Granulocyte Macrophage-Colony Stimulating Factor Reciprocally Regulates {alpha}v-Associated Integrins on Murine Osteoclast Precursors

Masaru Inoue, Noriyuki Namba, Jean Chappel, Steven L. Teitelbaum and F. Patrick Ross

Department of Pathology Washington University School of Medicine St. Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The integrins {alpha}vß5 and {alpha}vß3 are expressed reciprocally during murine osteoclastogenesis in vitro. Specifically, immature osteoclast precursors, in the form of bone marrow macrophages, contain exclusively {alpha}vß5, surface expression of which declines with commitment to the osteoclast phenotype, while levels of {alpha}vß3 increase concomitantly. The distinct functional significance of {alpha}vß5 is underscored by the integrin’s capacity, unlike {alpha}vß3, to mediate both attachment and spreading on ligand, of marrow macrophages, suggesting {alpha}vß5 negotiates initial recognition, by osteoclast precursors, of bone matrix. Northern analysis demonstrates changes in the two ß-subunits, and not {alpha}v, are responsible for these alterations. Treatment of early precursors with granulocyte-macrophage colony stimulating factor (GM-CSF) leads to alterations in ß3 and ß5 mRNA and {alpha}vß5 and {alpha}vß3, paralleling those occurring during osteoclastogenesis. Nuclear run-on and message stability studies demonstrate that while GM-CSF treatment of precursors alters ß5 transcriptionally, the changes in ß3 arise from prolonged mRNA t1/2. Similar to GM-CSF treatment, the rate of ß5 transcription falls during authentic osteoclastogenesis. In contrast to cytokine-induced {alpha}vß3, however, that attending osteoclastogenesis reflects accelerated transcription of the ß3-subunit. Thus, while GM-CSF may participate in modulation of {alpha}vß5 during osteoclast differentiation, signals other than those derived from the cytokine must regulate expression of {alpha}vß3.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The osteoclast is a multinucleated cell whose ability to degrade bone requires matrix attachment. Commitment to the osteoclast phenotype, by mononuclear precursors, involves substrate recognition, an event mediated largely by integrins. Integrins are a family of membrane-spanning, cell surface receptors consisting of noncovalently bound {alpha}- and ß-subunits which mediate cell-matrix binding. After attachment, integrins also transmit extracellular signals, thereby regulating intracellular events, including cytoskeletal reorganization, migration, proliferation, differentiation, and coagulation (1, 2, 3).

The {alpha}v-, ß1-, or ß2-subunits are the promiscuous components of integrin families, each combining with multiple partners, thereby generating heterodimers that effect cell anchoring (1). While mature osteoclasts express ß1- and {alpha}v-integrins (4, 5), it is the heterodimer {alpha}vß3 that plays a major functional role in these cells. Thus, a series of in vitro and in vivo experiments involving antibodies (5, 6), disintegrins (7, 8), peptides containing the integrin recognition motif Arg-Gly-Asp, RGD (9, 10, 11), or a small peptidomimetic (12) indicate that {alpha}vß3-blockade inhibits osteoclastic bone resorption. In fact, the capacity of an RGD peptide mimetic capable of recognizing {alpha}vß3 to arrest bone loss in the oophorectomized rat (12) raises the possibility that osteoclast integrin blockade may prevent postmenopausal osteoporosis.

