help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Biskobing, D. M.
Right arrow Articles by Rubin, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Biskobing, D. M.
Right arrow Articles by Rubin, J.
Endocrinology Vol. 138, No. 11 4852-4857
Copyright © 1997 by The Endocrine Society

ARTICLES

Induction of Carbonic Anhydrase II Expression in Osteoclast Progenitors Requires Physical Contact with Stromal Cells1

Diane M. Biskobing, Dongjie Fan, Xian Fan and Janet Rubin

Department of Medicine, Emory University School of Medicine and Veterans Administration Medical Center, Atlanta, Georgia 30033

Address all correspondence and requests for reprints to: Diane M. Biskobing, VA Medical Center - 111, 1670 Clairmont Road, Decatur, Georgia 30033. E-mail: dbiskob{at}emory.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Carbonic anhydrase II (CA II) expression is vital to normal osteoclast function. We and others have previously reported induction of CA II messenger RNA (mRNA) expression by 1,25(OH)2D3 in myelomonocytic cells and marrow culture. However, since 1,25(OH)2D3 stimulates osteoclast differentiation as well, we wished to separate direct effects of 1,25(OH)2D3 on the CA II gene from the differentiating effects of the hormone. Using primary murine mixed marrow cultures, we measured CA II mRNA expression by RT-PCR. 10 nM 1,25(OH)2D3 dose dependently induced expression of CA II mRNA (4.12 ± 0.68-fold) at day 4 in culture compared with control with an ED50 of 0.25 nM. When nonadherent marrow cells containing osteoclast progenitors were depleted of stromal cells and exposed to 10 nM 1,25(OH)2D3, CA II mRNA expression was decreased by more than 60%. Coculture of progenitors with ST-2 stromal cells for 3 days with 10 nM 1,25(OH)2D3 stimulated CA II expression by 22 ± 3.6-fold. 1,25(OH)2D3 stimulated CA II mRNA expression in progenitors separated from ST-2 cells by transwells was insignificant demonstrating that the two cell types must be in physical contact. PTH also stimulated CA II mRNA expression (4.91 ± 0.01-fold) to a similar degree as seen with 1,25(OH)2D3 treatment. These results demonstrate that induction of CA II in osteoclast progenitors requires their physical communication with stromal cells and is inseparable from the osteoclast differentiation process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS HEMATOPOIETIC precursors differentiate toward an osteoclast phenotype, they express proteins vital to osteoclast function (1). One of these proteins is carbonic anhydrase II (CA II), an enzyme that catalyzes the production of protons, necessary for bone resorption, from CO2 and H2O (2, 3). 1,25(OH)2D3 has previously been shown to stimulate CA II expression in cell culture and avian marrow cultures (4, 5, 6) and a vitamin D response element (VDRE) consensus sequence has been identified in the chicken CA II promoter (7). However, because 1,25(OH)2D3 also is known to promote osteoclast formation, the question remains whether stimulation of CA II expression in marrow culture is due to direct up-regulation of the CA II gene by 1,25(OH)2D3 or whether it arises during the 1,25(OH)2D3 induced program of osteoclastogenesis, which requires the presence of accessory stromal cells.

Regulation of CA II messenger RNA (mRNA) expression has previously been studied in myelomonocytic cell lines (4, 5, 6, 8). In the myelomonocytic HL-60 cell line and transformed avian myelomonocytes, CA II mRNA expression is increased by PMA and/or 1,25(OH)2D3 (ibid). While these myelomonocytic cell lines are models of colony forming units for granulocytes and macrophages, they do not readily form osteoclasts and may not be a good model for osteoclast differentiation (8, 9). In addition, CA II expression occurs in the absence of stromal cells that are known to be essential for full expression of the osteoclast phenotype (10). In avian marrow cells, CA II expression is detected within 24 h of exposure to 1,25(OH)2D3 and increases further as the cells differentiate toward mature, multinucleated osteoclasts (5). These authors suggest that 1,25(OH)2D3 may be directly increasing transcription of CA II via action on the promoter (ibid) and later demonstrated increased transcription in an avian myelomonocytic cell line (8). To separate possible effects of 1,25(OH)2D3 on the CA II promoter from the 1,25(OH)2D3 induced differentiation process, we studied CA II mRNA expression during osteoclastogenesis in primary mixed marrow.

