Teun J. de Vries

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock‐out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild‐type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild‐type and CD44 k.o. mice. Bone marrow cells from wild‐type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M‐CSF) and receptor activator of NF‐kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild‐type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild‐type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild‐type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild‐type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment. J. Cell. Biochem. 94: 954–966, 2005.

Osteoclast heterogeneity: lessons from osteopetrosis and inflammatory conditions

The multinucleated osteoclast has a unique function: degradation of mineralized tissues. It is generally taken that all osteoclasts are alike, independent of the skeletal site where they exert their activity. Recent data, however, question this view as they show that osteoclasts at different bony sites appear to differ, for example in the machinery responsible for resorption. Support for the notion that there may be heterogeneity in osteoclasts is obtained from studies in which osteoclast activity is inhibited and from observations in osteopetrosis and inflammatory bone conditions. In this review we discuss the available evidence and propose the existence of bone-site-specific osteoclast heterogeneity.

Gingival fibroblasts are better at inhibiting osteoclast formation than periodontal ligament fibroblasts

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast‐like cells. Here, we investigated whether a second population of tooth‐associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co‐cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1α,25dihydroxycholecalciferol (dex + vit D3) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co‐cultures with PLF than in GF‐PBMC co‐cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D3. In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D3. OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast‐like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast‐PBMC cultures with M‐CSF and RANKL. Our results suggest that tooth‐associated fibroblasts may trigger the formation of osteoclast‐like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts. J. Cell. Biochem. 98: 370–382, 2006.

Ae2(a,b)-Deficient mice exhibit osteopetrosis of long bones but not of calvaria

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pHi) is thought to be kept at a near‐neutral level by chloride/bicarbonate exchange. Here we show that the Na+‐independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the longbonesof Ae2a,b–/– mice (deficient in the main isoforms Ae2a, Ae2b1, and Ae2b2). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long‐bone marrow cells from Ae2a,b–/– mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2a,b–/– mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts—but not long‐bone osteoclasts—possess a sodium‐dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2a,b–/– mice.—Jansen, I. D. C., Mardones, P., Lecanda, F., de Vries, T. J., Recalde, S., Hoeben, K. A., Schoenmaker, T., Ravesloot, J.‐H., van Borren, M. M. G. J., van Eijden, T. M., Bronckers, A. L. J. J., Kellokumpu, S., Medina, J. F., Everts, V., Oude Elferink, R. P. J. Ae2a,b‐Deficient mice exhibit osteopetrosis of long bones but not of calvaria. FASEB J. 23, 3470–3481 (2009). www.fasebj.org

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis

OBJECTIVE Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src activity, could selectively interfere with both tumor and osteoclast activity. Here we explored the effects of AZD0530 on human osteoclast differentiation and activity. The effect on osteoclasts formed in vivo was assessed in mouse fetal calvarial explants and in isolated rabbit osteoclasts, where it dose-dependently inhibited osteoclast activity. Its effect on formation and activity of human osteoclasts in vitro was determined in cocultures of human osteoblasts and peripheral blood mononuclear cells. AZD0530 was most effective in inhibiting osteoclast-like cell formation when present at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial phase of osteoclast formation. Formation of active phosphorylated c-Src, which was highly present in osteoclast-like cells in cocultures and in peripheral blood mononuclear cell monocultures, was significantly reduced by AZD0530. Furthermore, it reversibly prevented osteoclast precursor migration from the osteoblast layer to the bone surface and subsequent formation of actin rings and resorption pits. These data suggest that Src is pivotal for the formation and activity of human osteoclasts, probably through its effect on the distribution of the actin microfilament system. The reversible effect of AZD0530 on osteoclast formation and activity makes it a promising candidate to temper osteoclastic bone degradation in bone diseases with enhanced osteoclast activity such as osteolytic metastatic bone disease. (Mol Cancer Res 2009;7(4):476–88)

Direct Cell-Cell Contact Between Periodontal Ligament Fibroblasts and Osteoclast Precursors Synergistically Increases the Expression of Genes Related to Osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell–cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell–cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co‐culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule‐1 (ICAM‐1) and of osteoclastogenesis‐related genes (RANKL, RANK, TNF‐α, and IL‐1β) was highly up‐regulated in the co‐cultures compared to mono‐cultures and the 5–10‐fold up‐regulation reflected a synergistic increase due to direct cell–cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)2VitD3 and dexamethasone. In case of indirect cell–cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast‐like cells that were formed in co‐culture with periodontal ligament fibroblasts was significantly augmented compared to mono‐cultures. Our data indicate that cell–cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts. J. Cell. Physiol. 222: 565–573, 2010.

