A Phage Display Technique Identifies a Novel Regulator of
Cell Differentiation*
Tzong-Jen
Sheu
,
Edward M.
Schwarz
,
Daniel A.
Martinez§,
Regis J.
O'Keefe
,
Randy N.
Rosier
,
Michael J.
Zuscik
, and
J. Edward
Puzas
¶
From the
Department of Orthopaedics, Center for
Musculoskeletal Research, University of Rochester School of Medicine
and Dentistry, Rochester, New York 14642 and the
§ Connective Tissue Physiology Laboratory, Department of
Biology and Biochemistry, University of Houston, Houston, Texas
77204-5001
Received for publication, August 13, 2002, and in revised form, October 24, 2002
 |
ABSTRACT |
The formation of new bone during the
process of bone remodeling occurs almost exclusively at sites of prior
bone resorption. In an attempt to discover what regulatory
pathways are utilized by osteoblasts to effect this site-specific
formation event we probed components of an active bone resorption
surface with an osteoblast phage expression library. In these
experiments primary cultures of rat osteoblasts were used to construct
a phage display library in T7 phage. Tartrate-resistant acid
phosphatase (type V) (TRAP) was used as the bait in a biopanning
procedure. 40 phage clones with very high affinity for TRAP were
sequenced, and of the clones with multiple consensus sequences we
identified a regulatory protein that modulates osteoblast
differentiation. This protein is the TGF
receptor-interacting
protein (TRIP-1). Our data demonstrate that TRAP activation of TRIP-1
evokes a TGF
-like differentiation process. Specifically, TRIP-1
activation increases the activity and expression of osteoblast alkaline
phosphatase, osteoprotegerin, collagen, and Runx2. Moreover, we show
that TRAP interacts with TRIP intracellularly, that activation of the
TGF
type II receptor by TRIP-1 occurs in the presence of TRAP and
that the differentiation process is mediated through the Smad2/3
pathway. A final experiment demonstrates that osteoblasts, when
cultured in osteoclast lacunae containing TRAP, rapidly and
specifically differentiate into a mature bone-forming phenotype.
We hypothesize that binding to TRAP may be one mechanism by
which the full osteoblast phenotype is expressed during the process of
bone remodeling.
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INTRODUCTION |
The formation of new bone during the process of bone remodeling
occurs almost exclusively at sites of prior bone resorption. As there
are 1-2 million active remodeling sites in an adult skeleton at any
point in time (1), this spatial localization of formation plays a key
role in maintaining skeletal architecture. Aberrant or disorganized
formation could lead to architectural changes that would weaken
skeletal structure.
The observation that osteoclastic bone resorption precedes osteoblastic
bone formation has been a well known characteristic of the cell
activity in the basal metabolic unit (BMU) described by Frost (2),
nearly 40 years ago. In a BMU, osteoclastic activation is followed by a
reversal phase in which the osteoclast either migrates to another bony
site or dies by apoptosis. Osteoblast bone formation then proceeds
directly on the reversal line and if the two activities are matched in
amount, a constant skeletal mass is preserved. Activation of
osteoprogenitor cells in the immediate area of a resorption event has
been attributed to the release of growth factors from the bone during
osteoclast activity. This process, first termed coupling by Harris
and Heany in 1969 (3, 4), accounts for the temporal sequence of
resorption preceding formation. However, what has not been appreciated
is that the formation occurs at the immediate sites were osteoclast activity occurred.
In work performed by Gray, Jones, and Boyde (5, 6) it was shown that
osteoblasts produce more mineralized matrix at sites of prior
resorption and in geometrical grooves created on bone and dentin
wafers. The authors suggest that this may be one signaling mechanism by
with these cells "even off" the contours of bone resorption
lacunae. It also appears that surface roughness on implants may
stimulate bone formation (7-10). However, since there are numerous
contours that persist in trabecular bone structure, it is unlikely that
surface geometry is the only driving factor in directing bone formation.