This report describes regulation of CA II mRNA expression in mixed murine marrow cultures. CA II mRNA expression was studied in osteoclast precursors at day 4 in culture before fusion. We found that induction of CA II mRNA in precursors is intrinsic to the differentiation pathway toward the osteoclast phenotype and requires physical contact with stromal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
C57BL/6NCR male mice were obtained from Frederick Cancer Center (Frederick, MD). The immortalized stromal ST-2 cell line was kindly provided by Dr. Ed Greenfield (Case Western Reserve, Cleveland, OH). L929 cells, derived from fibrosarcoma cells, was obtained from the American Type Culture Collection (Rockville, MD). 1,25(OH)2D3 was purchased from Biomol (Plymouth Meeting, PA). FBS and media were obtained from Atlanta Biological (Atlanta, GA). Molecular biology reagents were obtained from Promega (Madison, WI). Bovine PTH (1–34) was obtained from Calbiochem (La Jolla, CA). Transwells were purchased from Corning (Cambridge, MA). Other chemicals were obtained from Sigma (St. Louis, MO).

Cell culture
Primary marrow culture: 6- to 28-week-old mice were killed by cervical dislocation after CO2 anesthesia. Femorae and tibiae were aseptically removed and dissected free of adhering tissue. Bone ends were cut off and marrow cavity flushed with {alpha}-MEM using a 25G needle and marrow cells washed twice with {alpha}-MEM (11). Cells were cultured in {alpha}-MEM containing 10% FBS at 2 x 106 cells/well in 24-well plates. Twenty-four hours after plating, cultures were treated with 10 nM 1,25(OH)2D3 for 3 days. All cultures were maintained in a humidified atmosphere of 5% CO2 at 37 C.

Nonadherent marrow cells.
Primary marrow cells were incubated with 0.83% NH4Cl/Tris-HCl buffer for 5 min to burst red cells, washed, and plated overnight in {alpha}-MEM with 10% FCS. The nonadherent cells were removed and replated in a 24-well dish at a density of 1 x 106/well. All nonadherent marrow cultures were treated with 2.5% L929 conditioned media to maintain cell viability. These cultures exhibited 99% nonspecific esterase staining and were negative for alkaline phosphatase (data not shown).

Coculture with ST-2 cells.
ST-2 cells were plated overnight in 24-well plates at a density of 40,000/well in {alpha}-MEM with 10% FCS. 1 x 106 nonadherent cells were plated over ST-2 cells the next day. For transwell experiments, 60,000 ST-2 cells were plated in 12-well plates, and 24 h later a transwell placed in the well into which 1.5 x 106 nonadherent marrow cells were plated. All cocultures were treated with 10-7 M dexamethasone and when indicated 10-8 M 1,25(OH)2D3.

L929 conditioned media
L929 cells were cultured in RPMI media with 10% FCS. Conditioned media was collected from cells grown for 1 week. Macrophage colony stimulating factor (MCSF) levels in the conditioned media were assayed using the MCSF-dependent cell line M-NFS-60 bioassay as described previously (12).

