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Endocrinology Vol. 138, No. 10 4160-4166
Copyright © 1997 by The Endocrine Society

ARTICLES

Thrombopoietin Inhibits in Vitro Osteoclastogenesis from Murine Bone Marrow Cells

Takeshi Wakikawa, Atsushi Shioi, Masayuki Hino, Masaaki Inaba, Yoshiki Nishizawa, Noriyuki Tatsumi, Hirotoshi Morii and Shuzo Otani

Departments of Biochemistry (T.W., S.O.) and Clinical Hematology (M.H., N.T.) and Second Department of Internal Medicine (A.S., M.I., Y.N., H.M.), Osaka City University Medical School, Osaka 545, Japan

Address all correspondence and requests for reprints to: Atsushi Shioi, M.D., Second Department of Internal Medicine, Osaka City University Medical School, 1–5-7 Asahi-machi, Abeno-ku Osaka 545, Japan. E-mail: as{at}msic.med.osaka-cu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether thrombopoietin (TPO) can modulate the osteoclastic differentiation from hematopoietic stem cells, we investigated the effect of TPO on in vitro osteoclastogenesis by using the coculture of murine bone marrow cells with the stromal cell line (ST2) in the presence of 1{alpha},25-dihydroxyvitamin D3 and dexamethasone. Recombinant human TPO inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells in a dose-dependent manner (0.02–200 ng/ml). The effect of TPO on differentiation of bone-resorbing capacity was investigated by pit assay. TPO dose dependently decreased the areas of toluidine blue-stained resorption pits (2.0–200 ng/ml). To identify the cellular target of TPO, we used a variety of bone marrow/stromal cell coculture methods. Initially, we found that TPO mainly exerted its effect on the early stage of osteoclastic differentiation in delayed addition experiments. Consequently, the majority of TPO’s inhibition of osteoclastic cell formation was due to its effect on bone marrow cells. Finally, we examined whether transforming growth factor-ß (TGFß) and platelet-derived growth factor (PDGF), major cytokines produced by megakaryocytes, mediate the inhibitory effect of TPO. The addition of either anti-TGFß or anti-PDGF antibody to bone marrow cell culture completely antagonized the effect of TPO on osteoclastogenesis. Furthermore, treatment of bone marrow cells with TGFß or PDGF mimicked the inhibitory effect of TPO. These data suggest that TPO inhibits osteoclastogenesis through stimulating thrombopoiesis and that TGFß and PDGF mediate the effect of TPO by impacting on macrophage-lineage cells as osteoclast precursors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THROMBOPOIETIN (TPO) stimulates both megakaryocyte colony formation and megakaryocyte maturation (1, 2, 3). TPO is a novel cytokine composed of an N-terminal domain homologous to erythropoietin and a glycosylated carboxy domain of unknown function (1, 4, 5). TPO stimulates the formation of colony-forming unit-megakaryocytes both alone and in combination with early acting factors such as interleukin-3 and c-kit ligand (2). It also stimulates the production of megakaryocytes and functional platelets from enriched murine or human stem cell populations (6, 7). The protooncogene c-mpl was originally isolated as the cellular homology of v-mpl, the transforming gene of the mouse myeloproliferative leukemia virus (8). Characterization of c-Mpl, the receptor encoded by c-mpl, revealed structural homology with the hematopoietic cytokine receptor family and its involvement in megakaryocyte development (9, 10). Recently, TPO has been identified as a ligand for c-Mpl (1, 2, 3, 4, 5). The physiological significance of c-mpl in the regulation of thrombopoiesis in vivo was demonstrated by the generation of c-mpl-deficient mice (11). These mice exhibit a 85% reduction in peripheral platelet counts and in marrow and spleen megakaryocytes. Expression of c-mpl appears to be restricted to hematopoietic tissues, primitive hematopoietic stem cells, megakaryocytes, and platelets (12).