Osteoclasts are polykaryons derived from macrophages, the latter expressing the two closely related integrins, {alpha}vß3 and {alpha}vß5 (13, 14). Given the homology of their protein sequences, it is not surprising that {alpha}vß3 and {alpha}vß5 share a number of matrix ligands (1), including the RGD-containing proteins, vitronectin, osteopontin, and bone sialoprotein, all present in bone (15). Since osteoclasts derive from cells known to express the two {alpha}v-integrins, we examined expression of {alpha}vß3 and {alpha}vß5 in a murine model of osteoclastogenesis. We find {alpha}vß5 to be the sole {alpha}v-integrin on immature precursor cells and that this heterodimer is replaced by {alpha}vß3 during osteoclast differentiation. Furthermore, the two integrins serve separate functions in osteoclast precursors attached to the RGD-containing protein vitronectin. While {alpha}vß5 mediates both attachment and spreading, {alpha}vß3 regulates only attachment. Finally, the changes in {alpha}v-integrin expression during osteoclastogenesis are mimicked by treatment of precursors with the hematopoietic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). While the molecular mechanism by which GM-CSF diminishes ß5 mirrors that occurring during osteoclastogenesis, the pathways by which the cytokine enhances ß3 mRNA differs from that occurring in osteoclastogenic culture, an indication that molecules other than GM-CSF regulate {alpha}vß3 on bone-resorptive cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
mRNA Levels of the ß3- and ß5-Integrin Subunits Vary Reciprocally during Osteoclastogenesis
We examined expression of {alpha}v-integrins during murine osteoclastogenesis, using an in vitro system wherein osteoclast precursors, supported by the mouse stromal line ST-2, differentiate, with time, into functional osteoclasts. Most importantly, the macrophage-derived population is easily separated from stromal cells, during culture (16), thereby permitting analysis of integrin expression on osteoclast precursors as they differentiate. Northern analysis reveals that early osteoclast precursors express almost exclusively ß5 mRNA, with ß3 message virtually undetectable (Fig. 1Go). With increasing time of coculture [and with a concomitant rise in the number of osteoclasts formed (16)], levels of ß3 mRNA rise, while those of ß5 decline.



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Figure 1. Levels of mRNA for the Integrin ß3 and ß5 Vary Reciprocally during Murine Osteoclastogenesis

Adherent bone marrow macrophages (osteoclast precursors) were cultured for varying periods of time with the stromal line ST-2 in the presence of 1,25-(OH)2D3 and dexamethasone. Stromal cells were totally removed by collagenase digestion, and total RNA was extracted from the remaining adherent cells (osteoclasts and their precursors), electrophoresed, and transferred to nylon membranes. Northern analysis was performed with a murine ß5 CDNA. The same membranes were stripped and reprobed with ß3 cDNA and an oligomer specific for 18S ribosomal RNA, to show equal loading.

 
GM-CSF Reciprocally Regulates Expression of ß3- and ß5-Integrin Subunit mRNAs
To identify molecules that may play a role in regulating ß3 and/or ß5 expression, osteoclast precursors were treated with a range of hematopoietic cytokines. GM-CSF decreases steady-state ß5 mRNA (Fig. 2Go). Several cytokines, such as interleukin-3 (IL-3) or IL-6, are not as effective, while others fail to alter ß5 mRNA levels.



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Figure 2. Effects of Different Cytokines on ß5 mRNA Levels in Murine Osteoclast Precursors

Cells were stimulated by cytokines for 16 h, and total RNA was analyzed by Northern blot using a murine ß5 cDNA probe. Cells were treated with vehicle (C), IL-1ß (5 ng/ml), IL-3 (300 U/ml), IL-4 (50 U/ml), IL-6 (1000 U/ml), IL-8 (100 ng/ml), IL-11 (10 ng/ml), GM-CSF (10 ng/ml), ciliary neurotrophic factor (CNTF, 10 ng/ml) or oncostatin M (OSM, 20 ng/ml).

 
To characterize further the role of GM-CSF in regulating expression of {alpha}v-integrins, we examined the time course for changes in both ß3 and ß5 mRNA after treatment with a single concentration (10 ng/ml) of the cytokine. As shown in Fig. 3AGo, ß5 mRNA begins to decrease between 4 and 8 h and reaches its nadir after 48 h. In contrast to ß5, ß3 mRNA is detectably increased after 48 h of GM-CSF treatment and maximizes at 96 h.



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Figure 3. GM-CSF Reciprocally Alters ß3 and ß5 mRNA Levels in Osteoclast Precursors in a Time- and Dose-Dependent Manner

A, Adherent bone marrow macrophages were stimulated by a single addition of 10 ng/ml GM-CSF. Total RNA was isolated at the indicated times and analyzed by Northern blot. B, Adherent bone marrow macrophages were stimulated with the indicated doses of GM-CSF. After 96 h, total RNA was isolated and analyzed by Northern blot. The membranes were stripped and rehybridized with {alpha}v, ß3, and 18S probes.