RT-PCR
RNA was extracted by the method of Chomcynzski (13). Cells were lysed in 4 M guanidium isothiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol, at pH 7. To the lysate was added 1/10 vol of 2 M sodium acetate (pH 4), 1 vol phenol, and 2/10 vol chloroform/isoamyl alcohol. This mixture was cooled on ice for 15 min and centrifuged 15 min at 4 C. The RNA was precipitated, washed with 75% ethanol, and resuspended in 20 µl water containing 40 U RNAsin with 2 mM DTT. A quantity of 0.5 µg of the isolated RNA was added to a 10 µl RT reaction containing: 2.5 mM deoxy-NTPs, 1 x buffer, 2 µM reverse primer, recombinant RNAse H-moloney murine leukemia virus reverse transcriptase, RNAsin, and an RT primer specific for carbonic anhydrase II [CAII RT primer: 5'-CGCCAGTTGTCC-3' (734–723)], incubated for 30 min at 37 C. PCR was performed on the 10 µl of RT product. PCR: 200 µM deoxy-NTPs, 0.5 µM of PCR primers, PCR buffer (provided by Perkin-Elmer, Foster City, CA), 1.6 µM MgCl2, 10 µl of RT reaction is heated to 95 C for 2 min and then Taq polymerase added. The forward primer was end-labeled with 32P-{gamma}-ATP using T4 kinase to allow visualization and quantitation. Fifteen microliters of of PCR products were run on a 15% acrylamide gel, silver stained, and exposed to a phosphorimager to measure density. Glyceraldehyde dehydrogenase phosphate (GAP) was used as control. Data are expressed as CAII/GAP. The CA II reaction was carried out for 23 cycles, annealing at 56 C. Cycle experiments were carried out to ascertain optimal conditions; CA II RT-PCR product plateaued at 25 cycles, GAP at 23 cycles. PCR conditions were selected for log-phase expansion. Primers: CA II reverse primer: 5'-ATCCAGGTCACACATTCCAGC-3' (626–606), CAII forward primer: 5'-CTCTCAG-GACAATGCAGTGC-3' (216–235), GAP reverse primer: 5'-CAAAGTTGTCATGGATGACC-3' (545–526), GAP forward primer: 5'-TGTCATCAA-CGGGAAGC-3' (239–257).

Statistics
Differences between means were evaluated between groups in Figs. 2Go, 3Go, and 5Go, and Table 1Go by ANOVA. Differences between means in Figs. 1Go and 4Go were evaluated by Student’s t test.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 2. 1,25(OH)2D3 dose response. Mixed marrow cultures were treated for 3 days with increasing doses of 1,25(OH)2D3 and total RNA extracted for RT-PCR of CA II and GAP. A, A representative phosphorimage is shown with densities expressed as CA II/GAP in OD units normalized to control cultures. B, The figure shows pooled data from two separate experiments. Results demonstrate an ED50 of 0.25 nM 1,25(OH)2D3 for induction of CA II mRNA. *, P < 0.05 compared with control.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Time course for 1,25(OH)2D3 induced CA II mRNA expression. Mixed marrow cultures were treated with 10 nM 1,25(OH)2D3 for 1–3 days before RNA extraction. RT-PCR was performed for CA II and GAP. Pooled data from nine separate experiments is depicted, demonstrating that 3 days of treatment was required for a significant increase in CA II mRNA expression. Results are expressed as CAII/GAP as follows: control, 1 ± 0.05; 1 day, 1.02 ± 0.13; 2 days, 1.67 ± 0.2; 3 days, 4.12 ± 0.68; *, P < 0.001 compared with control.

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Physical contact with stromal cells is required for expression of CA II by osteoclast precursors. Coculture: mixed marrow cells were plated overnight in 10% FCS. The next day nonadherent cells were plated onto ST-2 cells and treated with 100 nM dexamethasone alone or combined with 10 nM 1,25(OH)2D3. Transwells: ST-2 cells were plated in 12-well plate. A transwell was placed in the well and nonadherent cells plated in the transwell to prevent physical contact with the ST-2 cells. Cultures were treated with dexamethasone alone or combined with 1,25(OH)2D3. Results pooled from three separate experiments show a 22 ± 3.6-fold increase in CA II expression in cocultures treated with 10 nM 1,25(OH)2D3, *, P < 0.001 compared with control. Separation of ST-2 and nonadherent marrow cells prevents the large increase in CA II seen with 1,25(OH)2D3 treatment.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Conditioned media does not stimulate increased CA II expression