Megakaryocytes and platelets can produce cytokines and growth factors such as transforming growth factor-ß (TGFß) and platelet-derived growth factor (PDGF), which may be involved in the development of myelofibrosis (13, 14, 15, 16). In this condition, osteosclerosis of trabecular bones has been often noted. Moreover, megakaryocytes express bone matrix proteins such as osteocalcin and osteonectin (17, 18), suggesting that they may play a role in skeletal metabolism.

Osteoclasts are derived from the hematopoietic progenitors, and various cytokines and colony-stimulating factors can modulate osteoclastogenesis. As the early stages of hematopoiesis and osteoclastogenesis proceed along similar pathways, it is likely that the same cytokines and colony-stimulating factors that are involved in hematopoiesis are also involved in the development of osteoclasts (19). Therefore, we hypothesized that TPO may modulate the osteoclastic differentiation from hematopoietic progenitors. To prove this hypothesis, we used an in vitro model of murine osteoclastogenesis by coculture of bone marrow cells with stromal cells (20, 21). In this study, we found that TPO inhibits osteoclastogenesis in vitro through acting on the early stages of osteoclastic differentiation and that the cellular target of TPO is present in bone marrow cells. Furthermore, we demonstrated that TPO exerts its inhibitory effect through stimulating thrombopoiesis and that TGFß and PDGF, major cytokines elaborated by megakaryocytes, mediate the effect of TPO by impacting on macrophage lineage cells as osteoclast precursors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and cell lines
C3H/HeN mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). ST2 cells (mouse stromal cell line) were obtained from RIKEN Cell Bank (Tsukuba, Japan), maintained in {alpha}MEM supplemented with 10% FCS containing 100 U/ml penicillin and 100 µg/ml streptomycin, passaged weekly using trypsin-EDTA (Sigma Chemical Co., St. Louis, MO), and used up to passage 10.

Reagents
Recombinant human TPO (22, 23) and affinity-purified rabbit anti-TPO polyclonal antibody (blocking type) were provided by Pharmaceutical Research Laboratory, Kirin Brewery Co. (Tokyo, Japan). Recombinant human macrophage colony-stimulating factor (M-CSF) was provided by Green Cross Co. (Osaka, Japan). Pronase was purchased from Boehringer Mannheim (Indianapolis, IN). 1{alpha},25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was provided by Chugai Pharmaceutical Co. (Tokyo, Japan). Mouse anti-TGFß monoclonal and rabbit antihuman PDGF polyclonal antibodies were obtained from Genzyme (Cambridge, MA). Human TGFß and PDGF were purchased from Calbiochem (La Jolla, CA). Unless otherwise indicated, all other reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Bone marrow cell preparation
Murine bone marrow cells were prepared with a slight modification of the method described previously (20, 21). Briefly, the bone marrow of C3H/HeN mice was flushed from femurs and tibiae with ice-cold {alpha}MEM. The cells were collected, pelleted, resuspended in {alpha}MEM containing 10% FCS, plated in a 150-mm tissue culture dish, and subsequently incubated for 24 h at 37 C in the presence of 5% CO2. The nonadherent cells were then collected, pelleted (1, 500 rpm, 7 min, 4 C), and resuspended (1 x 107 cells/ml) in pronase solution (0.02% pronase and 1.5 mM EDTA in PBS, pH 7.4). After a 15-min incubation at 37 C, the reaction was stopped by adding 200 µl FCS, and the suspension was layered onto 15 ml heat-inactivated horse serum and sedimented at 1 x g for 15 min at 4 C. The cell suspension from the top of the gradient was carefully transferred onto 15 ml of another heat-inactivated horse serum gradient and centrifuged for 10 min at 3000 rpm at 4 C, and the cell pellet was suspended in {alpha}MEM containing 10% FCS.

