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Endocrinology Vol. 141, No. 1 284-290
Copyright © 2000 by The Endocrine Society

ARTICLES

Tumor Necrosis Factor {alpha} Regulates {alpha}vß5 Integrin Expression by Osteoclast Precursors in Vitro and in Vivo1

Masaru Inoue, F. Patrick Ross, Jeanne M. Erdmann, Yousef Abu-Amer, Shi Wei and Steven L. Teitelbaum

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

Address all correspondence and requests for reprints to: Steven L. Teitelbaum, M.D., Department of Pathology, Washington University School of Medicine, Barnes-Jewish Hospital North, 216 South Kingshighway, St. Louis, Missouri 63110. E-mail: teitelbs{at}medicine.wustl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable {alpha}vß3 integrin, contain abundant {alpha}vß5 and attach to matrix in an {alpha}v integrin-dependent manner. Furthermore, {alpha}vß5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-{alpha}, (TNF) down-regulates {alpha}vß5 expression by BMMS via attenuation of ß5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the ß5 suppressive effect. The functional implications of TNF-mediated {alpha}vß5 down-regulation are underscored by the capacity of an {alpha}v inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant {alpha}vß5 while failing to impact those in which the integrin has been diminished by TNF. Finally, ß5 mRNA in BMMs of wild-type mice administered lipopolysaccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, ß5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the ß5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix attachment of osteoclast precursors and mature osteoclasts are governed by distinct {alpha}v integrins which are differentially regulated by specific cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS are physiological polykaryons, and the principal if not exclusive resorptive cells of bone. They are derived from macrophage precursors residing in diverse tissues, principally marrow (1).

While the precise means by which osteoclast precursors undergo differentiation are incompletely defined, attachment to matrix is probably essential. For example, macrophage polykaryons, including osteoclasts, fail to develop in suspension culture (Teitelbaum, S. L., unpublished observation) but form in abundance when in contact with matrix. Thus, the capacity of osteoclast precursors to recognize and attach to matrix is pivotal to generation of the resorptive phenotype.

Integrins, which are major negotiators of cell matrix-attachment, are transmembrane heterodimers consisting of noncovalently linked {alpha} and ß subunits. Given the importance of matrix recognition to osteoclast recruitment and function, attention has turned to identifying integrins that mediate these events. These experiments establish {alpha}vß3 as an integrin essential to osteoclast function. For example, antibody blockade of {alpha}vß3 blunts bone resorption in vitro and in vivo (2, 3, 4), and the ß3 integrin knockout mouse develops osteosclerosis due to osteoclast failure (5).

Given the abundance of {alpha}vß3 on multinucleated osteoclasts, it seems likely the integrin participates in bone degradation by the mature cell. Consistent with the this posture, {alpha}vß3 is progressively expressed as osteoclast precursors differentiate in culture (6). Moreover, cytokines and steroids that impact osteoclast differentiation (6) accelerate appearance of {alpha}vß3 on osteoclast precursors and do so by regulating the ß3 subunit (6, 7).

While these observations are in keeping with the hypothesis that {alpha}vß3 plays a pivotal role in the matrix degrading capacity of the mature polykaryon, they fail to address the means by which mononuclear osteoclast precursors recognize and attach to matrix. In this regard, we find {alpha}vß3 undetectable on early, marrow-derived osteoclast precursors, despite their capacity to bind to extracellular matrix protein recognized by this integrin (6). This conundrum is a reflection of the fact these {alpha}vß3 negative cells express the closely related integrin {alpha}vß5, suggesting the latter heterodimer mediates osteoclast precursor-matrix recognition. In fact, as marrow macrophages assume the osteoclast phenotype in culture, {alpha}vß5 disappears pari passu with emergence of {alpha}vß3 (6).

Given osteoclastogenesis is attended by diminution of {alpha}vß5, one would expect agents that accelerate osteoclastogenesis to down-regulate the integrin. Tumor necrosis factor (TNF) is a proinflammatory cytokine with potent bone resorptive capacity, exerting its effect by enhancing osteoclast recruitment (8, 9, 10, 11). We find this cytokine hastens disappearance, on osteoclast precursors, of {alpha}vß5, a reflection of accelerated degradation of ß5 subunit mRNA. The TNF effect on the ß5 integrin is mediated through the p55TNF receptor and, reflecting the in vitro situation, the TNF agonist lipopolysaccharide (LPS), when administered in vivo, rapidly diminishes marrow macrophage ß5 mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unless specified, all reagents were obtained from Sigma (St. Louis, MO).

