A Phage Display Technique Identifies a Novel Regulator of Cell Differentiation*

Tzong-Jen SheuDagger , Edward M. SchwarzDagger , Daniel A. Martinez§, Regis J. O'KeefeDagger , Randy N. RosierDagger , Michael J. ZuscikDagger , and J. Edward PuzasDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 TGFbeta receptor-interacting protein (TRIP-1). Our data demonstrate that TRAP activation of TRIP-1 evokes a TGFbeta -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 TGFbeta 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (TGFbeta receptor-interacting protein, TRIP-1) possess very high affinity for TRAP and is poised for activating the TGFbeta differentiation pathway in osteoblasts. TRIP-1 has been previously described in other cell types and has been shown to modulate TGFbeta signaling in both a stimulatory and inhibitory fashion (16, 17). In osteoblast systems TGFbeta signaling pathways control osteoblast differentiation (18-23). We show that this effect is modulated by the interaction of TRAP with TRIP-1.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Osteoblast differentiation at sites of bone remodeling is mediated by a number of regulatory factors. One of the key factors is TGFbeta . In most systems, ours included, TGFbeta is known to be a potent enhancer of the osteoblast phenotype (22, 23). Fig. 1 shows that in the presence of TGFbeta 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 TGFbeta on osteoblast phenotypic markers. The qualitative pattern of stimulation by TRAP and TGFbeta for markers of osteoblast differentiation is similar. The concentration of TRAP was 10 µg/ml and for TGFbeta 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.

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.

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.

We next sought to determine if TRAP could activate one of the TGFbeta signaling pathways. The data in Fig. 5 show that both TGFbeta and TRAP can strongly upregulate a reporter gene (P3TP-Lux) that is sensitive to the TGFbeta regulatory Smads 2 and 3. The effects of TGFbeta and TRAP are additive. As TRIP-1 is known to interact with the type II TGFbeta receptor we investigated whether TRAP activation of this pathway could be blocked in the presence of a dominant negative type II TGFbeta receptor expression vector. Fig. 5 also demonstrates that in cells that have been co-transfected with a dominant negative type II TGFbeta receptor we can block both TGFbeta 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 TGFbeta 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 TGFbeta (5 ng/ml) causes an activation of the Smad pathway. The effect of TRAP plus TGFbeta are additive. Co-transfection of the cells with a dominant negative TGFbeta type II receptor blocks all TRAP and TGFbeta 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).

When these experiments were repeated in a Smad4-deficient cell type (SW408 cells), neither TGFbeta 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 TGFbeta and TRAP signaling (Fig. 6). Thus, all of these pieces of evidence point to the activation of the TGFbeta /Smad pathway through the association of TRAP with TRIP-1.


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Fig. 6.   TRAP and TGFbeta 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 TGFbeta (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.

We also have direct evidence that TRIP-1 will interact with the type II TGFbeta receptor (TGFbeta 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 TGFbeta RII and Smad 2 were performed with commercially available antibodies. For these studies, all cells were transfected with the TGFbeta 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, TGFbeta RII, nor Smad 2 can be detected. However, when both His-TRIP and GST-TRAP were present the complex of the TGFbeta 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 TGFbeta receptor and Smad 2. 


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Fig. 7.   Demonstration of a TRIP-1, TGFbeta type II receptor, Smad2 complex with TRAP. 293T cells were transfected with TGFbeta 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 TGFbeta RII, and Smad2 can be found in close association with each other.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 TGFbeta 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 TGFbeta 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 TGFbeta response (16). It is a WD40 repeat-containing protein that is a phosphorylation substrate for the type II TGFbeta receptor. TRIP-1, when phosphorylated, represses TGFbeta -driven reporter activity from the plasminogen activator inhibitor-1 (PAI-1) promoter but has no effect on the TGFbeta -driven cyclin A promoter (16). Our data indicate that when TRAP associates with TRIP-1, there is an activation of TGFbeta 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 TGFbeta 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, TGFbeta receptor-interacting protein; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; HRP, horseradish peroxidase; GST, glutathione S-transferase; OPG, osteoprotegerin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Parfitt, A. M. (1983) in Bone Histomorphometry: Techniques and Interpretation. (Recker, R. R., ed) , pp. 143-223, CRC Press, Boca Raton, FL
2. Frost, H. M. (1966) J. Bone Joint Surg. 48, 1192-1203[Medline] [Order article via Infotrieve]
3. Harris, W. H., and Heaney, R. P. (1969) N. Eng. J. Med. 280, 253-259[Medline] [Order article via Infotrieve]
4. Harris, W. H., and Heaney, R. P. (1969) N. Eng. J. Med. 280, 303-311[Medline] [Order article via Infotrieve]
5. Jones, S. J., Gray, C., and Boyde, A. (1994) Anat. Embryol. 190, 339-349[Medline] [Order article via Infotrieve]
6. Gray, C., Boyde, A., and Jones, S. J. (1996) Bone 18, 115-123[CrossRef][Medline] [Order article via Infotrieve]
7. Schwartz, Z., Lohmann, C. H., Vocke, A. K., Sylvia, V. L., Cochran, D. L., Dean, D. D., and Boyan, B. D. (2001) J. Biomed. Mat. Res. 56, 417-426[CrossRef][Medline] [Order article via Infotrieve]
8. Boyan, B. D., Lohmann, C. H., Sisk, M., Liu, Y., Sylvia, V. L., Cochran, D. L., Dean, D. D., and Schwartz, Z. (2001) J. Biomed. Mat. Res. 55, 350-359[CrossRef][Medline] [Order article via Infotrieve]
9. Deligiann, D. D., Katsala, N. D., Koutsoukos, P. G., and Missirlis, Y. F. (2001) Biomaterials 22, 87-96[CrossRef][Medline] [Order article via Infotrieve]
10. Schwartz, Z., Lohmann, C. H., Oefinger, J., Bonewald, L. F., Dean, D. D., and Boyan, B. D. (1999) Adv. Dental Res. 13, 38-48[Abstract]
11. Hayman, A. R., Bune, A. J., and Cox, T. M. (2000) J. Anat. 196, 433-441[CrossRef][Medline] [Order article via Infotrieve]
12. Tiffee, J. C., and Aufdemorte, T. B. (1997) J. Oral Maxillofac. Surg. 55, 1108-1112[Medline] [Order article via Infotrieve]
13. Baron, R., Neff, L., Brown, W., Courtoy, P. J., Louvard, D., and Farquhar, M. G (1988) J. Cell Biol. 106, 1863-1872[Abstract]
14. Wergedal, J. E., and Baylink, D. J. (1969) J. Histochem. Cytochem. 17, 799-806[Medline] [Order article via Infotrieve]
15. Yamamoto, T., and Nagai, H. (1992) J. Bone Miner. Res. 7, 1267-1273[Medline] [Order article via Infotrieve]
16. Choy, L., and Derynck, R. (1998) J. Biol. Chem. 273, 31455-31462[Abstract/Free Full Text]
17. Chen, R. H., Miettinen, P. J., Maruoka, E. M., Choy, L., and Derynck, R. (1995) Nature 377, 548-552[CrossRef][Medline] [Order article via Infotrieve]
18. Kassem, M., Kveiborg, M., and Eriksen, E. F. (2000) Eur. J. Clin. Invest. 30, 429-437[CrossRef][Medline] [Order article via Infotrieve]
19. Yamada, T., Kamiya, N., Harada, D., and Takagi, M. (1999) Histochem. J. 31, 687-694[CrossRef][Medline] [Order article via Infotrieve]
20. Chung, C. Y., Iida-Klein, A., Wyatt, L. E., Rudkin, G. H., Ishida, K,., Yamaguchi, D. T., and Miller, T. A. (1999) Biochem. Biophys. Res. Commun. 265, 246-251[CrossRef][Medline] [Order article via Infotrieve]
21. Cheifetz, S., Li, I. W., McCulloch, C. A., Sampath, K., and Sodek, J. (1996) Connect. Tissue Res. 35, 71-78[Medline] [Order article via Infotrieve]
22. Harris, S. E., Bonewald, L. F., Harris, M. A., Sabatini, M., Dallas, S,., Feng, J. Q., Ghosh-Choudhury, N., Wozney, J., and Mundy, G. R. (1994) J. Bone Miner. Res. 9, 855-863[Medline] [Order article via Infotrieve]
23. Bonewald, L. F., Schwartz, Z., Swain, L. D., and Boyan, B. D. (1992) Bone Miner. 17, 139-144[Medline] [Order article via Infotrieve]
24. Ling, P., and Roberts, R. M. (1993) J. Biol. Chem. 268, 6896-6902[Abstract/Free Full Text]
25. Martinez, D. A., Zuscik, M. J., Ishibe, M., Rosier, R. N., Romano, P. R., Cushing, J. E., and Puzas, J. E. (1995) J. Cell. Biochem. 59, 246-257[Medline] [Order article via Infotrieve]
26. Ionescu, A. M., Schwarz, E. M., Vinson, C., Puzas, J. E., Rosier, R., Reynolds, P. R., and O'Keefe, R. J. (2001) J. Biol. Chem. 276, 11639-11647[Abstract/Free Full Text]
27. Sheu, T. J., Schwarz, E. M., O'Keefe, R. J., Rosier, R. N., and Puzas, J. E. (2002) J. Bone Miner. Res. 17, 915-922[Medline] [Order article via Infotrieve]
28. Bikle, D. D., and Halloran, B. P. (1999) J. Bone Miner. Metab. 17, 233-244[CrossRef][Medline] [Order article via Infotrieve]
29. Hirano, T., Turner, C. H., Forwood, M. R., Johnston, C. C., and Burr, D. B. (2000) Bone 27, 13-20[Medline] [Order article via Infotrieve]
30. Seeman, E., and Delmas, P. D. (2001) Trends Endocrinol. Metab. 12, 281-283[CrossRef][Medline] [Order article via Infotrieve]
31. Jiang, J., and Clouse, S. D. (2001) Plant J. 26, 35-45[CrossRef][Medline] [Order article via Infotrieve]
32. Hayman, A. R., Jones, S. J., Boyde, A., Foster, D., Colledge, W. H., Carlton, M. B., Evans, M. J., and Cox, T. M. (1996) Development. 122, 3151-3162[Abstract/Free Full Text]
33. Romano, P. R., Caton, J. G., and Puzas, J. E. (1997) J. Periodontal Res. 32, 143-147[Medline] [Order article via Infotrieve]
34. Peterkofsky, B., and Diegelmann, R. (1971) Biochemistry 10, 988-994[Medline] [Order article via Infotrieve]


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