Bone marrow/stromal cell coculture
Coculture of bone marrow cells and ST2 cells were performed in two different ways, both of which resulted in the formation of osteoclastic cells (21). In the first method, termed the continuous coculture method, fractionated nonadherent bone marrow cells (1 x 106 cells/well) were cocultured with ST2 cells (1 x 105 cells/well) in 24-well tissue culture plates in {alpha}MEM supplemented with 10% FCS in the presence of 10-8 M 1,25-(OH)2D3 and 10-7 M dexamethasone. The cultures were maintained for 10 days, with medium changed twice weekly. TPO and other reagents at the indicated concentrations were added at the times of coculture initiation and medium change.

In the second method, termed the two-step coculture method, the nonadherent bone marrow cells (1 x 106 cells/well) were grown on 24-well tissue culture plates in {alpha}MEM supplemented with 10% FCS in the presence of 5000 U/ml of M-CSF (macrophage differentiation; step 1). After 4 days, nonadherent cells were rinsed away, and the remaining adherent cells were cocultured with ST2 cells (105 cells/well) in {alpha}MEM supplemented with 10% FCS in the presence of 10-8 M 1,25-(OH)2D3 and 10-7 M dexamethasone. The cultures were maintained for additional 10 days, with medium changed twice weekly (osteoclast differentiation; step 2).

Bone resorption assay (pit assay)
Thin disc-shaped wafers (7 x 7 x 0.5 mm) of cortical bone were prepared from transverse slices of bovine femurs (diaphysis) with a low speed diamond saw (Maruto, Kyoto, Japan), cleaned by sonication, and stored in absolute ethanol at 4 C. Before use, the slices were washed with sterile PBS and placed in 48-well plate wells. The nonadherent bone marrow cells (1 x 106 cells/well) were cocultured with ST2 cells (1 x 105 cells/well) in {alpha}MEM containing 10% FCS in the presence of 10-8 M 1,25-(OH)2D3 and 10-7 M dexamethasone. After a 10-day incubation with medium changed twice weekly, the bone pits (resorption lacunae) were visualized using a slight modification of the previously described method (24). The slices were fixed with 10% formalin, washed with PBS twice, and exposed to 0.1 M sodium hydroxide for 1 h. The slices were then washed with PBS twice, sonicated twice for 30 sec to remove the cell layer from the bone, and stained with toluidine blue solution [1% (wt/vol) in 1% (wt/vol) sodium borate] for 10 min. The slices were air-dried and mounted on glass slides with Eukitt mounting medium (O. Kindler Co., Freiburg, Germany). Resorptive pits appeared as darkly stained, clearly marginated areas using light microscopy.

Cytochemical staining for tartrate-resistant acid phosphatase
The number of formed osteoclasts was evaluated by cytochemical detection of tartrate-resistant acid phosphatase (TRAP) using a commercial kit (Sigma Chemical Co.). TRAP-positive cells with three or more nuclei (TRAP-positive MNC) were counted as osteoclastic cells.

Image analysis
Toluidine blue-stained bone pits were viewed with an Olympus BX50 microscope interfaced with an image-analyzing system (OsteoMeasure, OsteoMetric, Atlanta, GA). Pit image was projected onto a television screen via a camera mounted on the microscope. Measurements of the total area of generated pits of consecutive 25 fields were made on the screen by tracing with the assistance of a digital tablet and image analysis software (OsteoMeasure version 2.2, OsteoMetric).

Statistics
In certain experiments, data were analyzed for statistical significance by ANOVA with post-hoc analysis, unless otherwise stated. These analyses were performed with the assistance of a computer program (StatView version 4.11, Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TPO effect on murine osteoclastogenesis
To investigate the effect of TPO on osteoclastogenesis, we used the continuous coculture method (1 in Fig. 1Go) (20, 21). The nonadherent bone marrow cells were cultured with ST2 cells in the presence of various concentrations of human TPO. After 10 days of coculture, osteoclastic cell number was determined. As shown in Fig. 2Go, TPO dose dependently inhibited osteoclast formation at a concentration range from 0.02–200 ng/ml; maximal inhibition (61%) occurred at 200 ng/ml, and the 50% inhibitory dose (ID50) of TPO was approximately 0.5 ng/ml.