Isolation and culture of osteoclast precursors
These procedures have been reported in detail (1, 12). Briefly, marrow cells were obtained from 6-week-old male C3H or transgenic mice by flushing femora and tibiae with {alpha}-modified Eagle’s medium ({alpha}-MEM). The cells were cultured in {alpha}-MEM supplemented with 10% FBS in the presence of 500 U/ml stage 1 macrophage (M)-CSF (13) for 24 h. The nonadherent population was collected and mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (500 x g, 15 min). The marrow- macrophage enriched population was recovered by centrifugation (500 x g, 7 min) and 5 x 106 cells plated in {alpha}-MEM supplemented with 10% FBS on 100-mm plates. The cells, maintained for 3–4 days, were supplemented each day with 500 U/ml M-CSF yielding a pure population of M-CSF dependent, adherent osteoclast precursors. In selected studies, the same adherent osteoclast precursors, maintained in the presence of 500 U/ml M-CSF throughout, were treated, with time, with indicated doses of murine recombinant TNF (Genzyme, Cambridge, MA).

Osteoclast generation
Nonadherent bone marrow macrophages and the murine stromal line ST2 were cultured at a ration of 10:1 (12). Cultures were fed every third day, at which time fresh steroids (10 nm 1,25 dihydroxyvitamin D3 and 100 nm dexamethasone) were added. Osteoclasts and their precursors were freed of ST2 cells by treatment with 0.1% bacterial collagenase, 0.1% BSA in {alpha}-MEM for 2 h at 37 C (11, 12).

Transgenic mice
C3H/HeN male mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) were used. Transgenic mice include: (a) mice in which the p55TNF receptor gene has been deleted (14) (provided by Dr. Warner Lesslauer, Hoffmann-La Roche, Basel, Switzerland); (b) those in which the p75TNF receptor gene has been deleted (15); (c) those in which both the p55 and p75TNF receptor genes have been deleted by interbreeding dominant negative p55TNF receptor knockout mice with their counterparts from p75TNF receptor knockout mice (provided by Dr. Mark Moore, Genentech, Inc., South San Francisco, CA), and (d) their respective wild-type mice.

Northern blot analysis
Total cellular RNA was isolated with TRIzol (Life Technologies, Inc., Gaithersburg, MD) and 5 µg per lane electrophoresed in 0.9% agarose gel containing formaldehyde and transferred to Hybond N (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was prehybridized with hybridization buffer [5 x SSPE, 5 x Denhardt’s solution, 50% formamide, 0.1% SDS, 1 x Background Quencher (Tel-Test Inc., Friendswood, TX) for at least 2 h at 42 C. Northern analysis was performed using mouse ß5 (16) and ß3 (17) integrin complementary DNAs (cDNAs) cloned in our laboratory, or human {alpha}v cDNA kindly provided Dr. Eric Brown (Washington University School of Medicine, St. Louis, MO), 32P labeled by the random primer method (Roche Molecular Biochemicals, Indianapolis, IN). After 16 h hybridization, membranes hybridized with ß5, ß3 or {alpha}v cDNAs were washed as previously described (18). For reprobing, membranes were submerged 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 (19). For determination of mRNA stability, cells in 100-mm dishes were cultured with or without 6.0 ng/ml TNF for 1 day. Actinomycin D (5 µg/ml) was added to all plates and total RNA isolated 0, 2, 4, and 6 h later. Thirty micrograms per lane of RNA was fractionated in agarose, 0.0 Northern analysis was performed using a ß5 cDNA and the results quantified by densitometry.

Nuclear run-on transcription assay
Adherent bone marrow macrophage were cultured for 24 h with or without TNF 6.0 ng/ml and washed twice with ice-cold PBS. Nuclear isolation and in vitro transcription were performed as previously described (18). After transcription the nuclei were harvested and RNA was isolated with TRIzol. Equal amounts of freshly transcribed RNA as determined by trichloroacetic acid (TCA)-precipitable counts were hybridized in hybridization buffer to denatured DNA (5 µg/slot ß5 cDNA, ß3 cDNA, vector DNA, and human G3PDH (CLONTECH Laboratories, Inc. San Diego, CA). After 36 h of hybridization, the membranes washed with 1 x SSPE 0.1% SDS for 20 min at 50 C, 0.1 x SSPE 0.1% SDS for 20 min at 60 C twice.