Vitamin B-12 deficiency stimulates osteoclastogenesis via increased homocysteine and methylmalonic acid

The risk of nutrient deficiencies increases with age in our modern Western society, and vitamin B12 deficiency is especially prevalent in the elderly and causes increased homocysteine (Hcy) and methylmalonic acid (MMA) levels. These three factors have been recognized as risk factors for reduced bone mineral density and increased fracture risk, though mechanistic evidence is still lacking. In the present study, we investigated the influence of B12, Hcy, and MMA on differentiation and activity of bone cells. B12 deficiency did not affect the onset of osteoblast differentiation, maturation, matrix mineralization, or adipocyte differentiation from human mesenchymal stem cells (hMSCs). B12 deficiency caused an increase in the secretion of Hcy and MMA into the culture medium by osteoblasts, but Hcy and MMA appeared to have no effect on hMSC osteoblast differentiation. We further studied the effect of B12, Hcy, and MMA on the formation of multinucleated tartrate-resistant acid phosphatase–positive osteoclasts from mouse bone marrow. We observed that B12 did not show an effect on osteoclastogenesis. However, Hcy as well as MMA were found to induce osteoclastogenesis in a dose-dependent manner. On the basis of these results, we conclude that B12 deficiency may lead to decreased bone mass by increased osteoclast formation due to increased MMA and Hcy levels.

Myeloid blasts are the mouse bone marrow cells prone to differentiate into osteoclasts

Cells of the myeloid lineage at various stages of maturity can differentiate into multinucleated osteoclasts. Yet, it is unclear which developmental stages of this lineage are more prone to become osteoclasts than others. We investigated the osteoclastogenic potential of three successive stages of myeloid development isolated from mouse bone marrow. Early blasts (CD31hi/Ly‐6C–), myeloid blasts (CD31+/Ly‐6C+), and monocytes (CD31–/Ly‐6Chi), as well as unfractionated marrow cells, were cultured in the presence of M‐CSF and receptor activator of NF‐κB ligand (RANKL), and the differentiation toward multinucleated cells and their capacity to resorb bone was assessed. Myeloid blasts developed rapidly into multinucleated cells; in only 4 days, maximal numbers were reached, whereas the other fractions required 8 days to reach maximal numbers. Bone resorption was observed after 6 (myeloid blasts and monocyte‐derived osteoclasts) and 8 (early blast‐derived osteoclasts) days. This difference in kinetics in osteoclast‐forming capacity was confirmed by the analysis of osteoclast‐related genes. In addition, the myeloid blast fraction proved to be most sensitive to M‐CSF and RANKL, as assessed with a colony‐forming assay. Our results show that osteoclasts can develop from all stages of myeloid differentiation, but myeloid blasts are equipped to do so within a short period of time.

Intercellular adhesion molecule-1 clusters during osteoclastogenesis

Adhesion between osteoblasts and osteoclast precursors is established via an interaction involving intercellular adhesion molecule-1 (ICAM-1) on osteoblasts and leukocyte function-associated antigen-1 (LFA-1) on osteoclast precursors. The latter cells also express ICAM-1, but little is known about the expression over time and its possible role during osteoclastogenesis. In the present study we investigated the expression of ICAM-1 on both human osteoblast-like cells and osteoclast precursors in a co-culture. The protein expression on osteoclast precursors strongly increased whereas the osteoblast-like cells became ICAM-1 negative. Interestingly, ICAM-1 on osteoclast precursors manifested as clusters which localized at the baso-lateral membrane. Furthermore, clustered ICAM-1 was associated with F-actin and remained present for several days. Our data suggest that osteoblastic ICAM-1 is mainly involved in the initial adhesion of osteoclast precursors whereas clustered ICAM-1 on osteoclast precursors and its association with F-actin suggest an involvement in cell movement at a later stage.