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. CA II mRNA expression in mixed marrow cultures. Mixed marrow cells were cultured for 4 days before total RNA extraction. 10 nM 1,25(OH)2D3 was added 24 h after plating. Total RNA was subjected to RT-PCR for CA II or GAP in separate reactions using a 32P-labeled forward primer to allow for visualization. PCR products were run on a 15% acrylamide gel and the gel exposed to a phosphorimager for quantitation. Densitometric readings expressed as CA II/GAP are normalized to control conditions. 1,25(OH)2D3 treated cultures showed a 4.12 ± 0.68-fold increase in CA II mRNA expression compared with control cultures, P < 0.05. The figures shows pooled data from seven separate experiments.

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. CA II mRNA expression in the absence of stromal cells. Mixed marrow cells were plated in 10% FCS overnight. Nonadherent cells were removed the next day and replated. Twenty-four hours later, 10 nM 1,25(OH)2D3 was added to indicated cultures. Four days after plating, total RNA was extracted and subjected to RT-PCR for CA II and GAP. Results expressed as CAII/GAP and normalized to control cultures are representative of pooled data from four separate experiments. The figure demonstrates a 64 ± 6% inhibition of CA II expression in 1,25(OH)2D3 treated cells compared with control, P < 0.01.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
1,25(OH)2D3 increased CA II mRNA expression in mixed marrow culture
The effect of 1,25(OH)2D3 on expression of CA II mRNA in marrow cultures was studied by semiquantitative RT-PCR. Treatment with 10 nM 1,25(OH)2D3 for 3 days beginning 24 h after plating mixed marrow cultures resulted in a 4.12 ± 0.68-fold increase in CA II mRNA expression compared with control (Fig. 1Go). 1,25(OH)2D3 did not change cell number: control cultures had equivalent number of cells (7.13 x 105/ml) as those treated with 10 nM 1,25(OH)2D3 (6.93 x 105/ml). Figure 2AGo shows a representative phosphorimage of PCR products demonstrating a dose response to 1,25(OH)2D3. Figure 2BGo depicts pooled data from two separate experiments, demonstrating that the ED50 for 1,25(OH)2D3 stimulation of CA II was 0.25 nM. To better understand the mechanism of CA II induction, we looked at the time course of increased expression. In HL-60 cells, we had previously shown increased expression at 12 h, but maximal stimulation required at least 48 h treatment (4). To determine the time course of CA II expression in this culture system, 10 nM 1,25(OH)2D3 was added to cultures for 24, 48, or 72 h before RNA extraction on day 4 of culture. Figure 3Go demonstrates that 1,25(OH)2D3 had no effect on cells exposed for only 24 h and required a full 3 days exposure to see significant effects.

1,25(OH)2D3 does not stimulate CA II mRNA expression in the absence of stromal cells
We next studied CA II in the absence of stromal elements. To deplete the marrow cultures of stromal elements, mixed marrow cells were plated overnight and the nonadherent fraction replated. These cells consist of monocytic precursors that exhibit 99% nonspecific esterase staining and are negative for alkaline phosphatase. Because these cells are of the monocytic lineage, they require MCSF for survival (14). 2.5% L929 conditioned media was used as a source of MCSF to achieve a final concentration of 875 U/ml. Cultures of nonadherent marrow cells treated with 1,25(OH)2D3 for 3 days did not show increased CA II expression and in fact showed a greater than 60% decrease in expression compared with control cultures (Fig. 4Go).

To ensure that the nonadherent marrow cells were precursors capable of forming mature osteoclasts, the cells were plated on ST-2 cells that support osteoclast formation in coculture (15). When the nonadherent marrow cells were plated on ST-2 cells in the presence of dexamethasone and 1,25(OH)2D3 for 7 days, tartrate resistant acid phosphatase positive multinucleated cells (TRAP + MNC) were formed, indicating that the nonadherent marrow cells contained osteoclast progenitors (data not shown). When these cells were plated on ST-2 cells for 4 days, 3 day treatment with 10 nM 1,25(OH)2D3 stimulated CA II expression 22 ± 3.6-fold (shown in the two left bars in Fig. 5Go), a significantly greater degree than that seen with primary cultures. ST-2 cells alone do not express CA II mRNA and do not form TRAP+MNCs in our hands (data not shown) and in the laboratories of others (14, 15).