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  		Figure 1. Cell culture methods used for osteoclastic cell development (see Materials and Methods). 1) Continuous coculture method. 2) Two-step coculture method. a and d, TPO treatment during coculture; b and c, treatment of ST2 and bone marrow cells with TPO, respectively. BMC, Bone marrow cells; OC, osteoclastic cells.

 


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  		Figure 2. The effect of TPO on in vitro osteoclastogenesis. The nonadherent bone marrow cells were cocultured with ST2 cells in the presence of 10-8 M 1,25-(OH)2D3 and 10-7 M dexamethasone as described. Various concentrations of TPO were added at the times of coculture initiation and medium change. After 10 days, the cultures were cytochemically stained for TRAP. Osteoclastic cells were counted as described and are presented as the mean (n = 4) ± SEM. The differences compared with untreated control were statistically significant (*, P < 0.05, by Fisher’s protected least significant difference test). CTL and TRAP(+)MNC indicate untreated control and TRAP-positive multinucleated cells, respectively.

 
To confirm the specificity of TPO effect, we used rabbit antihuman TPO polyclonal antibody that had been proven to inhibit TPO binding to its receptor (c-mpl; data not shown). This antibody dose dependently neutralized the inhibitory effect of TPO on osteoclastogenesis, whereas equivalent amounts of rabbit IgG had no effect (data not shown), confirming that the inhibitory effect is specific for TPO.

As osteoclasts are the principal cells for bone resorption, we next examined the effect of TPO on the differentiation of bone-resorbing capacity by pit assay. Osteoclastic cells were induced on the bone slices in the presence of various concentrations of TPO by the 10-day coculture described above. The pit areas were evaluated by image analysis after the bone slices had been appropriately stained. Resorption pits appeared as darkly stained, clearly marginated areas using light microscopy, and TPO dramatically inhibited bone resorption, as shown in Fig. 3Go. Quantitatively, the total areas of pits were dose dependently decreased by treatment with TPO, and maximal inhibition (80%) was observed at 200 ng/ml (Fig. 4Go). These results suggest that TPO inhibits the differentiation of bone-resorbing capacity as well.



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  		Figure 3. Photomicrographs of resorption pits (magnification, x74). a, Control; b, 2 ng/ml TPO; c, 20 ng/ml TPO; d, 200 ng/ml TPO. Osteoclastic cells were induced on the bone slices. After 10 days of culture, the slices were processed and stained to demonstrate resorption pits.

 


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  		Figure 4. Effect of TPO on formation of resorption pits. Osteoclastic cells were induced, and bone slices were processed and stained as described. Total pit area and total bone area were measured in each bone slice by image analysis, and the percent pit area in each group was calculated and plotted. Data are presented as the mean (n = 3) ± SEM, and the differences compared with untreated control were statistically significant (*, P < 0.05, by Fisher’s protected least significant difference test). CTL, Untreated control.

 
TPO cellular target in osteoclastogenesis
To clarify the target cells of TPO in osteoclastogenesis, we first examined whether its effect is dependent on the time of addition using the continuous coculture method. TPO was added at the time of culture initiation or on days 4 and 7 thereafter, and osteoclastic cell number was determined on day 10. As shown in Fig. 5Go, TPO inhibited osteoclast formation only when it was added at the time of coculture (day 1). The addition of TPO, when delayed to either day 4 or 7 of coculture, failed to impair osteoclastic differentiation. These results suggest that the principal effects of TPO occurred during the first 4 days of the coculture period.