We speculate that biochemical signals deposited on resorption lacunae
by osteoclasts may play a major role in directing the differentiation
of osteoprogenitor cells into a mature osteoblast phenotype. In order
to explore this hypothesis, we utilized an osteoblast cDNA
phage-display library in T7 phage to probe one of the components of the
lacunar surface, namely type V tartrate-resistant acid phosphatase
(TRAP).1 TRAP is an acid
hydrolase with optimum activity on phosphoester bonds at pH values
below 6.0. TRAP is produced in large amounts by osteoclasts and is a
hallmark of their activity (11, 12). For many years we have known that
TRAP remains firmly attached to resorption surfaces and that bone
formation can proceed directly on a TRAP-coated lacunae (13-15).
As we show in this study, an osteoblast protein (TGF
receptor-interacting protein, TRIP-1) possess very high affinity for TRAP and is poised for activating the TGF
differentiation pathway in
osteoblasts. TRIP-1 has been previously described in other cell types
and has been shown to modulate TGF
signaling in both a stimulatory
and inhibitory fashion (16, 17). In osteoblast systems TGF
signaling
pathways control osteoblast differentiation (18-23). We show that this
effect is modulated by the interaction of TRAP with TRIP-1.
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MATERIALS AND METHODS |
Purified type V TRAP was obtained as a generous gift from Dr.
R. M. Roberts, University of Missouri, Columbia, Missouri. This enzyme, also known as uteroferrin, shares identity with osteoclast TRAP
(24). Cortical bone wafers were obtained by cutting 4.0 × 4.0 × 0.3 mm sections from bovine femoral cortical bone obtained from a local abattoir.
Isolated osteoblasts were prepared from neonatal rat calvaria as
previously described (25). Alkaline phosphatase assays and standard
Western analyses were performed as previously described (26).
Construction of a T7 Primary Rat Osteoblast cDNA
Library--
A T7 phage display library of rat osteoblast cDNAs
was constructed from an existing primary rat osteoblast day 8 cDNA
plasmid library generated from primary isolated rat osteoblasts. The
cDNA inserts of the plasmid library were excised by digestion with EcoRI and NotI and inserted between the
corresponding sites of an equimolar mixture of T7Select 1-1 vector arms
(T7Select System Manual, Novagen). The resulting phage library
contained 5.6 × 107 independent clones/ml, as
determined by plaque assays. The library was amplified once by
infecting a mid-log phase Escherichia coli (BLT 5615 bacterial strain) culture (250 ml, OD600 ~0.6) with the
phage library at a multiplicity of infection of 0.001. After cell
lysis, the phage lysate was made in 0.5 M NaCl, clarified by centrifugation, and stored at
80 °C. The insert sizes of 24 individual clones as well as of the complete library were analyzed by
PCR with the forward primer 5'-GGAGCTGTCGTATTCCAGTC-3' and the reverse
primer 5'-AACCCCTCAAGACCCGTTTA-3'.
T7 Phage Clone Biopanning Procedure--
An aliquot of the
amplified phages (109 pfu) were allowed to bind to TRAP,
which was immobilized on an ELISA plate for 2 h while rotating
gently. Unbound phages were removed by washing ten times with 0.2 ml of
1 M NaCl, 0.1% Tween 20 in PBS, pH 7.2, further washed
twice in 0.2 ml of PBS, and finally resuspended in 100 µl of elution
buffer (Novagen).
Ten microliters of the supernatant was used to determine the amount of
detached phage in each round of selection. The remaining 90 µl of the
supernatant was added to a 10-ml culture (OD, 0.6) of E. coli (BLT5615). The bacteria had been induced with 100 µl of 100 mM IPTG 30 min before phage addition, to ensure production of the phage capsid protein. Approximately 2 h after phage
addition the bacteria were lysed, and the phage sublibrary was added to the ELISA plate (Nunc, Rochester, NY) coated with TRAP. After binding
and washing of the sublibrary, a new round of selection was started.