Induction of CA II mRNA by 1,25(OH)2D3 is not via a soluble factor
To determine whether the stromal cells induce CAII mRNA expression via a soluble factor, nonadherent marrow cells were treated with conditioned media. Two different sources of conditioned media were used: ST-2 culture media under conditions that induce CA II expression in coculture with nonadherent marrow cells (dexamethasone and 1,25(OH)2D3) and control conditions (dexamethasone alone) and primary marrow cultures treated ± 1,25(OH)2D3. 50% conditioned media from either primary or ST-2 cultures was added to cultures of nonadherent marrow cells and CA II mRNA expression measured after 3 days (Table 1Go). 1,25(OH)2D3 alone significantly decreased CA II expression in the nonadherent marrow cells as previously shown. The addition of 50% conditioned media from primary cultures (control or 1,25(OH)2D3 treated) had no significant effect on CA II expression. The increase in CA II expression seen with conditioned media from ST-2 cells treated with dexamethasone + 1,25(OH)2D3 was not significant. Treatment with 50% conditioned media from ST-2 cells treated with dexamethasone alone produced a small increase in CA II mRNA expression compared with control cultures not treated with conditioned media. However, CA II mRNA expression in cultures exposed to conditioned media treated with 1,25(OH)2D3 was not significantly different than cultures treated with conditioned media under control conditions.

Because the small increase in CA II expression seen in the presence of ST-2 cell conditioned media suggested a minor role for a labile soluble factor released by the cells, we performed experiments using transwells. Each experiment consisted of four groups: 1) a standard coculture with nonadherent marrow cells plated on ST-2 cells treated with dexamethasone alone; 2) standard coculture treated with dexamethasone and 1,25(OH)2D3; 3) dexamethasone treated coculture of ST-2 cells and nonadherent marrow cells separated by a membrane allowing flow of media and secreted substances; and 4) dexamethasone and 1,25(OH)2D3 treated coculture of ST-2 cells and nonadherent marrow cells separated by a membrane. As previously described, coculture with dexamethasone and 1,25(OH)2D3 resulted in 22 ± 3.6-fold increase in CA II mRNA expression compared with cultures treated with dexamethasone alone. However, when cellular contact was prevented by the use of transwells, treatment with 1,25(OH)2D3 resulted in a small rise (1.9 ± 0.7-fold) in CA II mRNA expression (Fig. 5Go). The increase was significantly less than that seen in coculture when cellular contact was maintained and was not significantly different than control.

PTH stimulates CA II mRNA expression in mixed cultures
We then asked if the CA II response was specific to 1,25(OH)2D3 or could be evoked by other agents known to promote osteoclast differentiation. Mixed marrow cultures were treated with 10 nM 1,25(OH)2D3, 25 ng/ml PTH, or neither for 3 days. CA II expression was similarly stimulated approximately 4-fold by either 1,25(OH)2D3 or PTH. Combined treatment did not cause an additive effect (Fig. 6Go).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 6. PTH stimulates CA II mRNA expression to a similar degree as 1,25(OH)2D3. Mixed marrow cells were cultured for 4 days before total RNA extraction. Either 10 nM 1,25(OH)2D3 or 25 ng/ml PTH was added 24 h after plating. Total RNA was subjected to RT-PCR for CA II or GAP and 32P-labeled PCR products run on an acrylamide gel. A representative phosphorimage is shown, demonstrating induction of CA II expression by 1,25(OH)2D3 or PTH. Combined treatment was not additive. The experiment was repeated showing similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate here that 1,25(OH)2D3 stimulates CA II mRNA expression in osteoclast precursors raised in mixed marrow cultures. Significant induction of CA II expression by 1,25(OH)2D3 requires at least 3 days of treatment indicating that the response to 1,25(OH)2D3 is not due to a direct effect on transcription. In fact, 1,25(OH)2D3 diminishes CA II expression in monocytic precursors cultured in the absence of stromal cells. Therefore, our results suggest that CA II expression results from effects of differentiating agents on accessory stromal cells, creating a microenvironment that promotes osteoclast differentiation.