 
Osteoclast precursors were treated with varying concentrations of GM-CSF and, after 96 h, analyzed for alterations of ß5 and ß3 mRNA. Levels of both subunits respond in a dose-dependent manner, with the effective minimum GM-CSF concentration required for change in both integrin subunits mRNAs being the same, namely, 0.3 ng/ml. In contrast to ß3 and ß5, {alpha}v is not substantially regulated by GM-CSF (Fig. 3BGo).

GM-CSF-Induced Changes in ß5 and ß3 mRNA Are Paralleled by Surface Expression of the Integrins {alpha}vß3 and {alpha}vß5
Having documented the reciprocal effect of GM-CSF on ß5 and ß3 mRNA levels, we asked whether these events are paralleled by surface expression of {alpha}vß5 and {alpha}vß3. Cells were surface labeled with 125I, lysed, and immunoprecipitated with antibodies that recognize ß5 or ß3 specifically. Reflecting the levels of mRNA, control cells express abundant {alpha}vß5 and little {alpha}vß3 (Fig. 4Go). GM-CSF induces surface expression of {alpha}vß3 while reducing that of {alpha}vß5. While the size of the upper band, about 150 kDa, is consistent with that for {alpha}v, the two associated ß-chains differ significantly, with ß3 being smaller than ß5, which displays the expected size of about 95 kDa.



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Figure 4. GM-CSF Reciprocally Regulates Surface Expression of the Integrins {alpha}vß3 and {alpha}vß5 on Cultured Bone Marrow Macrophages

Cells treated for 4 days with or without GM-CSF were surface radioiodinated and lysates immunoprecipitated with a monoclonal antibody to ß3 or a polyclonal rabbit antibody to ß5. The immune complexes were analyzed by SDS-PAGE.

 
The Integrin {alpha}vß5 Is Functional in Osteoclast Precursors
Regardless of the mechanism of expression, we find {alpha}vß5 to be the dominant {alpha}v-integrin on osteoclast precursors. However, in contrast to the resorptive importance of {alpha}vß3, the functional role, if any, of {alpha}vß5 in development of the osteoclast phenotype is unknown. We examined this issue by performing both attachment and spreading assays, using marrow macrophages whose predominant integrin is either {alpha}vß3 or {alpha}vß5. To obtain these respective populations, immature osteoclast precursors were cultured, nonadherent (Teflon beakers), for 3 days and treated with either vehicle (yielding cells expressing {alpha}vß5) or 10 ng/ml GM-CSF (in which case {alpha}vß3 is the dominant {alpha}v-integrin; see Fig. 4Go). As determined by Northern analysis, mRNA levels of ß3 and ß5 are similar under conditions of adherence or nonadherence (data not shown). Cells bearing one or other {alpha}v-integrin were allowed to adhere for 45 min to plates coated with either vitronectin or type 1 collagen. Cells expressing either {alpha}vß3 or {alpha}vß5 attached equally well to vitronectin, but neither cell type exhibited significant collagen binding (Fig. 5Go). These results indicate that integrins are capable of mediating attachment to the RGD-containing matrix protein vitronectin, but only those cells expressing {alpha}vß5 (i.e. untreated with GM-CSF) spread, an event inhibited by a peptidomimetic recognizing both {alpha}v-integrins (12) (Fig. 6Go).



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Figure 5. Macrophages Expressing Either {alpha}vß5 or {alpha}vß3 Attach to Vitronectin, but not Collagen

Macrophages were isolated and cultured in Teflon beakers with or without GM-CSF. Cells were added to plates coated with vitronectin or collagen I. After 45 min, plates were washed, stained with methylene blue and the cell-associated dye was solubilized. The absorbance of the wells was read at 650 nm.

 


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Figure 6. Spreading of Macrophages on Vitronectin Is Mediated by {alpha}vß5, and not {alpha}vß3, and Inhibited by an {alpha}v Antagonist

Cells were cultured in 48-well plates for 3 days with or without GM-CSF. Selected wells were treated with the {alpha}v antagonist SC56331. Wells were washed, fixed, and photographed with a standard microscope.

 
GM-CSF Regulates ß5 and ß3 mRNA Levels by Different Mechanisms
To determine the molecular mechanism by which GM-CSF alters ß-integrin subunit mRNA, we performed both nuclear run-on and message stabilization studies. Nuclei from control or GM-CSF-treated cells were isolated and used to measure ß3- and ß5-gene transcription in vitro. The cytokine decreases the rate of ß5 transcription, but has no effect on that of ß3 (Fig. 7Go). Given the failure of GM-CSF to impact ß3 transcription in the face of increased steady-state ß3 mRNA, we measured the cytokine’s impact on ß3 message stability, using actinomycin D as a specific inhibitor of RNA polymerase ll. As seen in Fig. 8Go, GM-CSF increases ß3 mRNA t1/2 from 6.5 to 38 h.



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Figure 7. GM-CSF Alters the Rate of Transcription of the ß5, but not ß3, Gene

Nuclei isolated from control or GM-CSF-treated cells were used for in vitro transcription in the presence of [32P]UTP. Transcripts containing equal amounts of trichloroacetic acid-precipitable counts were hybridized to excess ß3, ß5, GAPDH and empty vector cDNAs immobilized on a nylon membrane.

 


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Figure 8. GM-CSF Treatment Leads to Increased Stability of ß3 mRNA

Cells treated with or without GM-CSF for 4 days. At this time actinomycin D (5 µg/ml final concentration) was added to inhibit transcription of mRNA. After 0, 4, 8, and 12 h, total RNA was extracted and analyzed by Northern blot. The intensity of the ß3 specific band was normalized to the level of the 18S ribosomal RNA band. Results are expressed as the percentage of the normalized ß3 mRNA values obtained before the addition of inhibitor. The t1/2 of ß3 in untreated cells is 6.5 h, while that in cells treated with GM-CSF is considerably longer (assuming linear extrapolation of the experimental data, the value would be 38 h).

 
Transcription of the ß5 and ß3 Genes Is Altered during Osteoclastogenesis
To delineate the mechanisms by which steady-state levels of ß3 and ß5 mRNAs alter during in vitro osteoclastogenesis, we once again turned to nuclear run-on and mRNA stability studies. In this instance, we compared generated osteoclasts, purified by collagenase treatment, to a homogenous population of bone marrow macrophages maintained in the absence of GM-CSF and ST2 stromal cells, the latter essential for osteoclast differentiation. Similar to GM-CSF-treated cells (Fig. 8Go) the rate of ß5 transcription in cells undergoing osteoclast differentiation, normalized to that of the housekeeping gene, GAPDH, is 5.4-fold less than that occurring in untreated macrophages. In contrast to the stabilization of ß3-mRNA induced by the cytokine, no such alteration occurs with osteoclastogenesis, in which ß3-message is enhanced by a 2-fold increase in transcription (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reflecting development of experimental models in which osteoclasts may be isolated or generated in culture, a number of insights have been gained into the mechanisms by which these cells resorb bone. Among those molecules identified as crucial to the resorptive process is the {alpha}vß3-integrin. In osteoclasts, the heterodimer mediates attachment to bone matrix (5, 18), generation of matrix-derived intracellular signals (19, 20, 21, 22), and cytoskeletal reorganization (23). Thus, regulation of this heterodimer is an issue of importance. In contrast to {alpha}vß3, however, little is known concerning the function of {alpha}vß5 in osteoclast biology.

Our initial observation of reciprocal {alpha}vß3 and {alpha}vß5 expression in a murine model of osteoclast formation prompted us to determine whether any of a wide range of cytokines mediates changes in integrin appearance during precursor differentiation. While other cytokines are capable of decreasing ß5-mRNA, only GM-CSF, which is produced by macrophages and both endothelial and stromal cells (24), all components of the bone marrow, prompts a simultaneous rise in ß3. Our data demonstrate GM-CSF alters {alpha}vß3 and {alpha}vß5 expression in osteoclast precursors by different mechanisms. Whereas changes in ß5 mRNA levels, and hence expression of {alpha}vß5, are rapid and transcription dependent, those relating to {alpha}vß3 are slow and reflect enhanced ß3 mRNA stability. Similar to GM-CSF, osteoclastogenesis is accompanied by a decline in ß5 transcription. In contrast to marrow macrophages exposed to the cytokine, however, enhancement of ß5 in osteoclast formation is the product not of ß3 message stabilization, but of accelerated transcription. Thus, while GM-CSF is a candidate regulator of {alpha}vß5 in murine osteoclast precursors as they differentiate, other molecules must stimulate surface appearance of {alpha}vß3.

The integrins {alpha}vß3 and {alpha}vß5, while structurally related, appear to serve different functions, which may also be cell type specific. This is true in cells as diverse as human foreskin fibroblasts (25), smooth muscle cells (26), pancreatic carcinoma cells (27), hamster melanoma cells (28), or rabbit mesothelial cells (29). Finally, and of greater relevance, on human monocytes, the integrin {alpha}vß5 is involved in spreading on vitronectin, while {alpha}vß3 plays a role in migration on the same substrate (13).

Essential components of the osteoclast phenotype include multinucleation and formation of the cell’s resorptive organelle, its ruffled membrane. Because induction of both features requires cell matrix recognition, the manner in which precursors interact with substrate is critical to their progression into the mature resorptive cell (30). We find that osteoclast precursors expressing {alpha}vß5, but not {alpha}vß3, can both bind and spread on the RGD-containing protein vitronectin, and that these processes are blocked by a small molecule {alpha}v-integrin antagonist. Given that early osteoclast precursors express only {alpha}vß5, this integrin may play an important role in their initial attachment of precursors to bone matrix, an event critical to subsequent differentiation and fusion. Furthermore, our data indicate that these two integrins are expressed at different stages of osteoclast maturation. These findings, when taken together, suggest that, as in other cells, {alpha}vß5 and {alpha}vß3 play separate roles in osteoclasts and/or their precursors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation and Culture of Osteoclast Precursors
Except for cell attachment studies, which required use of cells in suspension, all other experiments were performed using adherent bone marrow macrophages, generated by culture of nonadherent precursors. These procedures have been reported elsewhere in detail (16, 31). Briefly, bone marrow was flushed from the tibiae and femurs of 6-week-old male C3H mice with {alpha}-modified Eagles medium ({alpha}-MEM). Cells were cultured for 24 h in {alpha}-10 ({alpha}-MEM supplemented with 10% FBS) in the presence of 500 U/ml macrophage colony stimulating factor (M-CSF). The nonadherent population was collected and mononuclear cells were isolated by Ficoll-hypaque gradient centrifugation (500 x g, 15 min). The macrophage-enriched population was recovered by centrifugation (500 x g, 7 min) and cultured for 3–4 days at 5 x 106 cells per 100-mm plates in {alpha}-10 supplemented daily with 500 U/ml M-CSF. This procedure yields a pure population of M-CSF-dependent, adherent osteoclast precursors. To generate osteoclasts, adherent bone marrow macrophages and the murine stromal line ST-2 were cocultured at a ratio of 10:1. Cultures were fed every third day, at which time fresh steroids [10 nM 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and 100 nM dexamethasone] were added. Osteoclasts and their precursors were freed of ST-2 cells by treatment with 0.1% collagenase, 0.1% BSA in {alpha}-MEM for 2 h at 37 C. In selected studies the same adherent osteoclast precursors, maintained in the presence of 500 U/ml M-CSF throughout, were treated with or without the indicated doses of GM-CSF for varying periods. We have demonstrated (32) that the procedure described for isolation of osteoclasts, or their precursors, does not lead to detectable contamination by the ST2 cells used during the coculture period. Thus, our findings of alterations in ß5 mRNA levels cannot be attributed to the presence of other cell types and must reflect changes in ß5 expression in the osteoclast precursors themselves.