Immunoprecipitation
Cells were washed with PBS and surface-labeled with 125I as described (18). Cells were lysed in buffer containing 2% Renex 30, 10 mM Tris pH 8.5, 150 mM NaCl, 1 mM CaCl2, 1 mM AEBSF and 0.02% NaN3. Each lysate, containing equal TCA-precipitable counts, was incubated with Gammabind (Amersham Pharmacia Biotech, Piscataway, NJ) and precleared again with Gammabind plus whole rabbit serum for ß5 or class matched monoclonal antibody for ß3. The precleared lysate was immunoprecipitated with a polyclonal rabbit anti-ß5 integrin subunit antibody kindly provided by Dr. Louis Reichardt (University of California, San Francisco, CA) or hamster monoclonal anti-ß3 integrin subunit antibody (PharMigen, San Diego, CA). The immunocomplex was bound to excess Gammabind. The precipitate was 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.

TNF assay
TNF in medium and serum was measured by ELISA (Genzyme, Cambridge, MA).

Cell morphology
Osteoclast precursors were cultured in 48-well plates in 500 U/ml M-CSF. At subconfluence, cells were treated with various combinations of a single dose of TNF, and daily additions of a small RGD peptide mimetic, SC56631 (10 µM) (Searle, Skokie, IL) known to block {alpha}vß3 and {alpha}vß5 (20). After 3 days the cells were fixed with 2% paraformaldehyde and photographed.

In vivo experiments
Four- to six-week-old mice were injected, ip, with 1 mg of LPG. Marrow macrophages were isolated, with time, and cultured for 24 h as described above. The cells were lysed and total RNA probed with {alpha}vß5 integrin cDNA. In some experiments TNF in tail vein blood was measured by ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF{alpha} specifically regulates ß5 integrin mRNA
BMMs were treated in a concentration-dependent manner with TNF for 24 h and probed, by Northern analysis, with {alpha}v, ß3, and ß5 cDNAs. As seen in Fig. 1Go, the quantity of ß5 messenger RNA (mRNA) progressively falls with increasing TNF. Attenuation of ß5 mRNA is detectable at cytokine concentrations as low as 1.8 ng/ml. In contrast to ß5, TNF fails to impact either {alpha}v or ß3 mRNA at concentrations as high as 60 ng/ml. ß5 mRNA declines as early as 4 h and nadir is reached 8 h after a single addition of 6.0/ml TNF-{alpha} (Fig. 2Go).



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  		Figure 1. TNF specifically regulates osteoclast precursor ß5 integrin mRNA in a concentration-dependent manner. Osteoclast precursors were treated for 24 h with increasing concentrations of TNF. Total RNA was extracted and probed, by Northern analysis, with murine {alpha}v, ß3, and ß5 integrin cDNAs. {alpha}v and ß3 autoradiograms were developed for 5 and 7 days, respectively, and ß5 autoradiogram, overnight.

 


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  		Figure 2. TNF regulates osteoclast precursor ß5 integrin mRNA in a time-dependent manner. Osteoclast precursors were maintained ± 6.0 ng/ml TNF. Cells were killed with time and total mRNA derived from TNF treated (+) and control (-) cells probed with a ß5 cDNA.

 
TNF-{alpha} decreases ß5 integrin mRNA stability
We next turned to the molecular mechanisms by which TNF regulates steady state ß5 mRNA levels, asking if the cytokine impacts transcription or message stability. To assess transcription, nuclei were isolated from TNF treated and control BMMs and run on studies performed. As seen in Fig. 3Go, TNF fails to alter transcription of the ß5 integrin gene.



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  		Figure 3. TNF fails to alter ß5 integrin gene transcription. Osteoclast precursors were maintained ± 6.0 ng/ml TNF for 24 h. Nuclei were isolated and incubated with 32P-UTP. Equal cpm were hybridized to excess ß3, G3PDH or ß5 cDNAs or empty vector (pCR11). (Representative of three experiments.)

 
ß5 mRNA stability was determined by exposing TNF-treated and virgin BMMs to the transcriptional inhibitor, actinomycin D and, using Northern analysis, assessing message levels with time. By densitometric quantitation, t 1/2 of ß5 mRNA in control and TNF-{alpha} treated cells are 5.1 and 1.9 h, respectively (Fig. 4Go).