The necessity of stromal cells during osteoclast differentiation has been demonstrated by other investigators. The formation of mature osteoclast-like cells does not occur when hematopoietic precursors are separated from stromal cells by a membrane (10, 15, 16, 17). Our results suggest that the accessory function of stromal cells begins at an early time point: mononuclear osteoclast progenitors are not induced to express CA II mRNA when separated from stromal cells. From these findings, it can be surmised that an unidentified factor presented by the stromal cell membrane influences monocytic entry into the osteoclastic differentiation pathway. A possible candidate is membrane bound MCSF, which is known to increase in response to 1,25(OH)2D3 (12) and PTH (18).

These findings contrast with the previously reported ability of 1,25(OH)2D3 to directly induce CA II mRNA expression in the HL-60 or avian monomyelocytic cell lines (4, 5, 6, 8). As well, we have been unable to show an effect of PMA on CA II expression in primary marrow cultures (data not shown) despite our previously reported synergistic action of 1,25(OH)2D3 and PMA on CA II expression in the HL-60 cell line (4). Thus, CA II expression in these cell lines does not adequately reflect the pattern of CA II expression by cells capable of becoming mature osteoclasts. In particular, in cultures with osteoclastogenic potential physical contact with stromal cells is required for induction of CA II expression. Because stromal cells are known to be required for osteoclast differentiation, we hypothesized that the increased CA II mRNA expression was a result of differentiation of the cell toward the osteoclast phenotype rather than a direct response to 1,25(OH)2D3. To investigate this question, we substituted PTH, which also induces osteoclastogenesis (19, 20), in the role of differentiator. PTH also significantly increased CA II mRNA expression, indicating that the effect of either 1,25(OH)2D3 or PTH on CA II mRNA expression cannot be separated from regulatory control of the differentiation program.

In fact, in the absence of stromal cells 1,25(OH)2D3 actually decreases CA II expression in monocytic precursor cells. The decreased expression of CA II seen when monoblasts are treated with 1,25(OH)2D3 in the absence of stromal cells may indicate that these precursors are stimulated to undergo macrophage differentiation (21). Commitment to the macrophage phenotype should decrease CA II expression as this isozyme is not found in macrophages (5, 22). In contrast, the previously reported ability of 1,25(OH)2D3 to increase CA II expression in monomyelocytic cell lines (4, 5, 6, 8) may be due to direct action on the CA II gene. A putative VDRE has been identified in the CA II gene (7), and it may be an effect on this cis element, which is responsible for the minimal increase in CA II expression seen in the transwell experiments in the presence of 1,25(OH)2D3 reported here, as well as the response of the monomyelocytic cell lines (4, 5, 6, 8). Alternatively, David et al. (23) have reported that 1,25(OH)2D3 treatment results in activation of an AP-1 site on the CA II promoter rather than direct activation of a VDRE. Stimulation of c-fos and c-jun transcription factors during progenitor maturation may account for the increased CA II expression seen during programmed differentiation.

The paradigm of interaction between stromal cells and osteoclast precursors or the mature osteoclast occurs throughout the culture period (24, 25). Increased osteoclastogenesis resulting from estrogen deficiency, for instance, is due to effects on the stromal cells rather than osteoclast precursors. Estrogen decreases stromal cell production of the osteoclast promoting cytokine IL-6 (26) and estrogen can alter MCSF production by stromal cells (27). Even in giant cell tumors of bone, an example of dysregulated osteoclast formation, this hierarchy is maintained: the giant osteoclasts arise due to stimulation from a neoplastic stromal cell (28). The ability of both PTH and 1,25(OH)2D3 to stimulate early CA II expression shown here, as well, depends on the physical presence of stromal cells. It has recently been shown that bone resorption by mature osteoclasts also requires the physical presence of stromal cells (29, 30). These numerous examples illustrate that bone resorption, i.e. osteoclast recruitment and activity, is a process that cannot be disassociated from other cells within the bone environment.

