synthesized by the cultured osteoblasts had a molecular weight of -66 kDa. This 66
kDa bone phosphoprotein (66 kDa BPP) was purified to homogeneity and was used
for antibody production. Application of this antibody in Western blot analysis
revealed that 66 kDa BPP was present only in protein extracts of mineralizing
cultured osteoblasts and was absent in cultures of non-mineralizing chondrocytes,
myoblasts, and tendon fibroblasts. The 66 kDa BPP in vitro accumulated continuously in the extracellular matrix in a manner that paralleled both phosphoprotein synthesis and total phospho-amino acid production. A comparison of the
results obtained in vitro to those from developing embryonic tibiae in vivo demonstrated a similar qualitative and temporal expression of phosphoprotein and a
continual accumulation of 66 kDa BPP in the matrix with advancing mineralization and developmental age. Ultrastructural immunocytochemistry using the 66
kDa BPP antibody and the protein A-gold technique revealed specific immunolabeling over electron-dense regions of mineralization in the cultures that appeared
identical to the distribution of labeling observed in vivo (McKee et al.: Connect.
Tissue Res., 21:21-29, 1989; Anat. Rec., 228:77-92, 1990). These results demonstrate that this major 66 kDa BPP was expressed concurrently with other differentiated osteoblast functions and suggests that it may play a role in the initiation
or regulation of mineralization.
Central to the processes of bone mineralization is the
prerequisite synthesis and assembly of a n extracellular
matrix which can facilitate mineral nucleation and
also serve as the superstructure into which mineral can
be deposited and accumulated (Glimcher, 1976). Extracellular matrix synthesis, assembly, and mineralization, which largely determine the unique structure and
physiological role of bone in skeletogenesis and ion homeostasis, are ultimately regulated temporally and
spatially by the osteoblasts of this tissue through cellmatrix interactions and secretion and interplay of noncollagenous matrix components with collagen.
One class of non-collagenous extracellular matrix
proteins found in all vertebrate mineralized tissues are
0 1990 WILEY-LISS, INC
the phosphoproteins. Their ubiquitous nature and
other considerations have led to the hypothesis that
this class of protein may play a crucial role in initiating
or controlling the spatial distribution of mineral in the
Received October 3, 1989; accepted December 19, 1989.
Address reprint requests to Dr. Louis C. Gerstenfeld, Department of
Orthopedic Research G11, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115.
Dr. M.D. McKee’s present address is Department of Stomatology,
Facuity of Dental Medicine, University of Montreal, Montreal, Quebec, H3C 357, Canada.
94
L.C. GERSTENFELD ET AL.
extracellular matrix of dentin and bone (Veis, 1978;
Veis and Sabsay, 1983; Glimcher, 1976, 1989). For
chicken bone, numerous phosphorylated polypeptides,
ranging in molecular weight from 6 kDa to 150 kDa
and containing both 0-phosphoserine (Ser-P) and 0phosphothreonine (Thr-P) residues, have been extracted and purified to homogeneity (Spector and Glimcher, 1972; Cohen-Solal et al., 1978; Lee and Glimcher,
1981; Uchiyama et al., 1986). The chicken bone phosphoproteins have been shown to be synthesized and
secreted by osteoblasts in bone tissue in vivo and by
cultured chicken bone organ cultures and cultured osteoblasts in vitro (Glimcher et al., 1982; Gotoh et al.,
1983, 1989; Landis et al., 1984; Gerstenfeld e t al.,
1989).
Recently, i t has been demonstrated that many of
the phosphoprotein species previously isolated from
chicken bone are the result of proteolysis of a major
phosphoprotein species having a n apparent molecular
weight of -66 kDa (66 kDa BPP; Gotoh et al., 1990).
The DEAE sephacel elution characteristics, the presence of sialic acid, the amino acid composition, and the
presence of a n Arg-Gly-Asp amino acid sequence
within 66 kDa BPP (Gotoh et al., 1990) would suggest
that this protein is similar to a recently identified class
of phosphorylated sialoproteins found in bovine, rat,
and human bone (Fisher et al., 1983, 1987; Franzen
and Heinegard, 1985; Oldberg et al., 1986; Prince et al.,
1987). It is likely that the mammalian phosphorylated sialoproteins and the phosphoproteins present in
chicken bone represent a similar class of acidic glycosylated phosphoproteins and play comparable functional roles with regard to bone formation. To date,
these proteins have been identified in the bones of 20
vertebrate species and all contain Ser-P, Thr-P, and
sialic acid and have high Glu and Asp contents (Glimcher, 1984).