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  		Figure 4. TNF accelerates ß5 mRNA degradation. Osteoclast precursors were maintained ± 6.0 ng/ml TNF for 24 h. Total RNA was isolated before (0) and 2, 4, and 6 h following addition of Actinomycin D (5 µg/ml). Northern analysis was performed with a ß5 cDNA and the autoradiograms densitometrically assessed. The data are presented as % ß5 mRNA present at time 0. No detectable signal was obtained from mRNA derived from BMMs treated for 6 h with TNF.

 
TNF-{alpha} regulates ß5 integrin subunit mRNA via the 55-kDA TNF receptor
To identify the receptor mediating TNF modulation of ß5 mRNA we took advantage of three strains of transgenic mice in which either the p55, p75 or both TNF receptors (TNFrs) are deleted. ß5 mRNA declines in BMMs derived from wild-type mice challenged with TNF, whereas the cytokine fails to exert this effect on cells of animals lacking both TNFrs (Fig. 5Go). Thus, TNF modulates ß5 integrin expression via a classical TNFr(s). Confirming that soluble TNF-induced ß5 down-regulation is mediated via the p55TNFr, BMMs of animals lacking this receptor, like those of double receptor knockout mice, fail to normally alter integrin mRNA levels in response to the cytokine. In contrast, p75TNFr-/- mutant cells behave substantially like wild-type.



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  		Figure 5. TNF regulates ß5 integrin mRNA via the 55 kDa TNF receptor. Osteoclast precursors derived from marrow of wild-type mice [p55(+)p75(+)] and those in which the p55 [p55(-)p75(+)] or p75 [p55(+)p75(-)] TNFr or both TNFrs [p55(-)p75(-)] are deleted were maintained for 8 h in increasing amounts of TNF. Total RNA was probed with a ß5 integrin cDNA.

 
TNF-{alpha} specifically decreases {alpha}vß5 while not effecting {alpha}vß3 expression
To determine if TNF reduction of ß5 mRNA is reflected by surface expression of integrin heterodimers, BMMs were surface labeled with 125I, lysed, and the lysate immunoprecipitated with anti-ß5 or ß3 antibodies. As seen in Fig. 6Go, {alpha}vß5 is expressed by these mononuclear osteoclast precursors while, by this technique, {alpha}vß3 is virtually undetectable. Moreover, exposure of these cells to TNF (6.0 ng/ml) for 48 h diminishes {alpha}vß5 abundance while failing to impact {alpha}vß3.



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  		Figure 6. TNF decreases {alpha}vß5 but does not effect {alpha}vß3. Osteoclast precursors, maintained 48 h ± 6.0 ng/ml TNF, were surface labeled with 125I and immunoprecipitated with anti-ß3 or ß5 integrin antibodies. The immuno-precipitate was subjected to SDS-PAGE in nonreducing conditions and the gels autoradiographed.

 
The TNF-induced decline in {alpha}vß5 expression is accompanied by distinct morphological changes in adherent BMMs plated in serum containing medium, rich in vitronectin (Fig. 7Go). Thus, untreated cells assume a spindle shape, whereas those exposed to the cytokine appear more polygonal. Furthermore, a blocking peptide mimetic recognizing {alpha}vß3 and {alpha}vß5 (20) prevents spreading of virgin cells while not altering the morphology of TNF-exposed BMMs lacking both integrins. The latter observation indicates spreading of early osteoclast precursors is mediated by {alpha}vß5, a phenomenon lost with TNF treatment.



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  		Figure 7. An {alpha}v integrin antagonist alters the morphology of virgin but not TNF-treated osteoclast precursors. Osteoclast precursors were maintained 72 h with or without 6.0 ng/ml TNF and/or the anti {alpha}vß3-{alpha}vß5 peptide mimetic SC56631. The cells were visualized by phase contrast microscopy. (- and + refer to absence or presence, respectively, of TNF or/56631).

 
TNF mRNA is expressed during osteoclastogenesis
To determine if TNF suppression of {alpha}vß5 expression is biologically relevant, we asked if the cytokine is expressed during basal osteoclastogenesis. To this end, osteoclastogenic cultures consisting of BMMs and ST2 stromal cells were established. The stromal cells were removed, with time, and the remaining cells, consisting entirely of mononuclear precursors or mature osteoclasts (11, 12), probed for TNF mRNA. As seen in Fig. 8Go, freshly isolated (day 0) BMMs lack detectable TNF message, which is abundantly expressed within the first day of osteoclastogenic culture. Given the fact osteoclasts emerge in this system on day 6–7 and progressively increase with time, TNF mRNA levels are ample in osteoclast precursors but incrementally decline with appearance of the mature resorptive polykaryon.



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  		Figure 8. TNF mRNA is expressed early in osteoclast differentiation. Osteoclastogenic cultures consisting of a pure population of marrow derived osteoclast precursors to which the ST2 murine marrow stromal line had been added, were established. Stromal cells were removed by collagenase digestion, with time, and residual total RNA derived from osteoclast precursors or mature osteoclasts, probed with a murine TNF cDNA. Day 0 represents pure population of osteoclast precursors before addition of ST2 cells.

 
LPS induces TNF expression by BMMs and decreases ß5 mRNA levels ex vivo
LPS is a potent inducer of TNF and such is the case regarding BMMs (11). While TNF in medium conditioned for 8 h by virgin BMMs is undetectable, the cytokine is abundant in that containing cells exposed to LPS (Fig. 9Go). Furthermore, ß5 mRNA content of BMMs isolated, with time, from LPS injected animals, and cultured for 48 h in LPS free conditions, falls with duration of LPS exposure in vivo. The effect is apparent in cells exposed to LPS for 20 min in vivo with the nadir reached at 2 h, after which ß5 mRNA levels begin to increase (Fig. 10Go). Suggesting LPS suppression of ß5 mRNA is negotiated by TNF, circulating levels of the cytokine measured 30 min earlier are inversely proportional to integrin message. To confirm down-regulation of ß5 mRNA, in vivo, is mediated by TNF we isolated BMMs from endotoxin-treated wild-type mice and those deleted of both the p55 and p75 TNFrs. Figure 11Go, and densitometric analysis of the data contained therein, reveal ß5 message falls to 7.4% of control only in cells obtained from LPS-treated mice in which TNFrs are intact. In contrast, ß5 mRNA in BMMs deleted of both TNFrs does not decline under the influence of endotoxin (109.3% of control). Furthermore, despite the abundance of ß5 mRNA in wild-type compared with mutant virgin BMMs, the message is least in cells derived from LPS-treated wild-type mice.



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  		Figure 9. LPS induces TNF secretion by osteoclast precursors. Osteoclast precursors (4 x 105/well) were maintained for 8 h in 24-well plates, ± 100 ng/ml LPS. TNF concentration in conditioned medium was determined by ELISA.

 


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  		Figure 10. LPS, in vivo, decreases ß5 mRNA. Top panel, LPS injected C3H mice were killed, with time, and osteoclast precursor total RNA probed with a ß5 cDNA. Bottom panel, Circulating TNF levels were measured, by ELISA, before and at various times after LPS administration.

 


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  		Figure 11. LPS inhibition of ß5 mRNA expression, in vivo, is mediated by TNF. LPS was administered to wild-type [p55(+)p75(+)] or double TNFr deleted [p55(-)p75(-)] mice. The animals were killed after 3 h and osteoclast precursor total mRNA probed with a ß5 cDNA

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bone resorption is reflective of the rate at which osteoclasts degrade bone and the efficiency of osteoclast precursor differentiation. The bone degradative process by the mature polykaryon depends upon creation, at the cell-bone interface, of a highly acidic, isolated microenvironment (21). The gradient between the pH of the general extracellular space and that extant where the osteoclast contacts bone indicates physical intimacy between cell and matrix is fundamental to the resorptive process, an event involving the integrin, {alpha}vß3 (2, 3, 4). Cell-matrix recognition is also essential to osteoclast differentiation but the means by which mononuclear precursors recognize substrate are unknown. We find osteoclast precursors, in the form of isolated marrow macrophages, contain no detectable ß5 mRNA or {alpha}vß5 (6). These immature cells can, however, attach to and spread on vitronectin (6). This observation prompted us to search for a related heterodimer, expressed by early osteoclast precursors, which also binds this {alpha}vß3 ligand. In fact, these immature cells contain an abundance of {alpha}vß5, an integrin structurally similar to {alpha}vß3 also recognizing the RGD amino acid sequence resident in a number of matrix proteins including osteopontin, bone sialoprotein and vitronectin (22, 23, 24). Osteoclast precursors, as they differentiate into mature resorptive polykaryons, however, lose {alpha}vß5 and, in a reciprocal fashion, express {alpha}vß3 (6). Taken together, these findings suggest matrix recognition and attachment by early osteoclast precursors is mediated by {alpha}vß5 whereas the role of {alpha}vß3 is confined to more committed cells.

{alpha}vß5, while structurally related to {alpha}vß3, appears to mediate distinct events such as uptake of vitronectin, facilitated entry of adenovirus type 2 and enhanced angiogenesis (25). Our finding that {alpha}vß5, and not {alpha}vß3, mediates osteoclast precursor spreading in an RGD dependent manner also indicates the two integrins functionally differ.

TNF, among the most potent of resorptive cytokines, dramatically increases osteoclastogenesis in man (10) and mouse (11) marrow culture. Given its effect on osteoclast differentiation, we asked if TNF also impacts {alpha}v integrin expression by isolated osteoclast precursors. {alpha}vß5 is indeed down-regulated on early osteoclast precursors exposed to the cytokine. This event is paralleled by changes in ß5 mRNA reflecting message destabilization and not attenuated transcription. Because TNF, in other circumstances, stabilizes macrophage mRNA (26) its impact on ß5 message is not a reflection of nonspecific, accelerated degradation.

In contrast to ß5, {alpha}v message remains unaltered by TNF. The fact ß5 associates only with {alpha}v, whereas {alpha}v partners with a number of ß subunits, indicates it is the monogamous ß, and not promiscuous {alpha} chain, which regulates expression of the heterodimer. This finding is in keeping with the capacity of cytokines such as IL-4 (18) and steroids such as retinoic acid (7) to modulate {alpha}vß3 via the ß3 subunit.

Precisely why {alpha}vß5, abundant on osteoclast precursors, disappears as the cells differentiate into resorptive polykaryons is unknown. It is of interest, however, that {alpha}vß5, in contrast to the mature osteoclast integrin {alpha}vß3, mediates cell spreading. In fact, TNF, by blunting {alpha}vß5 expression, diminishes the capacity of osteoclast precursors to spread. This observation is in keeping with reports that osteoclasts actively resorbing bone fail to assume the spread configuration observed in their inactive counterparts (27). Thus, resorption may require intermittent attachment of osteoclasts to bone with motility inhibited by cell spreading.

The p55TNFr has been viewed as the major transmitter of TNF-induced signals although recent evidence indicates both p55TNFr and p75TNFr are functional (28). For example, while p75TNFr mediates differentiation of early hematopoietic precursors, p55TNFr is active in late stages of the process. On the other hand, soluble, compared with membrane-residing TNF, targets primarily, if not exclusively, p55TNFr (29), through which it promotes osteoclastogenesis (11). To determine if down-regulation of {alpha}vß5 on BMMs, an event characterizing commitment of these cells to the osteoclast phenotype (6), is also mediated by p55TNFr, we turned to mice lacking combinations of the two TNFrs. Confirming the TNF-induced fall in ß5 is negotiated via a classical TNFr, osteoclast precursors derived from double receptor deficient mice fail to alter expression of the integrin in response to the cytokine. Most importantly, failure of p55 but not p75 TNFr-/- BMMs to meaningfully dampen ß5 mRNA in response to the soluble cytokine establishes the p55 species as the principal mediator of the integrin suppressive signal.

Perhaps our most compelling evidence supporting the biological significance of TNF-induced {alpha}vß5 down-regulation are our experiments involving LPS. We find LPS-exposed osteoclast precursors, like other macrophages (30, 31, 32), secrete levels of TNF comparable to those used in our in vitro experiments. Importantly, administration of this TNF agonist, to mice, dampens ß5 expression by osteoclast precursors, ex vivo. Interestingly, the time course of ß5 regulation by in vivo LPS mirrors commitment of BMMs to the osteoclast phenotype (11). Mice devoid of both TNFrs fail to down-regulate ß5 mRNA when administered LPS establishing the phenomenon is mediated by TNF. The rapid suppression of ß5 in the in vivo state may be reflective of the complex manner by which LPS induces expression of the cytokine (33). These events, involving transcriptional and posttranscriptional phenomena, are known to promote TNF secretion by macrophages within minutes of endotoxin exposure. Our experiments, particularly those performed in vivo, suggest TNF-mediated ß5 down-regulation may obtain in pathological states such as bacterial infection which, as a component of periodontal disease, prompts profound bone loss.


   Footnotes
 
1 Supported in part by NIH Grants DE-05413, AR-32788 (S.L.T.), AR-42404 (F.P.R.), and grants from the Barnes-Jewish Hospital Foundation and Monsanto (Y.A.) Back

Received June 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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