Mouse Integrin ß5 cDNA Cloning
A partial mouse integrin ß5 cDNA was generated by RT-PCR. Total RNA was isolated from 6-week-old mice kidney with TRIzol (GIBCO-BRL, Gaithersburg, MD), using the manufacturer’s instructions. Total RNA was reverse transcribed with a cDNA cycler kit (Invitrogen, San Diego, CA), using oligo dT as primer. To perform PCR, two oligonucleotides were synthesized for use as forward (5'-ATCCGGAGCCTGGGCACCAAGCT-3') and reverse (5'-CAGGAGAAGTTGTCGCACTCACA-3') primers. The sequences used were based on a short mouse integrin ß5 cDNA sequence (forward primer provided by Dr. Rick Brown, Department of Medicine, Washington University School of Medicine) and the published human integrin ß5 sequence (33). After denaturation for 3 min at 94 C denaturation, PCR was carried out for 40 cycles as follows: 94 C, 1 min; 50 C, 2 min; 72 C, 3 min 30 sec; followed by incubation at 72 C for 7 min. The PCR product was cloned directly into pCRII vector (TA cloning kit, Invitrogen) and sequenced at both ends using standard methods.

Northern Blot Analysis and mRNA Stability Assay
Osteoclast precursors were treated with either vehicle or cytokine (GM-CSF) for the indicated times. Total cellular RNA was isolated with TRIzol, and equal amounts (5 µg per lane) were electrophoresed in 1% agarose gel containing formaldehyde and transferred to Hybond N (Amersham, Arlington Heights, IL) with a vacuum blotter. The membrane was prehybridized (5x SSPE, 5x Denhardt’s solution, 50% formamide, 0.1% SDS, 1x Background Quencher (Tel-Test Inc., Friendswood TX)) for at least 2 h at 42 C. Northern analysis was performed using the partial mouse integrin ß5 cDNA, a mouse integrin ß3 cDNA, which was cloned in our laboratory (34), or human {alpha}v cDNA kindly provided Dr. Eric Brown, Washington University School of Medicine). All probes were 32P labeled by the random primer method, using a kit (Boehringer Mannheim, Indianapolis, IN). After 16 h hybridization, membranes hybridized with ß5 or ß3 cDNAs were washed with 1x SSPE, 0.1% SDS for 15 min at 50 C twice, 0.1x SSPE 0.1% SDS for 15 min at 50 C twice, while probing with {alpha}v cDNA was followed by washing with 2x SSPE 0.1% SDS for 20 min at room temperature twice, 2x SSPE 0.1% SDS for 20 min at 42 C. For reprobing, membranes were stripped by submerging in 0.1% SDS at 100 C. To normalize for RNA loading, blots were finally reprobed with an end-labeled oligonucleotide specific to 18S RNA, as previously described (35). For determination of mRNA stability, cells in 100-mm dishes were cultured with or without GM-CSF for 4 days. Actinomycin D (5 µg/ml) was added to all plates and total RNA was isolated 0, 4, 8, or 12 h later. To compare ß3-integrin subunit mRNA stability between osteoclasts and their precursors, cells were cultured for 7 days either alone in the presence of 500 U/ml M-CSF, or in coculture with ST2 cells and steroids. ST2 cells were removed from cocultures by collagenase treatment, and actinomycin D (5 µg/ml) was added to all cells. At zero time and 7 h later, total RNA was isolated for analysis. In all instances, 30 µg per lane of total RNA was fractionated in agarose, Northern analysis was performed using ß3 or ß5 cDNA, as appropriate, and the results were quantified with a phosphorimage analyzer, using levels of 18S and 28S RNA to normalize the data.

Nuclear Run-on Transcription Assay
For studies involving GM-CSF treatment, adherent bone marrow macrophages were cultured for 48 h with or without cytokine (10 ng/ml) and washed twice with ice-cold PBS. For comparison of the rates of gene transcription in osteoclast precursors and during osteoclastogenesis, the same adherent precursors were cultured for 7 days either alone, with the addition every third day of 500 U/ml M-CSF, or together with ST2 cells and optimal concentrations of 1,25-(OH)2D3 and dexamethasone (10-9 and 10-8 M, respectively). To isolate osteoclast-derived nuclei, ST2 cells were removed totally from cocultures by collagenase treatment. In all instances, isolation of nuclei and in vitro transcription were performed essentially as previously described (36). Briefly, cells were extracted with lysis buffer and nuclei were obtained by low-speed centrifugation. Isolated nuclei were resuspended in suspension buffer and mixed with a reaction mixture containing ATP, CTP, GTP, and [32P]UTP (3000 Ci/mmol). After collection of nuclei by centrifugation, RNA was isolated with TRIzol. Equal amounts of freshly transcribed RNA (determined by trichloroacetic acid-precipitable counts) were hybridized to denatured DNA [5 µg/slot murine ß5 or ß3 cDNA, vector DNA, or human GAPDH (purchased from CLONTECH, San Diego, CA)] in a slotblot device. After 48 h, membranes were washed with 1x SSPE 0.1% SDS for 20 min at 50 C, 0.1x SSPE 0.1% SDS for 20 min at 60 C twice and exposed to x-ray film. Quantitation was performed using a phosphorimage analyzer.

Immunoprecipitation of Surface-Expressed Integrins
Cells were washed with PBS and surface-labeled with 125I as described previously (36). Cells were lysed with a buffer containing 2% Renex 30, 10 mM Tris, pH 8.5, 150 mM NaCl, 1 mM CaCl2, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.02% NaN3. Each lysate, containing equal numbers of trichloroacetic acid-precipitable counts, was incubated with Gammabind (Pharmacia Biotech, Inc., Piscataway, NJ) and precleared again with Gammmabind plus whole rabbit serum (for ß5) or class-matched monoclonal antibody (for ß3). The polyclonal antibody was raised against the peptide sequence of the human ß5 cytoplasmic tail. Given the homology between the murine and human ß5 tail sequences (X. Feng, unpublished data), it is not surprising this antibody recognizes murine ß5. The precleared lysate was immunoprecipitated with a polyclonal rabbit anti-ß5-integrin subunit, kindly provided by Dr. Louis Reichardt, University of California, San Francisco, or hamster anti-ß3 integrin subunit (PharMingen, San Diego, CA). The immune complex was bound to excess Gammabind, the precipitate recovered by boiling the beads in electrophoresis sample buffer and subject to 7% SDS-PAGE) under nonreducing conditions. The gels were dried and subject to autoradiography.

Cell Attachment Assay
Ninety six-well microtiter plates were coated overnight at 4 C with collagen (10 µg/ml) or vitronectin (10 µg/ml). The plate was washed with PBS (BSA wells) or blocked with 1% BSA, followed by PBS washing (vitronectin wells) before use. To each well was added, in 100 µl of {alpha}-MEM supplemented with 0.1% BSA, a total of 4 x 105 cells, previously maintained in Teflon-coated plates with or without GM-CSF (10 ng/ml) for 96 h. Plates were incubated for 45 min at 37 C, after which they were rinsed three times with PBS. Cells were fixed with 2% formaldehyde for 20 min, rinsed twice with 10 mM borate buffer, pH 8.4, and stained with 1% methylene blue for 10 min. After washing three times with tap water, each well was filled with 100 µl of 0.1 M HCl and incubated for 1 h at room temperature, and absorbance at 650 nm was measured using a microtiter reader.

Cell Morphology
Cells were cultured in 48-well plates in the presence of 500 U/ml M-CSF. When cells reached subconfluency, wells were treated with GM-CSF (10 ng/ml) or vehicle (PBS). In selected wells a small integrin peptidomimetic (SC56331, Searle Corp., Skokie, IL), known to inhibit {alpha}v-integrin function (12), was added daily to a final concentration of 10 µM. After 3 days, cells were fixed with 2% paraformaldehyde and photographed with a light microscope.

Experimental Animals
All animal experimentation described herein was conducted in accord with the highest standards of humane animal care as outlined in the Guidelines for the Care and Use of Experimental Animals.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Louis Reichardt, for the anti-ß5 polyclonal antibody, Dr. Eric J. Brown, for the murine ß5 cDNA sequence, and Dr. Allen Nickols (Monsanto/Searle), for providing the peptidomimetic SC56631.


    FOOTNOTES
 
Address requests for reprints to: F. Patrick Ross, Ph.D., Department of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital, 216 South Kingshighway, St. Louis, Missouri 63110. E-mail: rossf{at}medicine.wustl.edu

Supported by NIH Grants AR42404 (F.P.R.), DE05413, and AR32788, and a grant from the Shriners Hospital for Crippled Children (St. Louis Unit) (S.L.T.).

Received for publication March 6, 1998. Revision received September 15, 1998. Accepted for publication September 16, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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