This laboratory has used cultured chicken osteoblasts a s a model system to study cellular differentiation and the mechanism(s) of bone matrix assembly
and mineralization. The approach, in using these cultures, has been to quantitate systematically the temporal expression of known matrix components secreted
by osteoblasts and determine the extent of their accumulation within the extracellular matrix as i t relates
to the ongoing extensive mineralization of these cultures in vitro. Previous studies using this system have
demonstrated a n induction of several differentiated osteoblast functions (Gerstenfeld et al., 1987) that are
identical to the induction seen for these functions in
vivo (Yoon et al., 1988).Extensive ultrastructural examination of the cultures has also demonstrated that
a n intrinsic property of the cultured osteoblasts is a n
ability t o direct the assembly of their synthesized collagen into both individual fibrils and a suprafibrillar
arrangement that is similar to that observed in vivo.
Once preliminary matrix assembly has occurred, apatitic mineral is spatially disposed throughout the matrix in a manner resembling that found in bone (Gerstenfeld et al., 1988). These initial studies, which show
numerous similarities between the processes of bone
formation observed in vivo and in vitro, demonstrate
the usefulness of such a culture system to aid in the
understanding of bone development and mineralization. The results of the present study, using biochemi-
cal and immunocytochemical techniques, extend these
initial observations and relate the expression of a specific 66 kDa chicken bone phosphoprotein (BPP) to
mineralization in vitro.
MATERIALS AND METHODS
Cell Culture
Osteoblasts were isolated by sequential trypsinicollagenase treatment of 17-day embryonic chicken calvariae and cells capable of expressing a n osteoblastic
phenotype were selected (Gerstenfeld et al., 1987). Cultures were initially grown for 3 wk in minimum medium and then subcultivated and grown for up to 36
days in BJG, Fitton-Jackson medium supplemented
with 50 pg/ml ascorbate and 10 mM P-glycerophosphate.
Protein Pulse Labeling Analysis
All radiolabeled compounds were obtained from the
Amersham Co., Inc. (Arlington Heights, IL). Cells were
separately labeled, without serum, in a total of 5 ml per
100-mm dish with either 250 pCi of (3H)-leucine (120
mCi/mmol) for 24 h r or with 250 pCi of (32P)-orthophosphate (3,000 mciimmol) for 6 hr. In experiments
where total phosphoprotein synthesis was to be monitored, cultures were double-labeled with 250 pCi (3H)serine (20-40 mCi/mmol) and 50 pCi (14C)-threonine
(250 mCi/mmol) for 24 hr.
Phosphoprotein Extraction and Purification From Tissue
Metatarsals, tibiae, and femurs from 14-wk-old postnatal chickens were used for the extraction of bone
phosphoproteins. The tissues were rapidly frozen in liquid N,immediately after dissection and powdered with
a mortar and pestle. Ten grams of powdered tissue
were suspended in 300 ml of deionized H,O containing
1mM p-hydroxymercurobenzoic acid, 1 mM phenylmethylsulfonyl fluoride, and 10 mM levamisole as protease and phosphatase inhibitors and homogenized at
0°C with a polytron apparatus (Tekman Co., Cincinnati, OH). The homogenate was brought to 0.3 N HC1
by addition of 1.0 N HC1 at 4°C. The tissue homogenate
was then extracted in the HC1-acidified solution at 4°C
for 48 h r while the pH was maintained a t -1.0. Insoluble material was removed by centrifugation a t 15,000
rpm in a n SS34 Sorvall rotor. The acid-extracted proteins were dialyzed against deionized H,O until a neutral pH was obtained, following which they were
lyophilized. Preliminary chromatography on DEAE
Sephacel and molecular sieving on Sephacryl S-300
(Schema 11, Uchiyama et al., 1986; Gotoh et al., 1990)
were carried out for protein purification. Protein purity
was determined by amino terminal sequence analysis
and was carried out either directly on samples obtained
from filtration through Sephacryl S-300 or after blotting onto PVDF membranes followed by the sequencing method a s described by Hunkapillar and Lujan
(1986). Both analyses yielded identical amino terminal
sequences of good quality, high repetitive yield, and
good recovery (70-100 pM), and no underlying secondary sequences were detected. These analyses were carried out on a protein sequencer (Model 470A; Applied
Biosystems, Inc., CA) equipped with a n on-line HPLC
(Model 120A; Applied Biosystems, Inc.) a t the Mi-
IMMUNOLOCALIZATION OF SYNTHESIZED PHOSPHOPROTEIN
crochemistry Laboratory, Harvard University, Cambridge, MA.
95
point dried. Following drying, the specimens were
coated by gold evaporation. Some cultures were viewed
directly along their top surfaces while others were
Phosphoprotein Analysis
viewed from a n angle that revealed the full cross-secTotal amino acid analysis and Ser-P and Thr-P con- tional depth of the tissue. The latter cultures were pretents were determined from 6 N and 4 N HCl hydroly- pared by manually fracturing the flexible plastic cirsates, respectively, on a Model 121M automatic amino cular coverslips on which tissue had been grown and
acid analyzer [Beckman Instruments, Inc., Palo Alto, then gold-coating the exposed surfaces of interest.
CA (Cohen-Solal et al., 197811. Radiolabeled ( 14C)-Thr- Samples were observed with a JEOL JSM 840 having
P and (,H)-Ser-P were quantitated by scintillation a n LaB, electron gun and operating a t 15 kV.
counting after collecting the Ser-P and Thr-P peaks
Immunocytochemistry was performed on 30-day
from the amino acid analysis. Protein profiles were an- mineralizing osteoblast cultures which were fixed overalyzed by SDS-polyacrylamide gel electrophoresis on night a t 4°C with 1% glutaraldehyde in 0.1 M sodium
5-15% continuous linear gradient gels (Laemmli, cacodylate buffer, pH 7.3, followed by post-fixation
1970) followed by staining with rhodamine B, a stain with reduced osmium tetroxide at 4°C. After washing
specific for phosphorylated bone proteins (Uchiyama et with 0.1 M sodium cacodylate buffer containing 4% sual., 1986), or with AgNO, by using a Silver Staining crose, pH 7.3, the cultures were dehydrated through a
Kit (Bio-Rad Laboratories, Richmond, CA). Fluorogra- graded ethanol series and embedded in Epon 812 (E.F.
phy of (,H)-labeled proteins was carried out by the Fullam, Inc., Latham, NY). The resin was polymerized
methods of Bonner and Laskey (1974) and fluoro- for 2 days at 60°C. Thin sections (80 nm) were cut and
graphs were prepared for quantitation by the method of immunocytochemical labeling, using a rabbit 66 kDa
Laskey and Mills (1975). Scanning densitometry was BPP polyclonal antibody and the protein A-gold techperformed by using a n LKB Ultrascan laser densitom- nique, was carried out as detailed by McKee et al.
eter (LKB, Broma, Sweden).
(1990). The protein A-gold complex was as described
by Bendayan (1984), using gold particles (14 nm diamAntibody Production and Characterization
eter) made according to Frens (1973). The specificity of
Purified 66 kDa BPP (1 mg/ml) was injected subcu- the immunocytochemical reactions was assessed by intaneously into New Zealand white rabbits by using a n cubating control sections with either pre-immune seinjection schedule and protocol previously published rum or antigen-adsorbed antibody prior to incubation
for the generation of antibodies to osteocalcin (Gund- with the protein A-gold complex. Following the immuberg et al., 1987). The titer of the rabbit anti-serum nocytochemical procedures, tissue sections were conwas evaluated by serial dilution “dot blotting.” Con- ventionally stained with uranyl acetate for 5 min and
trol, pre-immune serum prepared from the rabbit on lead citrate for 2 min and examined at 80 kV in a Philthe initial day of its innoculation with the 66 kDa BPP ips 410 transmission electron microscope.
was simultaneously examined. Animals were initially
RESULTS
bled a t 0 wk, 4 wk, and 10 wk. After 10 wk, the titer
Protein
Purification
and
Antibody Characterization
against BPP reached a maximal level with a detectable
colorimetric response on a dot blot assay a t 50 pg. UsThe major 66 kDa BPP was extracted from 14-wk
ing 11-wk rabbit anti-serum, immunoblotting was car- post-natal bone as described earlier. A representative
ried out as described by Towbin and Gordon (1984). purification scheme by DEAE ion exchange chromatogProteins were electroblotted onto nitrocellulose by us- raphy and S-300 molecular sieving is shown in Figure
ing a Trans Blot SD blotter (Bio-Rad Laboratories, 1A while the SDS polyacrylamide gel analysis of each
Richmond, CA). Initial tests demonstrated that only a purification step is shown in Figure 1B. As Figure 1B
short time of transfer (30 min a t 15 V) was needed for illustrates by silver staining, a single protein of -66
66 kDa BPP. Subsequent to blotting, the membranes kDa is obtained after the second S-300 Sephacryl chrowere stored and air dried at 4°C. The transferred pro- matographic procedure. Prior to antibody preparation,
tein was visualized immunochemically with alkaline- protein purity was assessed by amino terminal sephosphatase-conjugated goat anti-rabbit IgG followed quence analysis of the second S-300 chromatographic
by staining with nitro blue tetrazolium (Sigma Chem- peak or by electro-blotting onto a PVDF membrane. An
ical Co., St. Louis, MO). Non-specific antibody binding identical sequence of high quality and good yield was
was blocked by using 5% non-fat dry milk (Carnation obtained for both analyses at recoveries of 60-150
Co., Los Angeles, CA) in tris-HC1, pH 8.0, containing pmoles and no secondary sequence was observed
150 mM NaCl and 0.5% Tween 20 (Johnson et al., (Gotoh et al., 1990).
1984).
Antibody specificity to the 66 kDa BPP was assessed
by Western blot analysis of total protein extracts from
Electron Microscopy and lmmunocytochemistry
both embryonic bone and cultured osteoblasts. The anFor ultrastructural analysis by scanning electron tibody reacted strongly with a protein a t -66 kDa and
microscopy, 30-day mineralizing osteoblast cultures less strongly with a protein a t -45 kDa (Fig. 2A). The
were fixed overnight at 4°C with 3% glutaraldehyde in 45-kDa species is a proteolytic product of the 66-kDa
0.1 M sodium cacodylate buffer, pH 7.3, and post-fixed protein which accumulates in the extracellular matrix
with potassium ferrocyanide-reduced osmium tetrox- of the bone (Gotoh et al., 1989). Indirect immunopreide (Karnovsky, 1971) for 1 h r a t 4°C. The cultures cipitation of (,H)-leucine-labeled protein (Fig. 2A)
were then washed extensively with 0.1 M sodium ca- demonstrated that the -66 kDa phosphoprotein was a
codylate buffer containing 4% sucrose, pH 7.3, dehy- major secretory protein synthesized by the osteoblast
drated through a graded ethanol series, and critical- cultures. Two additional (3H) bands were observed in
O
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B
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kD6645-
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Stained
Immuno-
(d
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40
50
60
2nd
70
3(H) Leucine
Blot
0 0.5
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m
0
80
Fraction Number
1
2
3
B
66KD-
Fig. 1. Purification of the major 66-kDa chicken bone phosphoprotein (66-kDa BPP). A: Representative chromatography profiles of
each step of the 66-kDa BPP phosphoprotein purification. The DEAESephacel gradient was from 0-500 mM NaCl and the gradient was
initiated at fraction 20. Subsequent purification was performed on
two sequential S-300 Sephacryl columns. Bars represent pooled fractions used for each subsequent purification procedure. B: SDS polyacrylamide gel electrophoresis profiles of each purification step. Gels
were 5-10% linear gradients and proteins were visualized by silver
staining. 1: Total protein applied to DEAE Sephacel. 2 Fraction D of
DEAE Sephacel (top panel, A). 3: Second S-300 fraction.
Fig. 2. Characterization of the 66-kDa BPP polyclonal antibody. A
Analysis of antibody specificity for BPP in total protein extracts from
30-day cultured osteoblasts (0)or total HCl protein extracts from
14-wk bone (B). Total newly synthesized media protein from 30-day
cultures (TI was indirectly precipitated UP). Samples were resolved on
linear 5-10% gradient SDS polyacrylamide gels. The method by
which individual lanes were visualized is denoted a t the bottom of the
figure. B Immunoblot analysis of antibody specificity for 66-kDa BPP
in total protein extracts from 30-day osteoblasts (OB), tendon fibroblasts (Fibro), myoblasts (Myo), and 4-day sternal chondrocytes
(Chon).
the fluorograph, one a t -50 kDa, which has previously
been shown to be a result of non-specific (3H)-labeled
protein binding to the large subunit of the IgG used in
indirect immunoprecipitations (Gerstenfeld et al.,
1984),and a band a t -45 kDa, which presumably is the
proteolytic product described above. Several different
cell types of mesenchymal origin were examined for the
presence of 66 kDa BPP. A comparable quantity of extracted protein from cultured sternal chondrocytes,
myoblasts, and tendon fibroblasts was blotted and reacted with the 66 kDa BPP antibody. As Figure 2B
shows, the 66-kDa BPP was only detected in proteins
extracted from the mineralizing osteoblast culture.
IMMUNOLOCALIZATION OF SYNTHESIZED PHOSPHOPROTEIN
97
phosphorylated protein accretion within the culture
matrix, the nature of the specific phosphorylated proteins that were synthesized was assessed by polyacrylamide gel electrophoresis of (3H)-leucine and (32P)
labeling of proteins, and specific 66 kDa BPP accumulation was determined by Western blot analysis. The
results of hosphoprotein synthesis analyses, by labeling with (8P) are shown in Figure 5A. Relatively little
synthesis of (”P)-labeled 66 kDa protein was seen before day 18, whereas thereafter, synthesis remained
elevated and labeled protein was found associated with
both the cell layer and the media. It is important to
note the predominance of low molecular weight phosphoprotein species in the cell layer and the general
increase in their quantity a t later time points. Since
analysis of (32P)-labeled protein only examines posttranslational phosphorylation, and i t may not reflect
actual protein synthesis, cultures were additionally labeled with (3H)-leucine to generate data shown in Figure 5B. The time frame of induction seen for this analysis was identical to that observed for the (32P)
labeling, a result indicating that protein phosphorylation was coordinately regulated with the induction of
protein synthesis.
Days in Culture
The accumulation of 66 kDa BPP was monitored by
Western blot analysis of the culture cell layers a t varFig. 3. Mineral accumulation as a function of culture time. Total Ca
was determined at 6-day increments over a 36-day experimental peious times throughout the 30-day experimental period.
riod. Measurements represent the average of triplicate samples each
Figure 5C shows that the 66-kDa BPP accumulation
determined from three separate 100-mm dishes. Error bars denote
parallels
the total phosphoprotein content as detertotal range of variation.
mined by amino acid analysis. As seen for the (32P)labeled protein, a progressive increase in the lower molecular weight species was found at later time points
for the accumulated protein in the cell layer. This rePhosphoprotein Expression During Extracellular Matrix
sult further suggests progressive proteolytic breakMineralization In Vitro
down of the 66-kDa BPP as it accumulates with time in
The temporal relationships between extracellular the extracellular matrix.
matrix mineralization and phosphoprotein synthesis
and accumulation were examined in vitro. The total
lmmunocytochemical Localization of 66-kDa BPP in the
accumulation of calcium (Ca) as a measure of mineral
Extracellular Matrix of Cultured Osteoblasts
in the cell layer is depicted in Figure 3. An apparent
Immunocytochemistry using the 66-kDa BPP antibiphasic increase in total calcium was observed in the
36-day period. From day 6 to day 30, there was a n body was carried out to determine the microscopic loinitial very slow, 3-4-fold increase observed in min- calization of 66-kDa BPP and to compare its distribueral, subsequently followed by a very rapid 3.5-fold in- tion to that seen in vivo. Ultrastructural examination
crease between days 30 and 36. In order to assess the of the surface of a 30-day culture by scanning electron
relationship of total phosphoprotein synthesis by osteo- microscopy revealed numerous flattened osteoblasts
blasts in culture to the accumulation of mineral, cul- having long cell processes extending between cells and
tures were labeled with (3H)-Ser and ( 14C)-Thrfollowed throughout the layer of the extracellular collagen maby determination of the incorporation of these labels trix (Fig. 6A). Additional cell processes were observed
into phosphoprotein. Total phosphoprotein accretion in about the cells in regions not associated with matrix.
the matrix was simultaneously determined by a n anal- The extracellular matrix was comprised of a n extenysis of Ser-P and Thr-P accumulation. As shown in sive layer of interwoven and well-developed collagen.
Figure 4, total phosphoprotein synthesis rapidly in- In cross-sectioned “on edge” views of these cultures,
creased 3-4-fold after day 12 with (3H)-Ser-P levels osteoblasts were positioned between layers of extracelpeaking a t day 18 and (I4C)-Thr-Plevels reaching a lular matrix such that as many a s 4-6 alternating
maximum a t day 24. Thereafter, protein synthesis uti- layers of cells and matrix were present by 30 days of
lizing both phosphorylated amino acids decreased, but cell culture (Fig. 6B). Successive layers of collagenous
it remained 1.5 times greater than the initial measured matrix were approximately equal in thickness.
levels. Total accumulated phosphoprotein, as assessed Whereas a continuous monolayer of cells was generally
by phospho-amino acid incorporation, showed a slow present a t the upper surface of the culture, this was not
and continual 4-fold increase over the same 30-day pe- a consistent feature of the lower surface a s collagen
riod and subsequently remained constant between days fibrils could often be observed immediately adjacent to
the plastic Petri dish.
30 and 36 (Fig. 4).
immunocytochemistry of 30-day osteoblast cultures
While the accumulation of phosphorylated amino acids and incorporation of label into Ser-P and Thr-P showed a distribution of gold immunoreactivity, idenmeasured total phospho-amino acid synthesis and tifying the sites of 66-kDa BPP antigenicity, practi-
98
L.C. GERSTENFELD ET AL.
CULTURED CHICK OSTEOBLAST PHOSPHOPROTEINS
I
S y n t h e s i s (Media)
PSer
PThr
-a
' O0
Y
xZ
I
Accumulation
(cell L a y e r )
*OOt
150-
EI 9
/.
5 a$ 100ZQ
I
n
0
50-
0
./O
6
12
18
24
6
30
12
18
24
30
36
Days in Culture
Fig. 4. Analysis of total phospho-amino acid synthesis and accumulation as a function of culture time. Phospho-amino acid synthesis was
determined by amino acid analysis of pooled cell layers and media
protein from each time point. Ninhydrin, Ser-P, and Thr-P peaks were
collected and (3H)-Ser-P and (I4C)-Thr-P in each peak were determined. Each data point represents the average of two determinations.
Total accumulations were determined from the average of triplicate
amino acid analysis determinations.
cally identical to that observed in vivo (McKee et al.,
1990). Immunolabeling was found principally over extracellular matrix regions undergoing mineralization
(Fig. 7A-C). Gold particles were most often associated
with electron-dense patches of organic material in close
association with collagen fibrils within the matrix.
These immunolabeled matrix sites contained relatively
high levels of Ca and P, a conclusion based on electron
probe x-ray microanalysis of other sections of glutaraldehyde-fixed, unstained sections not subjected to the
immunocytochemistry (data not shown). Following the
immunocytochemical incubation procedures and staining of sections with uranyl acetate and lead citrate, Ca
and P were lost from the tissue. These results demonstrate that 66-kDa BPP is associated with mineralization foci in the culture extracellular matrices. Since the
tissue processing and immunocytochemical technique
used here retain the phosphoproteins (McKee e t al.,
1990) and the 66-kDa BPP is specifically localized to
the dense patches as shown by immunolabeling, i t appears that the highest concentrations of 66 kDa BPP
are to be found in such dense extracellular foci.
expression of bone phosphoproteins and their relationship with other osteoblast functions and extracellular
matrix proteins (such as, for example, osteocalcin, alkaline phosphatase, type I collagen, and fibronectin).
The work provides a very well-defined in vitro model
system of mineralization for correlation with the mineralization processes t h a t occur in vivo.
A schematic summary of the temporal pattern of
synthesis of 66-kDa BPP is presented in Figure 8 in
relation to the other proteins previously examined in
this model system. Several interesting correlations
may be obtained from this comparison. Phosphoprotein
expression increases concurrent with the expression of
other proteins associated with advanced osteoblast differentiation, such as osteocalcin and alkaline phosphatase, a result suggesting that phosphoprotein synthesis is restricted to more differentiated or mature
osteoblasts. In contrast, collagen and fibronectin,
which have a more generalized expression in many
connective tissues, are maximally expressed during the
period of rapid cell proliferation associated with early
culture times (Shalhoub e t al., 1989). These results
would suggest that connective tissue proteins such a s
collagen and fibronectin are necessary prerequisites for
the growth of osteoblasts and for the initial assembly of
the extracellular matrix. The subsequent expression,
then, of high levels of 66-kDa BPP, osteocalcin, and
alkaline phosphatase in bone are no doubt a reflection
of the extracellular matrix specialization. It is also interesting to note that phosphoprotein begins to accumulate concurrent with, or slightly preceding, the first
increases in mineral deposition. These results obtained
in vitro are qualitatively identical to the sequence of
expression seen in vivo for collagen type I (Moen et al.,
1979), osteocalcin (Hauschka and Reid, 1978), and
phosphoprotein (McKee e t al., 1990), in which collagen
synthesis temporally precedes both osteocalcin and
phosphoprotein synthesis during embryonic chicken
DISCUSSION
In a previous study from this laboratory of embryonic
chicken bone development in vivo, analysis of the ultrastructural localization and extracellular matrix accumulation of the same major 66-kDa BPP examined
here showed a n intimate association of this protein
with areas of mineralization and a n unequivocal quantitative correlation between increased phosphoprotein
accumulation and mineral deposition (McKee e t al.,
1989). In the present work, phosphoprotein synthesis,
extracellular matrix accumulation, and ultrastructural localization were examined in vitro in cultured
embryonic chicken osteoblasts and compared with the
results obtained in vivo. The investigation also was
carried out in order to define more clearly the temporal
99
IMMUNOLOCALIZATION OF SYNTHESIZED PHOSPHOPROTEIN
Days
Day
6
18
30
6
3
9
21 24 30
18 30
MWx103
116946645-
29Osteocalcin
A
MEDIA
CELL
12
18 24 30
66kDa-
C
MEDIA
CELL
bone development. Similar temporal profiles are found
for the expression of these same collagenous and noncollagenous extracellular matrix proteins [including
the rat phosphoprotein, osteopontin (Prince et al.,
1987)] during mammalian embryonic bone development (Yoon et al., 1987). An identical sequence of expression for these respective genes was also seen in
cultured primary rat osteoblasts (Stein e t al., 1989).
This commonality indicates t h a t closely related mechanisms of bone matrix assembly and mineralization
may be operative in all vertebrates.
The chicken 66-kDa BPP shares many biochemical
features with the rat phosphoprotein, osteopontin.
Both proteins are glycosylated and contain sialic acid,
have phosphorylated threonine and serine residues,
have similar DEAE chromatography elution characteristics, and have approximately the same apparent sizes
on gel electrophoresis (Franzen and Heinegard, 1985;
Prince e t al., 1987). Analysis of recent sequence data
for the chicken 66-kDa BPP (with -25% of the protein
sequence completed and 30% of the cDNA sequence
completed) shows that there is little homology with rat
osteopontin. However, chicken 66-kDa BPP contains a
nine-amino-acid sequence, with two conservative substitutions, that contains the Arg-Gly-Asp cell-binding
sequence of osteopontin (Gotoh et al., 1990) and has a
6 I?-
B
Fig. 5. Characterization of 66-kDa BPP synthesis as a function of
culture time. A Specific protein phosphorylation was examined by
(32P)labeling for 6 hr. Both cell layer and media were separately
analyzed and the days examined are denoted at the top of the figure.
A constant 50 pg of protein was resolved on a linear 510% gradient
SDS polyacrylamide gel and autoradiographic exposure was for 24 hr.
B Specific protein synthesis was examined by (3H)-leucine labeling
for 24 hr. The secreted proteins of the media were analyzed and the
days examined are denoted in the figure. A constant 5 x lo4 cpm of
(3H)-leucinewas applied for each sample and the proteins were resolved on a linear 5-18% gradient SDS polyacrylamide gel. Fluorographic exposure was for 3 days. The positions of osteocalcin, 66-kDa
BPP, collagen a chains, and procollagen are denoted in the figure. An
osteocalcin standard was included in the left lane as a reference. C :
66-kDa BPP accumulation detected by Western blot analysis. Both
cell layer and media were separately analyzed and the days examined
are denoted in the figure. A constant 50 pg of protein was resolved on
a linear 5-10% gradient SDS polyacrylamide gel.
nine-amino-acid sequence of continuous aspartatic acid
residues (Moore, Gotoh, and Gerstenfeld, unpublished
data). Indeed, whole embryo immunolocalization studies using the 66-kDa BPP antibody shows a n immunohistochemical distribution of staining in the intestine
and kidney (Bruder et al., 1990) that is similar to the
localization for osteopontin and osteopontin mRNA
(Mark et al., 1987a,b, 1988; Nomura et al., 1988). Furthermore, 66-kDa BPP promotes cellular adherence of
MG63 osteosarcoma cells (M. Pierschbacher, unpublished results), a property also observed for osteopontin
and periodontal fibroblasts (Somerman et al., 1987).
These results would suggest that 66-kDa BPP is either
the chicken homologue to the rat protein, but has undergone extensive sequence divergence during evolution, or is a very similar functionally related protein.
The presence of a n Arg-Gly-Asp sequence in the rat
phosphoprotein, osteopontin, and the cell attachment
studies of Oldberg e t al. (1986, 198813) and Somerman
et al. (1987) led these researchers to hypothesize that
this protein transmitted positional information directly from the osteoblast surface to the mineralized
bone matrix. The synthesis of fibronectin determined
in studies from this laboratory (Winnard et al., 1989)
during the early period of culture growth indicates that
osteoblasts synthesize multiple Arg-Gly-Asp-contain-
100
L.C. GERSTENFELD ET AL.
Figs. 6-7.
101
IMMUNOLOCALIZATION OF SYNTHESIZED PHOSPHOPROTEIN
ing proteins and that fibronectin serves to promote cell
attachment and spreading during osteoblast proliferation, while the later expression of 66-kDa BPP probably is related to a different function. Recent data indicate that the class of integrin receptor that recognizes
bone sialoprotein and osteopontin is more similar to
thevitronectinreceptor (Oldberget al., 1988a,b),aresult
thereby suggesting that osteoblasts may also have
multiple types of integrin receptors which impart different types of positional information.
In the present study in vitro and in the accompanying study in vivo (McKee et al., 1990), chicken 66-kDa
BPP was observed predominantly in the extracellular
matrix, was clearly associated with mineral, and was
well removed from the cell surface. These results indicate that any cell surface association of 66-kDa BPP is
probably only transient in nature. Several possible
types of positional information might be imparted by
such a transient cell surface association. Previous results indicated that cultured osteoblasts assemble their
collagen fibrils in a tissue-specific orthogonal array
(Gerstenfeld et al., 1988). Since 66-kDa BPP accumulates a t extracellular sites of mineralization in a n identical fashion both in vitro and in vivo, specific positional information may be needed during matrix
assembly to direct specific interactions of matrix proteins during their translocation from the cell to the
matrix. Other functions that may be transient in nature might involve the post-translational modification
of 66-kDa BPP by specific cell-surface-associated kinases, phosphatases, or proteases. Alternatively, the
localization of 66-kDa BPP in the kidney and intestine,
Fig. 6. General ultrastructural characteristics of a 30-day osteoblast
culture. A Scanning electron micrograph of the upper surface of a
30-day osteoblast culture. Osteoblasts (OB) generally cover the underlying collagenous matrix (Coll) and develop long cell processes
(asterisks) extending both among the numerous collagen fibrils and
into the space not associated with matrix just above the cells. B:
Scanning electron micrograph of a 30-day osteoblast culture fractured
open to reveal a n “on edge” view of the tissue. Layers of osteoblasts
(OB) alternate with layers of the collagenous matrix (Coll) to form 3
relatively thick bone-like tissue having a lamellar structure.
Fig. 7. Immunolocalization of 66-kDa BPP in a 30-day mineralizing
osteoblast culture, fixed with glutaraldehyde and osmium tetroxide,
embedded in Epon, and stained with uranyl acetate and lead citrate.
A Transmission electron micrograph after incubation of the tissue
section with the 66-kDa BPP antibody and the protein A-gold complex. The most intense specific immunolabeling is observed over areas
of extracellular matrix undergoing mineralization (arrowheads) and
is associated with electron-dense organic material in close proximity
to collagen fibrils (Coll). Some gold particles are also observed nonspecifically over areas of collagenous matrix not associated with detectable mineral. OB, osteoblast. B: Higher-magnification electron
micrograph of a site of mineralization in the extracellular matrix
(MM). In this region some collagen is visible in partial longitudinal
profiles and a specific immunocytochemical reaction is found over
electron-dense organic matrix in close association with the fibrils. The
electron-dense foci contain high levels of Ca and P detectable by x-ray
microanalysis, but these elements are lost following antibody incubation and staining with uranyl acetate and lead citrate. The observed
uptake of stain is indicative of an organic composition of the foci,
contributed in part by 66-kDa BPP. C: Electron micrograph of the
extracellular matrix of a 30-day mineralizing osteoblast culture in
which the collagen fibrils (Coll) are predominantly sectioned transversely. Numerous gold particles are observed specifically over the
electron-dense organic matrix associated with areas of mineralization
among the fibrils (arrowheads).
3
6
12
18
24
TIME IN CULTURE
30
Fig. 8. Schematic representation of the temporal profiles of osteoblast functions during mineralization in vitro. A summary comparison of the percent maximal of each culture component is depicted for
different osteoblast functions. Data for osteocalcin and alkaline phosphatase are from Gerstenfeld et al. (1987). Data for collagen are from
Gerstenfeld et al. (1988), for fibronectin from Winnard et al. (1990),
and for phosphoprotein and mineral from the present study.
and by analogy to the known vitamin D and PTH regulation of osteopontin expression in the rat and phosphoprotein expression in the chicken (Prince and Butler, 1987; Noda and Rodan, 1988; Lian et al., 19821,
might indicate that this class of proteins plays a generalized role in phosphate metabolism, functioning either in phosphate mobilization or phosphate transport.
However, neither the exact phosphorylation state of
this protein in these tissues nor its quantitative expression is currently known.
In summary, the data presented here demonstrate
that 66-kDa BPP is expressed only concurrently with
other differentiated osteoblast functions. The ultrastructural localization of this phosphoprotein in vitro is
identical to that seen in vivo, thereby indicating that
osteoblasts in vitro retain their phenotypic ability to
express proteins that appear to be important in regu-
102
L.C. GERSTENFELD ET AL.
lating the assembly and mineralization of their extracellular matrix. The chicken 66-kDa BPP is primarily
associated with the extracellular organic matrix and
does not accumulate on the osteoblast cell surface.
These data obtained in vitro and those of McKee et al.
(1989, 1990) from work in vivo possibly reflect on the
suggestion t h a t a n Arg-Gly-Asp-containing protein
may be only transiently associated with the cell surface
and may transmit positional information a t a distance
well removed from the cell surface.
its possible linkage to the organic matrix by protein-bound phosphate bonds. Philos. Trans. R. SOC.
Lond. [Biol.], B304:479-508.
Glimcher, M.J. 1989 Mechanisms of calcification in bone: Role of collagen fibrils and collagen-phosphoprotein complexes in uztro and
in uiuo. Anat. Rec., 224t139-153.
Glimcher, M.J., D. Brickley-Parsons, and D. Kossiva 1982 Proof that
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Gotoh, Y., M. Sakamoto, S. Sakamoto, and M.J. Glimcher 1983 Biosynthesis of 0-phosphoserine-containingphosphoproteins by isolated bone cells of mouse calvaria. FEBS Lett., 154:116-120.
Gotoh, Y., L.C. Gerstenfeld, and M.J. Glimcher 1990 Identification
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