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  		Figure 5. TPO’s inhibitory effect on osteoclastic cell formation requires early exposure. Bone marrow/ST2 coculture was established as described in Fig. 2Go, and TPO (200 ng/ml) was added at culture initiation or on days 4 and 7 thereafter. Osteoclastic cells were enumerated, and the results are presented as the mean (n = 4) ± SEM. The 1–10 group was statistically different from all other groups (*, P < 0.05, by Fisher’s protected least significant difference test). CTL and TRAP(+)MNC indicate untreated control and TRAP-positive multinucleated cells, respectively.

 
To further delineate the cell population impacted by TPO, we designed a two-step coculture experiment (2 in Fig. 1Go) (21). As we previously reported, a M-CSF-dependent adherent cell population consisting principally of macrophages was isolated by the treatment with 5000 U/ml M-CSF for 4 days (macrophage differentiation; step 1). Then, bone marrow macrophages were cocultured with ST2 cells in the presence of 1,25-(OH)2D3 and dexamethasone for additional 10 days, yielding multinucleated TRAP-positive cells with features identical to those generated in the previous experiments (osteoclast differentiation; step 2). By using this method, we examined the effects of TPO on ST2 cells, bone marrow cells, and the coculture of macrophages with ST2 cells. Pretreatment of ST2 cells with TPO (Fig. 1bGo) did not affect osteoclastogenesis (Fig. 6Go). Together with the data presented in Fig. 5Go, these findings suggest that the main effect of TPO is on macrophage differentiation from bone marrow cells. To confirm this hypothesis, we examined the effect of TPO on step 1 culture in osteoclastogenesis (Fig. 1cGo). Bone marrow cells were inoculated for 4 days with TPO (0.2–200 ng/ml) in the presence of M-CSF (5000 U/ml) and then cocultured for additional 10 days with ST2 cells in the presence of 1,25-(OH)2D3 and dexamethasone. As shown in Fig. 7aGo, osteoclastogenesis was dose dependently inhibited by TPO (0.2–200 ng/ml), and maximal inhibition was 39.4% at 200 ng/ml. To exclude the possibility that the differentiation of adherent bone marrow macrophages into osteoclasts is affected by TPO, we examined the effect of TPO on step 2 (Fig. 1dGo). TPO did not exert its inhibitory effect on osteoclastogenesis from bone marrow macrophages (Fig. 7bGo). These data suggest that the main target of TPO in osteoclastogenesis is the differentiation stage from bone marrow cells into macrophages in the presence of M-CSF.



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  		Figure 6. Pretreatment of ST2 with TPO has no effect on osteoclastogenesis. ST2 cells were pretreated for 72 h with the indicated concentrations of TPO. After treatment, nonadherent bone marrow cells were added to the ST2 cell layer and cultured for an additional 10 days as described in Fig. 2Go. Osteoclastic cells were counted as described and are presented as the mean (n = 3) ± SEM. No statistical difference was observed compared with the control. CTL and TRAP(+)MNC indicate untreated control and TRAP-positive multinucleated cells, respectively.

 


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  		Figure 7. TPO’s effect on osteoclastogenesis in two-step coculture. Nonadherent bone marrow cells were cultured for 4 days in the presence of 5000 U/ml M-CSF (macrophage differentiation; step 1). After 4 days, the adherent bone marrow cells were cocultured with ST2 cells for an additional 10 days as described. The number of osteoclastic cells was determined, and the results are presented as the mean (n = 4) ± SEM. TPO treatment was performed in step 1 (a) and step 2 (b) cultures, respectively. The differences compared with the control were statistically significant (*, P < 0.05, by Fisher’s protected least significant difference test). CTL and TRAP(+)MNC indicate untreated control and TRAP-positive multinucleated cells, respectively.