Following two rounds of selection, 40 plaques were arbitrarily isolated
from LB plates and each dissolved in phage extraction buffer (100 mM NaCl, 20 mM Tris-HCl, pH 8.0, and 6 mM MgSO4). In order to disrupt the phages, the
dissolved material was mixed 1:1 with 10 mM EDTA, pH 8.0, and heated at 65 °C for 10 min. The phage DNA was then amplified by
PCR, using T7 SelectUP and T7 SelectDOWN primers (T7Select Cloning kit,
Novagen). After amplification, the PCR fragments were purified by
adding 1 ml of 100% ETOH to precipitate the PCR product. The purified
PCR fragments were then sequenced using ABI 377 Big Dye autosequence kit (Applied bioscience). Based on the sequence results, the predicted amino acid sequence displayed on the T7 phage capsid can be determined. The candidate clones were amplified and used to check the affinity to
TRAP with an ELISA method.
Phage ELISA and Far-Western--
Small-scale phage preparations,
obtained from single colonies of the third round of affinity biopanning
were analyzed for binding to TRAP by phage ELISA. Briefly, in this
method, selected phage at increasing titers were incubated for 2 h
at room temperature in TRAP- or BSA-coated wells. Phage that bound to
immobilized TRAP were detected by incubation with HRP-conjugated
anti-T7 antibody (Novagen), followed by incubation with HRP substrate
(ABTS Sigma A1888). The absorbance was read at an OD of 410 nm.
A Far-Western technique was used to document that the selected phage
were indeed binding to TRAP. In this procedure, two concentrations of
TRAP and control proteins (bovine serum albumin, 5 ug) were loaded in a
10% SDS-PAGE gel and electrophoresed. They were then transferred to
polyvinylidene difluoride membranes (PerkinElmer Life Sciences) and
incubated with 1010 M13 phage particles from a GPC4 phage
(Clone 5), which have been shown to have high affinity and high
specificity for TRAP (27). The membrane was washed in PBS with 0.5%
(v/v) Tween 20 four times. An anti-M13 phage peroxidase-conjugated
antibody (Amersham Biosciences) at a dilution of 1:15000 was added and
gently swirled at room temperature for 1 h. In the last washing
procedure, the membrane was incubated in PBS without Tween 20. Detection of the phage/antibody complex was accomplished using ECL-plus
(Amersham Biosciences) with the membrane exposed to Kodak Biomax MR
film for 30 s.
Osteoprotegerin (OPG) Sandwich ELISA--
ELISA plates were
coated by overnight incubation with 0.1 ml of carbonate buffer (15 mM Na2CO3, 35 mM
NaHCO3, 0.02% NaN3, pH 9.6) containing 1 µg/ml of anti-OPG antibody at 4 °C per well. The plates were
blocked with 0.2 ml of 5% dry milk in PBS per well for 1 h at
37 °C. 200 µl of medium prepared from different treatment cells as
described above were added and incubated for 1h at 37 °C. Samples
and serial dilution of OPG standards were loaded in triplicate to the
plates (0.2 ml/well). After washing with PBS-Tween (0.1%), the bound
OPG was quantified by successive incubation with another detection
antibody conjugated with biotin (1 h each at 37 °C). After
incubation, the plate was washed with PBS-Tween ten times and incubated
with 100 µl of streptavidin with HRP-conjugated (1:10000 dilution
from stock) for 30 min at room temperature. After incubation, the
plates were washed with PBS-Tween. 0.2 ml of 2,2'-azinobis
(3-ethylbenzthiazolinesulfonic acid) solution (ABTS Sigma A1888) per
well was added for reaction with horseradish peroxidase. The plate was
then read at 405 nm in an ELISA reader.
Glutathione S-Transferase (GST) Fusion Protein Preparation and
Pull-down Assay--
GST-TRAP and GST-TRIP fusion proteins, and GST
control protein were purified as instructed by the manufacturer
(Amersham Biosciences). Briefly, plasmids containing GST fusion protein
expressing cDNA were transformed into a BL21(DE3)pLysS bacteria
strain and selected for ampicillin- and chloramphenicol-resistant
colonies. Selected colonies were grown in LB medium at 30 °C until
the OD600 reached 0.6-1. Then 0.1 mM IPTG was
added to the medium for 3 h. Bacteria were lysed by B-PER (Pierce)
with 1 mM phenylmethylsulfonyl fluoride. Lysed bacteria
were spun down and the supernatants were collected. The GST fusion
proteins were pulled down by glutathione-coated beads (Amersham
Biosciences) in 4 °C for 1 h then washed three times with NETN
buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 6 mM MgCl2, 1 mM EDTA, 0.5% Nonidet
P-40, 1 mM dithiothreitol, 8% glycerol, and 1 mM phenylmethylsulfonyl fluoride). The purified GST fusion proteins and beads were suspended in 100 µl of NETN buffer.
Resuspended GST proteins and beads were incubated with 5 µg of
purified TRAP or radioimmune precipitation assay (RIPA) buffer-lysed
transfected cell lysate. After incubating for 1 h at 4 °C with
agitation, the glutathione-coated beads were washed with NETN buffer
four times, and then the protein complexes were loaded in SDS-PAGE and
visualized using the ECL-plus method.
Mammalian Two-hybrid Assay--
Transfections were performed
using the Panver LT-1 reagent method (Panvera) as described in the
product sheet. Briefly, 1.5-3 × 105 cells were
plated on 35-mm dishes for 24 h, and the medium was changed to
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
2 h before transfection. Cells were transfected with 0.5 µg of
plasmids expressing a Gal4-DBD (DNA binding domain) fused with a
full-length TRIP-1 cDNA or an antisense TRIP-1 cDNA and a
VP-16AD (activation domain) fused with a TRAP cDNA as indicated. A
Gal4 response element controlled firefly luciferase expression plasmid,
pG5-Luc, was used as reporter gene. A Renilla luciferase expression plasmid pRL-SV40 was used as an internal control for transfection efficiency. The total amount of DNA was adjusted to 5 µg
with pCMX vectors.
Statistics--
All data are presented as the mean ± 1 S.E. Statistical significance was determined by analysis of variance.
 |
RESULTS |
Osteoblast differentiation at sites of bone remodeling is mediated
by a number of regulatory factors. One of the key factors is TGF
. In
most systems, ours included, TGF
is known to be a potent enhancer of
the osteoblast phenotype (22, 23). Fig. 1
shows that in the presence of TGF
isolated osteoblasts show an
increase in alkaline phosphatase activity and an increase in Runx 2, OPG, and collagen protein synthesis. In an exactly analogous fashion,
TRAP demonstrates the same effect. Control phosphatases such as
myokinase and ATPase have no effect on the cells (data not shown).

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Fig. 1.
Effect of TRAP and TGF
on osteoblast phenotypic markers. The qualitative pattern of
stimulation by TRAP and TGF for markers of osteoblast
differentiation is similar. The concentration of TRAP was 10 µg/ml
and for TGF was 2 ng/ml for all determinations. Alkaline phosphatase
(Alk. Phos.) was measured in a direct biochemical assay.
Runx2 and osteoprotegerin (OPG) were quantified in an ELISA
assay using commercially available antibodies. Collagen was measured by
determining the amount of collagenase-digestible protein present in
prelabeled osteoblasts (34). All data are expressed as a percent of the
control, untreated cells. All data points are the mean ± one S.E.
*, p 0.05; **, p 0.01.
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The effect of TRAP on osteoblasts may be one mechanism by which the
cells are induced to differentiate only at sites of prior bone
resorption. In order to define what osteoblast proteins may be involved
in this mechanism we probed immobilized TRAP with T7 phage that were
expressing proteins from an osteoblast cDNA library. After three
rounds of biopanning 40 phage clones with very high affinity for TRAP
were sequenced and analyzed. Three of the clones contained sequences
that may be involved in osteoblast differentiation. They were type I
collagen, Sox9, and TRIP-1. However, the sequences for type I collagen
and Sox9 were from the 3'-untranslated region of the messages. The
TRIP-1 sequence, however, was from the coding region of the protein.