In summary, our findings show that hematopoietic stem cell contact with bone marrow stromal cells is essential for the early entry of monocytic precursors into the osteoclast lineage. We provide further evidence that stromal membrane bound factors are critical to the differentiation and expression of osteoclast functional machinery such as CA II. These finding suggest that restriction of osteoclast formation and function to the bone marrow environment evolves from the symbiotic relationship between osteoclasts and bone stromal cells.


   Footnotes
 
1 This work was supported by NIH Grant AR-01945–02 (to D.M.B.), NIH Grant AR-42360 (to J.R.), and a VA Merit award (to J.R.). Back

Received March 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Prallet B, Male P, Neff L, Baron R 1992 Identification of a functional mononuclear precursor of the osteoclast in chicken medullary bone marrow cultures. J Bone Miner Res 7:405–414[Medline]
  2. Blair H, Schlesinger P, Ross F, Teitelbaum S 1993 Recent advances toward understanding osteoclast physiology. Clin Orthop Relat Res 294:7–22
  3. Gay C, Mueller W 1974 Carbonic anhydrase and osteoclasts: localization by labeled inhibitor autoradiography. Science 183:432–434[Abstract/Free Full Text]
  4. Biskobing D, Nanes M, Rubin J 1994 1,25(OH)2D3 and phorbol myristate acetate synergistically increase expression of carbonic anhydrase II in a human monomyelocytic cell line. Endocrinology 134:1493–1498[Abstract]
  5. Billecocq A, Emanuel J, Levenson R, Baron R 1990 1,25-dihydroxyvitamin D3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells. Proc Natl Acad Sci USA 87:6470–7474[Abstract/Free Full Text]
  6. Shapiro L, Venta P, Yu Y, Tashian R 1989 Carbonic anhydrase II is induced in HL-60 cells by 1,25-dihydroxyvitamin D3: a model for osteoclast gene regulation. FEBS Lett 249:307–310[CrossRef][Medline]
  7. Quelo I, Kahlen J, Rascle A, Jurdic P, Carlberg C 1994 Identification and characterization of a vitamin D3 response element of chicken carbonic anhydrase II. DNA Cell Biol 13:1181–1187[Medline]
  8. Lomri A, Baron R 1992 1,25-dihydroxyvitamin D3 regulates the transcription of carbonic anhydrase II mRNA in avian myelomonocytes. Proc Natl Acad Sci USA 89:4688–4692[Abstract/Free Full Text]
  9. Yoneda T, Alsina M, Garcia J, Mundy G 1991 Differentiation of HL-60 cells into cells with the osteoclast phenotype. Endocrinology 129:683–689[Abstract]
  10. Takahashi N, Akatsu T, Udagawa N, Sasaki T, Yamaguchi A, Moseley J, Martin T, Suda T 1988 Osteoblastic cells are involved in osteoclast formation. Endocrinology 123:2600–2602[Abstract]
  11. Biskobing D, Fan X, Rubin J 1995 Characterization of MCSF-induced proliferation and subsequent osteoclast formation in murine marrow culture. J Bone Miner Res 10:1025–1032[Medline]
  12. Rubin J, Fan X, Thornton D, Bryant R, Biskobing D 1996 Regulation of murine osteoblast macrophage colony-stimulating factor production by 1,25(OH)2D3. Calcif Tissue Int 59:291–296[CrossRef][Medline]
  13. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  14. Jimi E, Shuto T, Koga T 1995 MCSF and IL-1 maintain survival of osteoclast-like cells. Endocrinology 136:808–811[Abstract]
  15. Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin T, Suda T 1990 Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA 87:7260–7264[Abstract/Free Full Text]