 
TGFß and PDGF mediate the inhibitory effect of TPO on osteoclastogenesis
As bone marrow cells prepared with the described method contain stem cells capable of differentiating into megakaryocytes, it is likely that the effect of TPO on step 1 culture in osteoclastogenesis may be exerted by the formation of megakaryocytes. In fact, TPO stimulated megakaryocyte formation during macrophage differentiation (step 1 culture) as detected by May-Giemsa staining (data not shown). Megakaryocytes produce cytokines and growth factors, such as TGFß and PDGF, which are known to impact on skeletal metabolism (25, 26). Therefore, it is likely that TGFß and/or PDGF mediate the inhibitory effect of TPO on osteoclastogenesis as the result of increased thrombopoiesis in step 1 culture (Fig. 1cGo). To test this hypothesis, we used mouse anti-TGFß monoclonal and rabbit antihuman PDGF-BB polyclonal antibodies to antagonize the effect of TPO on step 1 culture in osteoclastogenesis. Mouse anti-TGFß monoclonal antibody has been proven to neutralize mouse TGFß1, -ß2, and -ß3 (27). Neutralization of mouse PDGF-BB and PDGF-AB with rabbit antihuman PDGF-BB antibody has also been reported (28). Anti-TGFß and anti-PDGF antibodies dose dependently antagonized the inhibitory effect of TPO on osteoclastogenesis (Figs. 8aGo and 9aGo, respectively). Interestingly, high doses of anti-TGFß and anti-PDGF antibodies (25 and 5 µg/ml, respectively) almost completely restored the osteoclast-forming potential. Moreover, neither anti-TGFß nor anti-PDGF antibodies affected megakaryocyte formation in the presence of TPO (data not shown). Furthermore, to confirm mediation of the effect of TPO by TGFß and PDGF, we examined the effects of TGFß and PDGF on step 1 culture in osteoclastogenesis. TGFß and PDGF dose dependently inhibited osteoclast formation, and maximal inhibition occurred at 41.9% (1.0 ng/ml) and 47.9% (100 ng/ml), respectively (Figs. 8bGo and 9bGo). Additionally, neither TGFß nor PDGF stimulated megakaryocyte formation from bone marrow cells in step 1 culture (data not shown). These data evidently suggest that TPO exerts its inhibitory effect on osteoclastogenesis through stimulating thrombopoiesis and that TGFß and PDGF, major cytokines produced by megakaryocytes, mediate the effect of TPO by impacting on macrophage-lineage cells as osteoclast precursors.



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  		Figure 8. TGFß mediates the effect of TPO on osteoclastogenesis. Osteoclastic cells were generated by the two-step coculture method. The step 1 culture was treated with TPO, mouse anti-TGFß monoclonal antibody, and mouse IgG1 (a) or TGFß (b) as indicated, respectively. The number of osteoclastic cells was counted as described, and the results are presented as the mean (n = 3) ± SEM. The differences compared with the control were statistically significant (*, P < 0.05, by Fisher’s protected least significant difference test). TRAP(+)MNC indicates TRAP-positive multinucleated cells.

 


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  		Figure 9. PDGF mediates the effect of TPO on osteoclastogenesis. Osteoclastic cells were generated by the two-step coculture method. The step 1 culture was treated with TPO, rabbit anti-PDGF polyclonal antibody, and rabbit IgG (a) or PDGF (b) as indicated, respectively. The number of osteoclastic cells was counted as described, and the results are presented as the mean (n = 3) ± SEM. The differences compared with the control were statistically significant (*, P < 0.05, by Fisher’s protected least significant difference test). TRAP(+)MNC indicates TRAP-positive multinucleated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that the inhibitory effects of TPO on osteoclastic cell formation using the continuous coculture model of osteoclastogenesis occurred early in the culture period. Based on the two-step coculture experiments, the TPO cellular target was present in the bone marrow cells. As the expression of TPO receptor (c-mpl) is restricted to primitive hematopoietic stem cells, megakaryocytes, and platelets (12), we hypothesized that TPO exerts its inhibitory effect on osteoclastogenesis through stimulation of thrombopoiesis as the result of acting on early hematopoietic progenitor cells. TPO actually stimulated megakaryocyte formation in step 1 culture, as detected by May-Giemsa staining (data not shown). Moreover, we confirmed that the expression of c-mpl in bone marrow cells, but not in ST2 cells, was detected by reverse transcription-PCR (data not shown). In adherent bone marrow macrophages, c-mpl was barely expressed (data not shown). Therefore, it is likely that the inhibitory effect of TPO on osteoclastogenesis is associated with megakaryocyte formation. Finally, we have demonstrated that TGFß and PDGF, major cytokines elaborated by megakaryocytes, mediate the inhibitory effects of TPO on osteoclastogenesis.

There is a possibility that TPO may inhibit the proliferation or accelerate the death of osteoclast precursors by impacting on bone marrow cells. In this regard, we observed that there is no remarkable difference in the number of adherent macrophages present in step 1 culture between TPO-treated and -untreated groups (data not shown). Furthermore, it is worth noting that the inhibitory effect of TPO on the bone-resorbing capacity of osteoclasts, as evidenced by pit assay, was greater than its effect on the number of in vitro generated osteoclast, suggesting that TPO inhibits osteoclastogenesis as well as the function of osteoclasts. Therefore, it is necessary to clarify the effect of TPO on mature osteoclasts regarding bone-resorbing capacity.

Megakaryocytes produce TGFß and PDGF, which may be involved in the development of myelofibrosis (13, 14, 15). Recently, Yan et al. demonstrated that plasma levels of TGFß1 and PDGF increase in TPO-overexpressing mice and that these mice develop myelofibrosis and osteosclerosis (16). It is, therefore, suggested that TPO may play an important role in the development of these pathological conditions. Osteosclerosis is often noted in myelofibrosis and is thought to be induced mainly by increased osteogenic activity (29, 30). However, whether derangement of osteoclastic bone resorption is involved in the development of osteosclerosis remains unclear. The inhibitory effect of TPO on osteoclastogenesis as shown in this study may contribute to the pathogenesis of osteosclerosis. Additionally, whether TPO can prevent bone loss in certain pathological conditions with increased bone resorption also remains to be clarified.

As we have shown in this study, TGFß and PDGF mediate the inhibitory effect of TPO on osteoclastogenesis and impact mainly on macrophage lineage cells in the bone marrow cell population. TGFß has been shown to inhibit the formation of osteoclast-like cells from human and mouse bone marrow cells (31, 32) and may induce granulocyte-macrophage CFU to differentiate preferentially to cells of the granulocytic lineage (31). Additionally, TGFß has been recognized as a negative regulator of several macrophage functions, including nitric oxide production and immunological responses (33, 34, 35). Therefore, it is likely that TGFß can impair the osteoclast-forming potential of macrophage-lineage cells. However, the mechanism of TGFß action on macrophage lineage cells still remains unclear.

On the other hand, PDGF has been reported to stimulate osteoclast-like cell formation and bone resorption (36, 37). However, in these studies PDGF exerts its stimulatory effect on osteoclastogenesis in the coexistence with osteoblastic cells (26, 36). Surprisingly, as shown in this study, PDGF inhibited osteoclastogenesis by directly acting on bone marrow cells in the absence of stromal/osteoblastic cells. Therefore, PDGF may directly impair the osteoclast-forming potential of macrophage lineage cells.

Finally, anti-TGFß and anti-PDGF antibodies, as we have shown in this study, each completely antagonized the effect of TPO on macrophage lineage cells in osteoclastogenesis, suggesting that these two factors interdependently act on these cells. However, the relationship between TGFß and PDGF in their actions on osteoclastogenesis remains to be clarified.


   Acknowledgments
 
We thank Kirin Brewery Co., Chugai Pharmaceutical Co., and Green Cross Co. for providing recombinant human TPO, rabbit antihuman TPO polyclonal antibody, 1,25-(OH)2D3, and human M-CSF, respectively.

Received March 24, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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