Fig. 2A demonstrates that the
TRIP-1-expressing phage show a dose-dependent affinity for
TRAP. Fig. 2B is a compilation of data that indicate the
binding of TRIP-1 to TRAP is specific and of high affinity. For these
experiments we prepared a plasmid construct with human TRIP-1 cDNA
fused to glutathione S-transferase (GST-TRIP). As a control
protein we utilized the GST vector alone. GST-TRIP and TRAP were
incubated for 1 h, and any proteins associated with the GST-TRIP
were extracted from the reaction mixture by incubation with
glutathione-coated beads. The proteins were analyzed by Far-Western
analysis utilizing a phage clone with high affinity for TRAP (27). In
lane A of Fig. 2B we show that the control fusion
protein does not have any affinity for TRAP as no TRAP protein can be
detected. Lane B demonstrates that the GST-TRIP fusion
protein has no affinity for any of the components of bovine serum
albumin and is not recognized in the Far-Western. Lanes C
and D show that GST-TRIP has a dose-dependent
affinity for TRAP. Lane E is a positive control without
glutathione bead extraction, demonstrating that TRAP can be detected in
this Far-Western. These data document the affinity of TRIP-1 for TRAP
and prove that the association does not depend on post-translational
modifications of TRIP-1 as the protein was produced in bacteria and
that the human sequence of TRIP has affinity for TRAP in the same way
as the molecule from the rat cDNA library.

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Fig. 2.
Specificity and affinity of TRAP binding to
TRIP-1. Panel A demonstrates that wild type phage
(containing no osteoblast proteins) have little affinity for TRAP that
has been immobilized in a culture dish. TRIP-1-containing phage show a
dose-dependent increase in TRAP binding. Phage titer is
expressed as particles/ml. Phage binding was detected with HRP-anti-T7
phage antibody in a sandwich ELISA assay. All TRIP-1 phage data points
are the mean ± 1 S.E. **, p 0.01. Panel
B demonstrates the specificity of TRIP-1 binding to TRAP. Human
TRIP-1 was synthesized as a GST fusion protein. The GST vector alone
served as a control protein. Different amounts of TRAP and other
proteins (i.e. bovine serum albumin, BSA) were
incubated with the GST-TRIP-1 fusion protein, and the molecules
associating with the GST-TRIP-1 were extracted by exposure to
glutathione beads. The "pulled-down" proteins were separated on a
denaturing gel, and the level of TRAP was measured with Far Western
analysis (27). Lane A shows that GST has no affinity for
TRAP. Lane B shows that TRIP-1 does not bind to any
molecules in BSA that would be detected in the Far Western. Lanes
C and D show that increasing amounts of TRAP can be
captured by the GST-TRIP-1 in a dose-dependent fashion.
Lane E is a control lane loaded with TRAP to demonstrate the
detection of this system.
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In order to demonstrate that TRIP-1 and TRAP can interact inside of
cells we utilized a mammalian two hybrid system. In these experiments
we transfected 293T cells with fusion proteins composed of TRIP-1 with
a Gal4-DNA binding domain (DBD) and TRAP with a VP-16-activation domain
along with a luciferase reporter gene. Single transfections with either
fusion protein showed no increase in reporter activity; however,
co-transfection with both fusion proteins showed a 20-fold increase in
reporter activity (Fig. 3). Moreover, in
a control experiment where antisense TRIP-1 was substituted in the
Gal4-DNA binding domain, there was also no stimulation of the reporter
gene. These data demonstrate that TRIP-1 and TRAP can interact with
each other in a highly specific manner in the cytosol of cells. As a
further confirmation that TRAP and TRIP-1 interact in the same cell
compartment we performed fluorescent labeling co-localization studies.
293T cells were transfected with TRIP-1 tagged with red fluorescent
protein (TRIP-RFP) and TRAP tagged with a green fluorescent protein
(TRAP-GFP). The cells were examined for TRAP-GFP and TRIP-RFP under
fluorescence confocal microscopy. The images from the two wavelengths
were digitally superimposed and where red and green pixels overlapped we created a merged pseudo-color image. This image demonstrates that at
the resolution of the microscope and digital image, TRAP and TRIP-1
co-localize inside of cells (Fig. 4).

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Fig. 3.
Two-hybrid demonstration of TRAP association
with TRIP-1. A mammalian two-hybrid system was utilized to
demonstrate an association between TRAP and TRIP-1. 293T cells, when
individually transfected with a Gal4-DBD-TRIP-1 or VP-16-AD-TRAP fusion
protein, showed no activation of the luciferase reporter. However,
co-transfection of the cells with both constructs allowed for
interaction of TRAP with TRIP-1. This association permitted the
assembly of the transcription machinery for luciferase expression.
Substitution of an antisense form of TRIP-1 as the Gal4-DBD fusion
protein, as expected, did not show any association with TRAP. All data
are the mean of four determinations ± S.E. **, p 0.01 as compared with Gal4-BD-TRAP.
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Fig. 4.
Co-localization of TRAP with TRIP-1 in 293T
cells. TRAP and TRIP-1 were synthesized as a green fluorescent
fusion protein (GFP) and red fluorescent fusion protein
(RFP), respectively. When 293T cells were co-transfected
with TRAP-GFP and TRIP-1-RFP and analyzed with dual wavelength confocal
microscopy and digital imaging, it was possible to demonstrate
co-localization of TRAP and TRIP-1. Digital images of cells at each
wavelength were superimposed and where green and red pixels overlapped
we created a pseudo-color yellow image. This experiment was performed
three separate times. In each experiment over 100 cells were observed
and all of them demonstrated some level of co-localization. The image
in this figure represents a single cell. The bar indicates a
distance of 20 microns.
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We next sought to determine if TRAP could activate one of the TGF
signaling pathways. The data in Fig. 5
show that both TGF
and TRAP can strongly upregulate a reporter gene
(P3TP-Lux) that is sensitive to the TGF
regulatory Smads 2 and 3. The effects of TGF
and TRAP are additive. As TRIP-1 is known to
interact with the type II TGF
receptor we investigated whether TRAP
activation of this pathway could be blocked in the presence of a
dominant negative type II TGF
receptor expression vector. Fig. 5
also demonstrates that in cells that have been co-transfected with a
dominant negative type II TGF
receptor we can block both TGF
and
TRAP activation of the pathway. These results were obtained with both
osteoblast cell lines, MG-63 and SaOS2. The data with the MG-63 cell is
what is shown.

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Fig. 5.
TRAP and TGF
activate the Smad signaling pathway. P3TPLux is a Smad
2/3-sensitive reporter. Transfection of this reporter into either of
the osteoblast cell lines, SaOS2 or MG-63, and exposure of the cells to
TRAP (10 µg/ml) or TGF (5 ng/ml) causes an activation of the Smad
pathway. The effect of TRAP plus TGF are additive. Co-transfection
of the cells with a dominant negative TGF type II receptor blocks
all TRAP and TGF signaling. Each bar is the mean of at
least four determinations ± S.E. *, p 0.05 and
**, p 0.01 as compared with control. The data for
MG-63 cells are shown. The data for the SaOS2 cells are the same (data
not shown).
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When these experiments were repeated in a Smad4-deficient cell type
(SW408 cells), neither TGF
nor TRAP could activate the reporter gene
(Fig. 6). As Smad 4 is a requisite
co-factor for Smad 2 and 3 signaling, these results are consistent with
TRAP working through the Smad pathway. Restoration of Smad4 and TRIP-1 protein in these cells, restores TGF
and TRAP signaling (Fig. 6).
Thus, all of these pieces of evidence point to the activation of the
TGF
/Smad pathway through the association of TRAP with TRIP-1.

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Fig. 6.
TRAP and TGF
signaling in a Smad4-deficient cell line. SW408 cells are
deficient in Smad4, a requisite co-factor for Smad2 and 3 signaling.
The data in this figure demonstrate that neither TRAP nor TGF (nor
the combination of factors) can activate the Smad signaling pathway in
SW408 cells. However, when the cells are transfected with both Smad4
and TRIP-1, restoration of Smad signaling can occur. All values are the
mean ± S.E. *, p 0.05 and **,
p 0.01 as compared with control.
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We also have direct evidence that TRIP-1 will interact with the type II
TGF
receptor (TGF
RII) when TRAP is present in the cytosol. These
data are presented in Fig. 7. These
experiments utilized a His-tagged TRIP-1 (His-TRIP) and a GST-tagged
TRAP (GST-TRAP). Detection of His-TRIP was performed with anti-His antibodies and detection of the TGF
RII and Smad 2 were performed with commercially available antibodies. For these studies, all cells
were transfected with the TGF
RII. In the first experiment we
co-transfected these cells with His-TRIP for 18 h, and then exposed them to exogenously added GST-TRAP for 12 h. The cells were lysed and the lysate incubated with glutathione beads. The beads
were extensively washed, and the proteins interacting with the beads
were analyzed with Western analysis. Fig. 7 demonstrates that if the
cells were not exposed to GST-TRAP (column A) or they were
not co-transfected with His-TRIP (column C) neither TRIP, TGF
RII, nor Smad 2 can be detected. However, when both His-TRIP and
GST-TRAP were present the complex of the TGF
RII, His-TRIP, and Smad
2 was be extracted and detected (column B). These data provide evidence that in osteoblasts exogenously added TRAP can interact with cytosolic TRIP and that this complex associates with the
type II TGF
receptor and Smad 2.

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Fig. 7.
Demonstration of a TRIP-1,
TGF type II receptor, Smad2 complex with
TRAP. 293T cells were transfected with TGF RII. Some of these
cells were then co-transfected with a His-tagged TRIP-1 (His-TRIP-1).
In lane A, in which both the receptor and TRIP-1 were
present, but GST-TRAP was not added, no parts of the complex could be
pulled-down with glutathione-coated beads. The same was true if
His-TRIP-1 was not transfected into the cells. However, if the
receptor, TRIP-1 and GST-TRAP were all present, a protein complex
composed of the receptor, TRIP-1 and Smad 2 could be extracted with
glutathione-coated beads. These data indicate that TRAP, TRIP-1, the
TGF RII, and Smad2 can be found in close association with each
other.
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As a final test for the ability of osteoblasts to differentiate within
osteoclast lacunae containing TRAP, we cultured osteoblasts on cortical
bovine bone wafers on which we had previously created resorption
lacunae with authentic osteoclasts. The lacunae have been shown to
contain substantial amounts of TRAP (27). After 7-10 days of culture,
only osteoblasts residing within the lacunae had differentiated to the
point of producing histochemically detectable alkaline phosphatase
(Fig. 8). This model effectively
recapitulates the bone remodeling process in vitro and
verifies that osteoblasts can express a more differentiated phenotype
when exposed to molecules within osteoclast lacunae.

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Fig. 8.
Osteoblast differentiation within osteoclast
lacunae. Osteoclast lacunae were created by culturing osteoclasts
on cortical bone wafers in the presence of parathyroid hormone and
vitamin D. The osteoclasts were removed by gentle scraping. The margin
of the lacunae are visible in panel A. Osteoblasts were then
cultured on these wafers for 10 days, and alkaline phosphatase-positive
cells were identified with histochemical methods. Only the osteoblasts
residing within the lacunae demonstrated high levels of alkaline
phosphatase (panel B). The bar indicates a
distance of 100 microns.
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 |
DISCUSSION |
Maintenance of trabecular bone architecture is necessary to
withstand mechanical forces and stresses and to resist skeletal fractures. Mechanical properties of bone (reviewed in Ref. 28), microcracks (29), endocrine regulators (reviewed in Ref. 30), and
remodeling events are all likely factors involved in controlling the
amount of bone at any particular skeletal site. In the present work we
provide evidence that a non-traditional paracrine factor (i.e. TRAP) can act as a differentiating agent for
osteoblasts. Moreover, TRAP appears to participate in the spatial
orientation of where bone is formed during skeletal remodeling.
Our data demonstrate that the type V osteoclast tartrate resistant acid
phosphatase can activate the TGF
signaling pathway in osteoblasts to
enhance their differentiation. The interaction between TRAP and a
previously described, but not well characterized molecule, TRIP-1,
allows for activation of Smad signaling through the type II TGF
receptor. We show that exogenous TRAP can bind to TRIP-1 with high
affinity in the cytosol of osteoblasts. This complex then induces the
cells to differentiate into a more mature phenotype. As TRAP is present
in high concentrations on the surface of resorption lacunae, this may
be one mechanism by which bone formation can be directed to sites of
prior osteoclastic activity. Thus, we hypothesize that TRAP
participates in the spatial control of trabecular bone architecture.
The experiments reported under "Results" were performed with two
sources of TRAP. One was from a purified protein extraction. This
molecule would presumably be modified by post translational reactions.
The second source of TRAP was material we created as a GST fusion
protein. Both sources of TRAP behaved similarly in our binding and
activity assays. This finding argues that it is likely to be a specific amino acid sequence in the TRAP molecule that binds to TRIP-1 rather
than a post-translationally added carbohydrate moiety.
The idea of a site-directing mechanism for cell differentiation is not
a new concept. A number of properties of an extracellular matrix have
been implicated in controlling cell function. Molecules such as
fibronectin, osteopontin, laminin, the selectins and others have long
been recognized in controlling cell activity. However, TRAP, would be
considered a novel molecule in this regard because of its lysosomal
nature and its site-specific selectivity in bone. Nevertheless, TRAP is
perfectly positioned for this purpose. A hallmark of osteoclast bone
resorption is the creation of a resorption space between the cell and
the bone surface. Protons are actively pumped into this compartment
along with a varied list of lysosomal enzymes. The low pH is
responsible for mineral dissolution and the enzymes are responsible for
collagen degradation. Some investigators have likened this compartment
to an extracellular lysosome. However, when the osteoclast migrates to
another site or undergoes apoptosis, the return of pH to physiological
levels within the lacunae inactivates the enzymes and stops mineral
mobilization. Yet the enzymes remain adhered to the surface of the
resorption lacuna. Our findings suggest that one of these enzymes,
TRAP, may participate in subsequent bone formation.
TRIP-1 has been characterized as a modulator of the TGF
response
(16). It is a WD40 repeat-containing protein that is a phosphorylation
substrate for the type II TGF
receptor. TRIP-1, when phosphorylated,
represses TGF
-driven reporter activity from the plasminogen
activator inhibitor-1 (PAI-1) promoter but has no effect on the
TGF
-driven cyclin A promoter (16). Our data indicate that when TRAP
associates with TRIP-1, there is an activation of TGF
signaling.
Although the mechanistic pathway by which this occurs is not known at
this time, it is possible that the TRAP/TRIP-1 association may block
phosphorylation and allow a full expression of the TGF
signaling
pathway. TRIP-1 homologs have also been identified in plants. Their
function in these systems is not known, however, speculation that they
may be involved in cell cycle activity is supported by their similarity
to a translation initiation factor (31).
The role of TRAP in skeletal cell signaling is a novel concept. We have
known for a number of years that mice deficient in osteoclast TRAP
demonstrate skeletal abnormalities (32). As expected, TRAP-null mice
have a compromised ability to resorb bone through defective osteoclast
activity. This is manifested as a mild osteopetrosis, i.e.
skeletal density is slightly increased. However, a closer examination
of the bone reveals a haphazard and disorganized microarchitecture.
This is exactly what would be predicted if osteoblastic bone formation
was not targeted to sites of prior bone resorption. Additional evidence
that removal of TRAP and other site-directing signals leads to
inappropriate bone formation has been found at sites of inflammation
and infection in bone, such as in periodontal disease (33). Bacterial
and inflammatory cell activity at alveolar bone sites could very well destroy site-directing signals and prevent normal osteoblast function. Thus, in summary, our data support the concept that specific signals are involved in directing bone formation during the remodeling process
and that these signals may be molecules deposited by osteoclasts. Disruption of the site-directing signal would lead to a disorganized type of bone cell activity.
 |
ACKNOWLEDGEMENTS |
We thank Janet Cushing for technical support
and Dr. R. H. Chen for providing the TRIP-1 plasmids.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants RO1 DE 12011 and RO1 ES 08121 (to J. E. P.).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
Orthopaedics, 601 Elmwood Ave., University of Rochester School of
Medicine and Dentistry, Rochester, NY 14642. Tel.: 585-275-7664; Fax:
585-756-4727; E-mail: edward_puzas@urmc.rochester.edu.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M208292200
 |
ABBREVIATIONS |
The abbreviations used are:
TRAP, type V
tartrate-resistant acid phosphatase;
TRIP, TGF
receptor-interacting
protein;
ELISA, enzyme-linked immunosorbent assay;
PBS, phosphate-buffered saline;
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
HRP, horseradish
peroxidase;
GST, glutathione S-transferase;
OPG, osteoprotegerin.
 |
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