  16. Quinn J, McGee J, Athanasou N 1994 Cellular and hormonal factors influencing monocyte differentiation to osteoclastic bone-resorbing cells. Endocrinology 134:2416–2423[Abstract]
  17. Kodama H, Nose M, Niida S, Yamasaki A 1991 Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J Exp Med 173:1291–1294[Abstract/Free Full Text]
  18. Yao G, Insogna K, Weir E 1995 Osteotropic agents regulate expression of the cell-surface form of colony-stimulating factor-1 (CSF-1) in osteoblasts. J Bone Miner Res [Supp 1] 10:50 (Abstract)
  19. MacDonald B, Takahashi N, McManus L, Holahan J, Mundy G, Roodman G 1987 Formation of multinucleated cells that respond to osteotropic hormones in long term human bone marrow cultures. Endocrinology 120:2326–2333[Abstract]
  20. Takahashi N, Yamana H, Yoshiki S, Roodman D, Mundy G, Jones S, Boyde A, Suda T 1988 Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122:1373–1381[Abstract]
  21. Fan X, Biskobing D, Fan D, Hofstetter W, Rubin J 1997 Macrophage colony stimulating factor downregulates MCSF-receptor expression and entry of precursors into the osteoclast lineage. J Bone Miner Res 12:1387–1395[CrossRef][Medline]
  22. Sundquist K, Leppilampi M, Jarvelin K, Kumpulainen T, Vaananen H 1987 Carbonic anhydrase isoenzymes in isolated rat peripheral monocytes, tissue macrophages, and osteoclasts. Bone 8:33–38[Medline]
  23. David J, Rincon M, Baron R 1995 AP1 regulation of the chicken carbonic anhydrase II promoter. J Bone Miner Res [Supp 1] 10:M563
  24. Shevde N, Anklesaria P, Greenberger J, Bleiberg I, Glowacki J 1994 Stromal cell-mediated stimulation of osteoclastogenesis. Proc Soc Exp Biol Med 205:306–314[Abstract]
  25. Martin T, Ng K 1994 Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity. J Cell Biochem 56:357–366[CrossRef][Medline]
  26. Girasole G, Jilka R, Passeri G, Boswell S, Boder G, Williams D, Manolagas S 1992 17ß-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest 89:883–891
  27. Kimble R, Srivastava S, Ross F, Matayoshi A, Pacifici R 1996 Estrogen deficiency increases the ability of stromal cells to support murine osteoclastogenesis via an interleukin-1 and tumor necrosis factor-mediated stimulation of macrophage colony-stimulating factor production. J Biol Chem 271:28890–28897[Abstract/Free Full Text]
  28. Robinson D, Einhorn T 1994 Giant cell tumor of bone: a unique paradigm of stromal-hematopoietic cellular interactions. J Cell Biochem 55:300–303[CrossRef][Medline]
  29. Jimi E, Nakamura I, Amano H, Taguchi Y, Tsurukai T, Tamura M, Takahashi N, Suda T 1996 Osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell contact. Endocrinology 137:2187–2190[Abstract/Free Full Text]
  30. James I, Dodds R, Lee-Rykaczewski E, Eichman C, Connor J, Hart T, Maleet B, Lackman R, Gowen M 1996 Purification and characterization of fully functional human osteoclast precursors. J Bone Miner Res 11:1608–1618[Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. Rubin, D. M. Biskobing, L. Jadhav, D. Fan, M. S. Nanes, S. Perkins, and X. Fan
Dexamethasone Promotes Expression of Membrane-Bound Macrophage Colony-Stimulating Factor in Murine Osteoblast-Like Cells
Endocrinology, March 1, 1998; 139(3): 1006 - 1012.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Biskobing, D. M.
Right arrow Articles by Rubin, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Biskobing, D. M.
Right arrow Articles by Rubin, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals