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Endocrinology Vol. 138, No. 2 657-666
Copyright © 1997 by The Endocrine Society

Articles

Effects of Retinol on Activation of Latent Transforming Growth Factor-ß by Isolated Osteoclasts1

L. F. Bonewald, R. O. C. Oreffo, C. H. Lee, S. Park-Snyder, D. Twardzik and G. R. Mundy

Department of Medicine, Division of Endocrinology, University of Texas Health Science Center (L.F.B., R.O.C.O., C.H.L., S.P.-S., G.R.M.), and Audie L. Murphy Memorial Veteran’s Hospital (L.F.B.), San Antonio, Texas 78284; and Bristol-Myers Squibb Pharmaceutical Research Institute (D.T.), Seattle, Washington 98121

Address all correspondence and requests for reprints to: Dr. L. F. Bonewald, Department of Medicine, Division of Endocrinology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7877.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The multifunctional cytokine, transforming growth factor-ß (TGFß), is found in many tissues in a latent or inactive form. The nature and composition of the latent complex can vary depending on tissue type. The release of active TGFß from its latent complex is a potentially important mechanism for regulation of TGFß activity. We have shown previously that osteoclasts activate latent TGFß produced by bone and that bone cells produce a 100-kDa latent complex that lacks the latent TGFß-binding protein. Here we investigated the effects of retinol on osteoclast activation of various forms of latent TGFß. Two sources of osteoclasts were used that provide either mature avian osteoclasts or avian osteoclast precursors. Whereas both cell populations activate latent TGFß, only mature osteoclasts respond to retinol with an increase in activation of latent TGFß over basal levels. Activation could not be ascribed to pH changes in conditioned medium. Nonacid-dissociable 100-kDa latent complex, which is also produced by bone cells, was added to mature osteoclasts and to osteoclast precursors, but no activation was observed. Platelet latent TGFß, which contains the 130-kDa latent TGFß-binding protein, was activated by both osteoclast populations. Conditioned medium from the precursor population activated latent complex, whereas conditioned medium from mature cells did not. Activation of latent TGFß by retinol-treated mature cells was not blocked by inhibitors of plasmin, nor was activation by conditioned medium from precursor cells. These data suggest that retinol-induced activation of latent TGFß by osteoclasts is dependent on the stage of differentiation of these cells and the presence of other cell types, and that unlike other cell systems, the plasmin-plasminogen activator mechanism is not involved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSFORMING growth factor-ß (TGFß) appears to have important biological functions in bone. The largest source of TGFß in the body is in bone (1), and TGFß is released in an active form during bone resorption (2). TGFß appears to play a pivotal role in bone formation and fracture repair. Injections of TGFß over calvariae of mice result in accelerated bone formation (3, 4), and a single injection of TGFß will induce complete healing of a nonhealing skull defect (5). It has been suggested that TGFß acts as a coupling factor in bone, which couples resorption to formation (for review, see 6 . Low concentrations of TGFß inhibit osteoclast formation (7), and high concentrations inhibit mature osteoclastic bone resorption, possibly through the mechanism of apoptosis (8). Therefore, TGFß released by resorbing osteoclasts acts to both stimulate new bone formation and limit the extent of further bone resorption. Because TGFß has such potent biological effects, it is important that its activity be tightly controlled, and this may be achieved through activation of the latent complex.

TGFß is secreted by cells in culture in biologically latent forms that can be activated in vitro by transient acidification, alkalinization, or the actions of chaotropic agents (9, 10). The release of mature TGFß 25-kDa homodimer from the latent complex is necessary for TGFß to exert its effects on target cells. Many cell types have been shown to activate latent TGFß (LTGFß) depending upon other factors present in the cellular environment. Cell types that have been shown to activate LTGFß include macrophages, mesenchymal cells, endothelial cells, cocultures of endothelial cells with pericytes or smooth muscle cells, vascular smooth muscle cells, fetal fibroblasts, and human osteoblast-like cells (for review, see Refs. 11 and 12). We and others have shown that osteoclasts will activate LTGFß (13, 14).

In contrast to the in vitro cell culture systems used to study activation of LTGFß, we have shown the activation of LTGFß in vivo in lungs of mice infected with the pathogen Chlamydia trachomatis (15). Also, we have described a noncellular mechanism by which LTGFß is activated by extracellular organelles called matrix vesicles (16). Matrix vesicles found in cartilage are associated with matrix calcification. Alone, these organelles have no effect on LTGFß; however, upon pretreatment of these organelles with 1,25-dihydroxyvitamin D3, both recombinant LTGFß1 (rLTGFß1) and rLTGFß2 are activated. Matrix vesicles have been shown to contain proteases such as plasminogen activator, and therefore, the nongenomic effect of 1,25-hydroxyvitamin D3 on the membranes of these organelles may be the release of TGFß-activating proteases.

The composition of LTGFß secreted by cells appears to vary depending on tissue type. The first latent complex described is produced by platelets (17, 18). This complex contains TGFß1 precursor or latency-associated protein (LAP) noncovalently associated with mature TGFß and a truncated form of the LTGFß-binding protein (LTBP) covalently attached to one of the precursor chains. Fibroblasts, liver fat-storing cells, and other cell types produce a similar complex, but with the nontruncated LTBP (19, 20, 21). Bone cells produce at least three forms of LTGFß: one composed of only the TGFß1 precursor noncovalently associated with mature TGFß of 100 kDa (also known as small LTGFß), one that contains the TGFß precursor covalently associated with mature TGFß of 100 kDa (also known as the noncleaved, nonacid-dissociable form), and a third complex containing the 100-kDa form covalently bound to the nontruncated form of LTBP similar to that described in fibroblasts (also known as large LTGFß) (22, 23). The complex lacking LTBP appears identical to rLTGFß. Kidney cells appear to secrete five different forms of LTGFß (21). Although not completely characterized, several of these complexes appear to be composed of TGFß1 and of TGFß2, which is in contrast to the bone, fibroblast, and liver fat-storing cells, which produce 90–100% TGFß1 and little if any TGFß2. It is not clear at present why various tissues produce different LTGFß complexes. However, in bone, the LTGFß complex containing the nontruncated LTBP appears to be important in storing and releasing LTGFß from bone matrix (24).

High concentrations of plasmin have been shown to activate small LTGFß by proteolytically cleaving the precursor at multiple sites (25). However, low concentrations of plasmin can cleave pro-TGFß1 at the dibasic cleavage site without activating the complex. Earlier it was reported by the same group that plasmin only appears to activate the pool of LTGFß that is activated by mild pH (4.5) and not that activated by strong acidic pH (26). We have shown that high concentrations of plasmin (0.1–0.5 U/ml) are necessary to activate small LTGFß, whereas much smaller concentrations of plasmin (0.05 U/ml) will release large LTGFß from the bone matrix by cleavage of the LTBP (27). We have also shown that release of LTGFß by osteoblasts stimulated with PTH is completely blocked by aprotinin, whereas aprotinin has no effect on the release of LTGFß by isolated avian osteoclasts (27).

Retinoids increased activation of LTGFß in endothelial cell/smooth muscle cell cocultures by increasing membrane-associated plasminogen activator/plasmin levels (28, 29). In UMR-106-01 cells and neonatal mouse calvarial cells, PTH increases the activity of tissue-type and urokinase-type plasminogen activators, which are responsible for the activation of LTGFß (30). Keratinocytes when treated with retinoic acid secrete active TGFß2 (31). Therefore, retinoids and the plasminogen activator system appear to play an important role in the activation of LTGFß in a number of cell systems.

In the present study, the 100-kDa latent complex was mainly tested because bone cells secrete approximately 50% of their LTGFß complexes in this form. We sought to determine which forms of LTGFß could be activated by osteoclasts, whether the truncated LTBP would influence activation, and whether osteoclasts could activate nonacid-dissociable recombinant material. Although osteoclasts activate LTGFß (13, 14), how this process is regulated is not clear. We have shown previously that when isolated osteoclasts are treated with vitamin A or bone particles, they release active TGFß from the LTGFß complexes produced by bone organ cultures (13). The present studies were conducted to determine whether activation of LTGFß by vitamin A-treated cells is mediated through the plasmin/plasminogen activator system. To accomplish this, osteoclasts were prepared using two different techniques, one yielding homogeneous populations of osteoclast precursors, as described by Alvarez and co-workers (32), and a second, less homogeneous population of mature osteoclasts, as described by Zambonin-Zallone and co-workers (33). Here we show that these two populations of osteoclasts activate LTGFß differently and respond differently to retinol, and that the activation of LTGFß by these cells does not appear to be mediated via the plasmin/plasminogen activator system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dulbecco’s {alpha}MEM, FBS, penicillin-streptomycin solution, and tissue culture reagents were obtained from Life Technologies (Grand Island, NY). Vitamin A (retinol), aprotinin, {epsilon}-amino-n-caproic acid (EACA), and the diagnostic kit for tartrate-resistant acid phosphatase were purchased from Sigma Chemical Co. (St. Louis, MO). Neutralizing antibodies for TGFß1 and TGFß2 were purchased from R&D Systems (Minneapolis, MN).

Isolation of avian osteoclasts: Zambonin-Zallone technique
Avian osteoclasts were isolated from medullary bone of hypocalcemic egg laying white leghorn hens Gallus domesticus (Kingswheel, OH) as previously described (33). The cells were plated at 2.5 x 106 cells/well in 48-well plates. On day 2 after isolation, the cells were washed and incubated with bone particles (55 µm size) or vitamin A (retinol; 1 µM) and 2% FCS for 24 h, after which the cells were again washed before the addition of LTGFß in 1% BSA. After 48 h (at which time the cell number was approximately 1.7 x 104 cells/well), the osteoclast-conditioned medium was removed and assessed for TGFß activity in the alkaline phosphatase microassay as described below. To assess the effects of protease inhibitors on TGFß activation, aprotinin (50 µg/ml) and/or EACA (50 µg/ml) were added with the LTGFß.

Isolation of avian osteoclast precursors
Isolation of precursor cells was performed by the procedure described by Alvarez et al. (32). Tibiae and femora were dissected from egg-laying white leghorn hens Gallus domesticus (Kingswheel, OH) maintained on a low calcium diet (Purina Test Diets, Richmond, IN) for 21–28 days. The cells were isolated and plated as described previously (32). At 72 h, at which time the mononuclear cells have begun to fuse (cell number is ~2.1 x 104 cells/well), retinol (1 µM) was added for 24 h, after which time the medium was removed, and fresh {alpha}MEM-1% BSA with or without rLTGFß1 or -ß2 was added. After 24-h incubation, the medium was tested for TGFß activation in the ROS 17/2.8 microassay.

Alkaline phosphatase microassay for quantitating TGFß bioactivity
TGFß activity was assessed by its capacity to increase alkaline phosphatase activity in ROS 17/2.8 cells described previously (13, 16, 22). This technique uses the quantitation of alkaline phosphatase technique described by Majeska and Rodan (34), but modified to be performed in 96-well microtiter plates. A TGFß standard curve (5.0–0.019 ng/ml) was included in each assay. TGFß activation after incubation of LTGFß with osteoclasts was converted to TGFß equivalents in nanograms per ml. Specificity for TGFß was confirmed by neutralization of activity with antibodies to TGFß1 and TGFß2 (R&D Systems).

Acid-activation of LTGFß to be tested for total TGFß activity
One hundred microliters of sample were used in the alkaline phosphatase microassay (22). To acid-activate a 100-µl sample of LTGFß, 6 µl 1 M HCl were added, followed by 20 µl 10 mg/ml BSA and 10 µl 0.5 M NaOH for neutralization, followed by sterile filtration using Centrex (Schleicher and Schuell, Keene, NH) or Spin-X (Costar, Cambridge, MA) filters.

Purification of platelet LTGFß
Platelet material was purified using outdated platelets as described previously (22). The platelets were lysed and centrifuged, and the supernatant was dialyzed and applied to gel filtration, followed by application of the high mol wt fraction to fast pressure liquid chromatography using a preparative Mono Q anion exchange column (Pharmacia, Piscataway, NJ). The LTGFß from platelets elutes at 0.3 M NaCl, which results in a major band of approximately 235 kDa (22). This material was used for the osteoclast activation experiments.

Source of rLTGFß1 and rLTGFß2
Except for the experiments comparing acid-dissociable to nonacid-dissociable rLTGFß1, all experiments were performed using (NH4)2SO4-precipitated protein derived from conditioned medium from Chinese hamster ovary (CHO) cells transfected with either the gene coding for simian TGFß1 (35) or TGFß2 (36).

Preparation of acid-dissociable and nonacid-dissociable rLTGFß1
Simian rTGFß1 is expressed in high levels by transfected CHO cells (35). The simian TGFß1 precursor is secreted by transfected CHO cells in two forms, an acid-dissociable complex that can yield biologically active TGFß, and a nonacid-dissociable precursor complex. The acid-dissociable form of LTGFß precursor has been cleaved inside the cell, before secretion, at position 279, whereas the nonacid-dissociable latent precursor has not been proteolytically cleaved (see Fig. 4Go). The rTGFß1 precursor is glycosylated and phosphorylated at three sites (37, 38), and these sites can mediate binding to the insulin-like growth factor II/mannose 6-phosphate receptor (39).



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  		Figure 4. Platelet LTGFß was incubated with and without mature osteoclasts treated with and without retinol. A, Diagram of platelet LTGFß depicting the truncated form of LTBP covalently associated with one of the latency-associated precursor proteins. B, Platelet LTGFß incubated at 37 C in 5% CO2 for 24 h without cells, with untreated osteoclasts, and with retinol-treated (10-6 M) osteoclasts is shown. Crossed bars are without neutralizing antibody to TGFß1; solid bars are with neutralizing antibody. Control is medium (2% FBS in {alpha}MEM) alone. Mature osteoclasts were isolated using the technique of Zambonin-Zallone et al. (33). The results are presented as the mean ± SE (n = 4). P < 0.05, using Student’s t test. *, Significantly different from medium without osteoclasts. **, Significantly different from osteoclasts not treated with vitamin A.

 
Simian rTGFß1 precursor polypeptides were purified from CHO cells using ammonium sulfate precipitation and Concanavalin A-Sepharose chromatography with {alpha}-methylmannoside elution, as described previously by Gentry et al. (35). This is termed acid-dissociable material. The rTGFß precursor was treated with acid, and mature TGFß was separated from the nonacid-dissociable precursor using a TSK-sizing column on HPLC. The nonacid-dissociable material was pooled, lyophilized, resuspended to 1 µg/ml, and applied to isolated osteoclasts.

Western blots of acid-treated and nonacid-treated rTGFß1 precursor
Acid-treated and nonacid-treated material was applied to 15% SDS-PAGE and electrophoresed under nonreducing conditions. The gel was transblotted onto nitrocellulose overnight using a Tris-glycine transblotting buffer as described previously (22). The nitrocellulose membrane was removed, blocked with 5% BSA, washed, and blotted with antibody specific for mature TGFß1 or the precursor (LAP) region. The block for the anti-TGFß1 antibody was TGFß1, and that for the antiprecursor antibody was peptide 244–267. The secondary antibody was alkaline phosphatase conjugated, and the blots were developed using Bio-Rad developer (Bio-Rad Laboratories, Richmond, CA) containing 5-bromo-chloro-3-indoyl phosphate and nitroblue tetrazolium. The primary antibodies and their specific peptide blocks were generously supplied by Dr. Lalage Wakefield (NIH).

Incubation of osteoclast precursor-conditioned medium with proteinase inhibitors
To determine whether osteoclast precursor-conditioned medium would activate LTGFß, 100 µl conditioned medium were added to 100 µl LTGFß for 24 h at room temperature or 37 C before assaying for active TGFß. The conditioned medium was collected from precursor cells isolated using the Alvarez technique after 72 h of culture and pretreatment with retinol for 24 h. To determine whether this activation could be inhibited by serine protease inhibitors, EACA or aprotinin was added at a final concentration of 50 µg/ml to the conditioned medium and thoroughly mixed before the addition of LTGFß.

Statistical analysis
Data were analyzed using Student’s t test using a statistical package for the IBM PC (SAS Institute, Cary, NC). All data are presented as the mean ± SE (n = 4 wells). All experiments were repeated at least twice with similar results (Fig. 4Go) or more than 12 times (Fig. 2Go).



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  		Figure 2. Medium containing rLTGFß1 was incubated with mature osteoclasts isolated using the technique described by Zambonin-Zallone et al. (33). Retinol (10-6 M)-treated osteoclasts activate more of the latent material than nonretinol-treated osteoclasts. The crossed bars are without neutralizing antibody, and the solid bars are with neutralizing antibody. P < 0.05, using Student’s t test. *, Significantly different from medium without osteoclasts. **, Significantly different from osteoclasts not treated with vitamin A.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cellular composition of avian osteoclast isolations
Zambonin-Zallone and co-workers (33) were the first to describe the isolation of an enriched population of avian osteoclasts. These cells were isolated mechanically from the medullary bone of egg-laying hens. This was the technique used to isolate cells to examine the activation of LTGFß produced by bone (13). As shown in Fig. 1Go, A and B, these cells display a variety of morphologies from very large multinucleated cells to smaller precursors and a 5–10% contamination with marrow cells. These cells were stained for tartrate-resistant acid phosphatase according to manufacturer’s instructions (Sigma).



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  		Figure 1. Photographs of the avian cells used for the TGFß activation studies. A and B, Cells isolated using the Zambonin-Zallone technique after 5 days of culture. C and D, Cells isolated using the Alvarez technique 4 days after replating. Note the greater variety of cell size and the presence of small mononuclear cells in the mature osteoclast population (A and B). The mononuclear precursor cells isolated from avian marrow yield a more homogeneous population (C and D). Magnification: A and C, x180; B and D, x90. Bar = 100 µm.

 
Alvarez and co-workers (32) developed the isolation procedure for progenitor cells that fuse in culture to resemble avian osteoclasts. A uniform population of nonspecific esterase-positive, tartrate-sensitive, acid phosphatase-positive mononuclear cells is obtained from the marrow of egg-laying hens. When cultured, these cells fuse to form polykaryons, produce tartrate-resistant acid phosphatase and develop an osteoclast morphology. These cells form a relatively homogeneous cell layer in culture (see Fig. 1Go, C and B). Both populations will form resorption lacunae on bone slices.

Activation of rLTGFß by isolated mature osteoclasts
As we had shown previously that isolated mature osteoclasts would activate LTGFß isolated from bone organ cultures (13) and that bone organ cultures produce a form of LTGFß essentially identical to rTGFß1 (22), rLTGFß1 was applied to osteoclasts isolated using the technique described by Zambonin-Zallone et al. (33) with and without retinol pretreatment or with and without bone particle treatment. Mature osteoclasts alone significantly activated rLTGFß1 and pretreatment with retinol or bone particles increased this activation (Figs. 2Go and 3Go). rLTGFß was added for 48 h before assay for active TGFß. The medium containing rLTGFß was added at 1.5 µg protein/ml, which contains 20–30 ng/ml biologically active TGFß after acid activation. Therefore, 1–4% of the total available TGFß that was acid activatable was released by these osteoclasts. The specificity of the assay for TGFß was verified using a neutralizing antibody for TGFß1 (AB-101-NA, R&D Systems) as described previously (13). Ten micrograms per ml of this antibody efficiently blocks 1 ng/ml TGFß1, as determined by titration studies using the ROS 17/2.8 alkaline phosphatase microassay. These cells also activate rLTGFß2 (data not shown). Neutralizing antibody to TGFß (R&D Systems AB-112-NA) was used that efficiently neutralizes 1 ng/ml TGFß2 at 1 µg/ml antibody. To determine whether changes in the pH of osteoclast-conditioned medium were responsible, the pH of the conditioned medium was measured (see Table 1Go). No significant changes were observed.



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  		Figure 3. Medium containing rLTGFß1 was incubated with mature osteoclasts with and without bone particles. Bone particles also stimulate these cultured osteoclasts to activate rLTGFß. P < 0.05, using Student’s t test. *, Significantly different from medium without osteoclasts. **, Significantly different from osteoclasts not incubated with bone particles.

 

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  		Table 1. Changes in pH of the conditioned medium do not appear responsible for osteoclast-mediated activation of rLTGFß

 
Effects of osteoclast-conditioned medium from mature osteoclasts on rLTGFß
To determine whether a factor or enzyme produced or secreted by these osteoclasts might be responsible for the release of active TGFß from the recombinant complex, medium harvested from cultured osteoclasts was incubated with rLTGFß1. No detectable TGFß activity was found in any coincubation experiments using osteoclast-conditioned medium obtained from isolated avian osteoclasts with or without retinol pretreatment with rLTGFß.

Activation of platelet LTGFß by isolated mature osteoclasts
Platelets contain a 235-kDa latent complex composed of mature TGFß1 (25-kDa dimer) noncovalently associated with the remainder of the TGFß1 precursor proregion (75-kDa dimer), which, in turn, is bound through a disulfide bond to a protein of 135 kDa that is a truncated form of LTBP (17, 18). The presence of LTBP has also been shown to be necessary for cell surface activation of the latent complex (40). To determine whether LTBP would interfere with or enhance the LTGFß activation process, platelet LTGFß was incubated with isolated mature osteoclasts treated with and without retinol. The platelet material contained approximately 40 ng/ml TGFß activity after acid activation. The material was diluted 1:10 and incubated with mature osteoclasts from the technique of Zambonin-Zallone et al. (33) for 24 h at 10% CO2. As shown in Fig. 4Go, little, if any, autoactivation was observed. Significant activation was found with untreated cells compared to no cells, but greater activation occurred with retinol-treated cells. A maximal activation of approximately 8.7% of the total available TGFß was observed.

Incubation of mature osteoclasts with purified nonacid-dissociable rTGFß
The simian TGFß1 precursor secreted by transfected CHO cells is secreted in two forms, an acid-dissociable complex that yields biologically active TGFß and a nonacid-dissociable complex ( Figs. 5–7GoGoGo). The acid-dissociable form of LTGFß precursor has been cleaved at position 279 before secretion from the cell, whereas the nonacid-dissociable latent precursor has not been cleaved. Plasmin was found to convert this noncleaved, nonacid-dissociable form to the cleaved, acid-dissociable form by Lyons and co-workers (25, 26). We questioned whether osteoclasts have the capacity to activate this noncleaved, nonacid-dissociable form, as bone cells also appear to produce this form in relative abundance (22).



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  		Figure 5. Diagram of the two recombinant forms of TGFß produced by CHO cells. A, Uncleaved precursor that is nonacid-dissociable and migrates as a 100-kDa band by SDS-PAGE under nonreducing conditions. B, rLTGFß that is acid-dissociable to yield biologically active TGFß. By SDS-PAGE, this material gives a 100-kDa band, but once acid-treated, a 70- to 75-kDa band (precursor or LAP) and the 25-kDa TGFß homodimer appear under nonreducing conditions. See Figs. 6Go and 7Go.

 


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  		Figure 6. rLTGFß1 is composed of acid-dissociable and nonacid-dissociable complex as determined by SDS-PAGE. SDS-PAGE (15%) was performed of rLTGFß1 (left lane) and after reduction (right lane) upon staining with Coomassie blue. The intact material gives a band of approximately 100 kDa, whereas the reduced material results in a band of approximately 48 kDa, the noncleaved monomeric precursor or LAP; 35 kDa, the cleaved monomeric precursor; and the 12.5-kDa monomeric TGFß1. The sizes of the Mr markers are 68, 45, 30, and 14 kDa.

 


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  		Figure 7. Western blot of rLTGFß1 with and without acid treatment. This shows the dissociable and nondissociable complex. The nondissociable form remains at 100 kDa, whereas the acid-dissociable material migrates at 25 kDa, which is homodimeric TGFß, and at 70–75 kDa, which is the homodimeric LAP. The reduced Mr markers are 110, 84, 48, 33, 24, and 16 kDa.

 
Isolated mature osteoclasts activated the acid-dissociable, but not the nonacid-dissociable, complex, suggesting that osteoclasts do not possess plasmin or the enzyme necessary for cleavage between precursor and mature TGFß, or that this enzyme is not available extracellularly (Fig. 8AGo). Combining retinol with bone particles did not further enhance the amount of LTGFß activated or activate nonacid-dissociable rLTGFß1 (Fig. 8BGo). The release of active TGFß again appeared to be independent of osteoclast acid production (data not shown). Medium harvested from cultured mature osteoclasts was incubated with nonacid-dissociable complex for 48 h. No release of active TGFß occurred. Osteoclast precursors isolated using the technique of Alvarez et al. (32) also did not activate the nonacid-dissociable material (data not shown).



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  		Figure 8. Medium alone, medium plus nonacid-dissociable rTGFß1, or medium containing acid-dissociable rLTGFß1 were added to mature osteoclasts treated with retinol (A) or with retinol plus bone particles (B). The crossed bars are without neutralizing antibody, and the solid bars are with neutralizing antibody. The nonacid-dissociable material was not activated under any of these conditions. Mature osteoclasts were isolated using the technique of Zambonin-Zallone et al. (33). P < 0.05, using Student’s t test. *, Significantly different from nonacid-dissociable rLTGFß.

 
Effect of protease inhibitors on activation of LTGFß by retinol-treated mature osteoclast cells
As retinol stimulated these cells to activate LTGFß we sought to determine whether this mechanism of activation could be through the generation of plasmin. Neither aprotinin nor EACA had significant effects on retinol-induced activation of LTGFß by these cells (see Table 2Go). In experiments in which plasmin was added to rLTGFß1 and rLTGFß2 for 4 h at room temperature, 100% was activated with 0.5 U/ml plasmin, 50–75% with 0.1 U, and 2% activation was detectable with 0.01 U (data not shown). Although some activation was observed after 24 h of incubation of rLTGFß with mature osteoclasts, more significant activation was observed at 48 h as shown in Figs. 2Go and 3Go. After 48 h, only 1–4% of the total acid-activatable TGFß available was detected. As 50 µg/ml aprotinin will inhibit 50% of the activity of 3.5–8 U plasmin under optimal conditions, it is highly likely that if the TGFß activation were due to plasmin in these cultures, it would be inhibited. Although a slight decrease was observed, these effects were not statistically significant. Experiments using 2% FCS that had been applied to a lysine-Sepharose affinity column to remove plasminogen showed no significant difference from nonstripped FCS in these activation experiments (data not shown).


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  		Table 2. Effects of the protease inhibitors, aprotinin (50 µg/ml) and EACA (50 µg/ml), on activation of rLTGFß by avian osteoclast-like cells

 
Incubation and activation of rLTGFß1 and rLTGFß2 by avian osteoclast precursors
Using the technique described by Alvarez and co-workers (32), osteoclast precursors were isolated from bone marrow, separated using Ficoll-Hypaque, and plated overnight, and the adherent mononuclear cells were removed using EDTA and replated for study. These cells fuse to form large multinucleated cells in culture.

After avian osteoclast precursors had been plated for 3 days, retinol was added for 24 h, then removed, LTGFß was added, and the conditioned medium was assayed at 24 and 48 h. These cells significantly activated both rLTGFß1 and rLTGFß2 after 24 h without the addition of retinol (see Fig. 9AGo). This was similar to data described by Oursler et al. (14). Approximately 10 ng/ml total acid-activatable TGFß1 and 9 ng/ml total acid-activatable TGFß2 were available, showing that approximately 8% of the rLTGFß1 and 9% of the rLTGFß2 were activated by these cells. The addition of retinol to these osteoclast precursors had no effect on the activation process, in contrast to that in mature cells isolated using the technique of Zambonin-Zallone et al. (33). A greater percentage of the available LTGFß was activated by precursor cells compared to mature osteoclasts.



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  		Figure 9. rLTGFß1 and rLTGFß2 were added to osteoclast precursors isolated using the technique described by Alvarez et al. (32) (A) and incubated with conditioned medium from these cells (B). The crossed bars are factors with cells; the solid bars are the factors without cells. In contrast to the mature osteoclasts isolated using the technique of Zambonin-Zallone et al. (33), 5 times more TGFß was activated by these cells (A), and the conditioned medium harvested from precursor cells (B) also activated rLTGFß. P < 0.05, using Student’s t test. *, Significantly different from cultures without cells.

 
Incubation of rLTGFß1 and rLTGFß2 with conditioned medium from avian osteoclast precursors
After these cells had been plated for 3 days, retinol was added, and the conditioned medium was collected 24 h later. This conditioned medium was added to rLTGFß and incubated at 37 C for 24 h before assay of active TGFß. As shown in Fig. 9BGo, the osteoclast-conditioned medium alone was sufficient to activate rLTGFß1 and rLTGFß2. Again, preincubation with retinol had no effect.

Approximately 6.2 ng/ml rTGFß1 and 3.5 ng/ml rTGFß2 were available for activation. These experiments show that 13% of rLTGFß1 and 23% of rLTGFß2 were activated by incubation with precursor-conditioned medium.

Effects of protease inhibitors on activation of rLTGFß by osteoclast precursor-conditioned medium
As conditioned medium from isolated osteoclast precursors would activate LTGFß, and as it is known that plasmin activates LTGFß, the broad spectrum serine protease inhibitors EACA and aprotinin were added to conditioned medium incubated with rLTGFß1 and rLTGFß2. The inhibitors were added at a final concentration of 50 µg/ml, which has been reported previously to be effective at blocking LTGFß activation (28, 29, 40) and was tested and found to have little effect on the bioassay for TGFß. This experiment was repeated three times, with no significant inhibition of activation observed (see Table 3Go).


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  		Table 3. Comparison of the properties of osteoclasts isolated using the technique of Zambonin-Zallone et al. (33) (column 1) with those isolated using the technique of Alvarez et al. (column 2)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Avian osteoclasts were isolated using two different techniques, one that yields mature osteoclasts containing a small component (5–10%) of adherent marrow cells and one that yields a relatively homogeneous population of osteoclast precursors. Both populations activate rLTGFß1 and rLTGFß2. Platelet LTGFß, which contains a truncated form of LTBP, was also activated by these cells. The activation was not due to pH changes in the conditioned medium. Neither population activated the nonacid-dissociable recombinant complex, which was not cleaved between precursor and mature TGFß. Whereas vitamin A (retinol) and bone particles enhanced the capacity of the mature population of osteoclasts to activate LTGFß, retinol had no effect on the homogeneous precursor population. The homogeneous precursor population activated 5 times more LTGFß than the mature heterogeneous population. Conditioned medium harvested from the homogeneous precursor population activated LTGFß, but conditioned medium from the heterogeneous population containing the mature cells did not. This activation of LTGFß by conditioned medium was not blocked by inhibitors of plasmin. These data suggest that retinol-induced activation of LTGFß by osteoclasts is dependent on the stage of differentiation of these cells and perhaps on the presence of other cell types within the cell population.

The two techniques for isolation of osteoclasts used in this study gave quite different results with respect to activation of LTGFß. The major differences between the two techniques used to isolate the two osteoclast-like cell populations are the following. To obtain the mature osteoclasts, the marrow is scraped from the bone and allowed to adhere. The nonadherent cells are removed by washing before treatment. To obtain precursors, only the marrow is used, the bone is not scraped, the isolated cells are allowed to adhere then are washed, and the adherent cells are removed with EDTA and allowed to readhere. This additional removal and readherence step deletes several unknown cell types and multinucleated mature osteoclasts from the population. It is not clear at this time why these two populations would give different results, as similar numbers of tartrate-resistant acid phosphatase multinucleated cells are present between 3–4 days of culture. Mature osteoclasts were regulated by retinol and bone particles, whereas osteoclast precursor cells were not regulated by retinol. This heterogeneous population of mature cells may be more representative of how LTGFß is activated by osteoclasts in vivo. Perhaps other cell types in these cultures are important in the regulation of mature osteoclasts, whereas lack of these cells leads to the formation of activated multinucleated cells.

Oursler (14) showed that avian osteoclast-like cells isolated using a monoclonal antibody that recognizes the 121F antigen produce and activate LTGFß. The 121F antigen is a protein on avian osteoclasts related to superoxide dismutase (41, 42). The conditioned medium from these cells also activated different sources of LTGFß, and it was speculated that these cells were already activated because they were attached to bone particles. No significant inhibition of LTGFß activation was observed with a series of protease inhibitors, including aprotinin. However, a combination of five different inhibitors significantly inhibited LTGFß activation. The freshly isolated 121F-positive cells used for LTGFß activation by Oursler (14) are similar to the fused mononuclear precursors used in this present study, in that conditioned medium from both cell populations activated LTGFß. However, no bone particles were present in the population used in this study compared to those used by Oursler (14), so it remains unclear how the fused mononuclear precursor cells are activated.

It is well known that an acidic zone exists under the resorbing osteoclast. The osteoclast secretes protons into this sealed zone or resorption lacunae, an isolated extracellular compartment that has been compared to an extracellular lysosome (43, 44). As LTGFß is stored in bone and LTGFß is activated by acidification, it has been hypothesized that osteoclasts may activate LTGFß by this mechanism. In the present study, as in previous ones (13), no significant changes were observed in the pH of the osteoclast-conditioned medium. This observation does not rule out the possibility that osteoclasts use this mechanism. Osteoclasts cultured on plastic surfaces still exhibit a reduced pH in the attachment zone, as determined by the use of microelectrodes (45). In theory, if the soluble LTGFß in solution could be transported to this zone, activation would occur. However, this theory remains to be proven.

It has been proposed that differences between latent complexes of the TGFß isoforms may necessitate different mechanisms of activation of the latent complexes (46). Seventy-one percent homology exists between the mature regions of TGFß1 and -ß2, but only 31% homology exists between their respective precursors, suggesting that the amino-terminals may provide distinct functions (47). The TGFß2 precursor contains 59 more amino acids, and multiple TGFß2 messenger RNAs are observed using Northern analysis. The precursor region of TGFß2 also contains three N-glycosylation sites, but lacks the cell adhesion recognition site (Arg-Gly-Asp) present in the TGFß1 precursor (48). In the present studies, we observed no differences in the activation of LTGFß1 or LTGFß2, suggesting that the precursor regions are not sufficiently different or that the precursor portions are not playing a significant role in the activation process.

Fibroblasts and many other cell types produce LTGFß, which contains a single chain 190-kDa protein, LTBP, that is disulfide linked to one of the precursor or LAP chains (19, 20). Platelet LTGFß contains a truncated form of this protein of 130 kDa that appears to be a proteolytically processed form (17, 18). LTBP has no covalent linkage with mature TGFß and does not confer latency to the complex (19, 22, 35). Bone contains several latent forms of TGFß (23), including a 100-kDa form that lacks the LTBP (22). In the present studies, the presence of the truncated form of LTBP had little effect on the activation process.

The plasminogen activator/plasmin system, which plays such an important role in activation of LTGFß in other cellular systems (30), does not appear to be the mediator for osteoclastic activation of LTGFß. Plasmin has been ascribed important roles in tissue remodeling because of its ability to activate latent collagenase, activate LTGFß1, liberate insulin-like growth factor I from its binding protein, and promote osteoblast and osteoclast mobility (for review, see 49 . As retinoids and plasmin appear to play a role in the activation of LTGFß in a number of cell systems (29, 30, 31) and as both tissue-type plasminogen activator and urokinase were localized by immunohistochemistry to osteoclasts (50), the assumption was made that plasmin was also involved in the activation of LTGFß by osteoclasts. However, inhibitors of plasmin shown to block activation of LTGFß in other cell systems had no significant effect in the present studies and suggest that osteoclasts use another mechanism to activate LTGFß.

Vitamin A influences bone development and bone remodeling. It has long been known that vitamin A deficiency leads to increased bone thickness, whereas vitamin A excess leads to bone resorption (51). Our present data suggest that vitamin A or retinol has an effect on osteoclasts that lead to activation of LTGFß. Regulation of LTGFß activation by retinoids has also been reported in cultured keratinocytes (31) and bovine endothelial cells (29). Excessive levels of vitamin A or retinol have catabolic effects on articular cartilage, opposing the anabolic effects of TGFß (52). However, retinoic acid enhances the effects of TGFß on DNA synthesis induced by growth factors in keratinocytes (53). Clearly, there is a complexity of interactions between retinoids and members of the TGFß family (54). The specific regulatory sequences involved in these interactions are unknown, given the multitude of actions of both retinoids and TGFß in development and osteogenesis. Retinol and TGFß are probably necessary for regulation of homeostasis in bone, which, in turn, may be mediated via the osteoclast.


   Acknowledgments
 
We acknowledge the excellent secretarial skills of Thelma Barrios in preparation of this manuscript. We also thank Kumiko Moriyama and Jennifer Rosser for performing the studies using the protease inhibitors, and Dr. Sarah Dallas for thoughtful discussions.


   Footnotes
 
1 This work was supported by NIH Grant RO1-764217 and the V.A. Research Service. Back

Received July 25, 1996.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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