Ton Schoenmaker

Transcription factor C/EBPβ isoform ratio regulates osteoclastogenesis through MafB

Disequilibrium between bone‐forming osteoblasts and bone‐resorbing osteoclasts is central to many bone diseases. Here, we show that dysregulated expression of translationally controlled isoforms of CCAAT/enhancer‐binding protein β (C/EBPβ) differentially affect bone mass. Alternative translation initiation that is controlled by the mammalian target of rapamycin (mTOR) pathway generates long transactivating (LAP*, LAP) and a short repressive (LIP) isoforms from a single C/EBPβ transcript. Rapamycin, an inhibitor of mTOR signalling increases the ratio of LAP over LIP and inhibits osteoclastogenesis in wild type (WT) but not in C/EBPβ null (c/ebpβ−/−) or in LIP knock‐in (L/L) osteoclast precursors. C/EBPβ mutant mouse strains exhibit increased bone resorption and attenuated expression of MafB, a negative regulator of osteoclastogenesis. Ectopic expression of LAP and LIP in monocytes differentially affect the MafB promoter activity, MafB gene expression and dramatically affect osteoclastogenesis. These data show that mTOR regulates osteoclast formation by modulating the C/EBPβ isoform ratio, which in turn affects osteoclastogenesis by regulating MafB expression.

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

Calvarial Osteoclasts Express a Higher Level of Tartrate-Resistant Acid Phosphatase than Long Bone Osteoclasts and Activation Does not Depend on Cathepsin K or L Activity

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.

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.

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.

IL-1β favors osteoclastogenesis via supporting human periodontal ligament fibroblasts.

The balance between bone formation and bone resorption in inflammatory diseases is often disturbed. Periodontitis, a chronic inflammation of the tooth gums, leads to unwanted bone loss as a response to inflammatory compounds such as interleukin‐1β (IL‐1β). This excessive bone loss reflects an increased osteoclast formation and activity. Osteoclast formation is a multistep process driven by osteoclastogenesis supporting cells such as periodontal ligament fibroblasts. The inflammatory factors can induce osteoclastogenesis, probably also by affecting the periodontal ligament fibroblast. In this study we investigated how pre‐culture of periodontal ligament fibroblasts with IL‐1β affected osteoclastogenesis. Fibroblasts were pre‐cultured with IL‐1β and/or dexamethasone, a commonly used anti‐inflammatory compound, before being co‐cultured with peripheral blood mononuclear cells (PBMCs). Pre‐culture with IL‐1β (1–100 ng/ml) resulted in an increased number of adhered PBMCs as well as an increased mRNA expression of intercellular adhesion molecule‐1 (ICAM‐1), macrophage colony stimulating factor (M‐CSF) and IL‐1β. Pre‐culture with IL‐1β also caused retraction of fibroblasts and an augmented formation of TRACP+ multinucleated cells. Our data suggest that stimulation of fibroblasts with IL‐1β has a long‐lasting effect, leading to a significantly increased osteoclastogenesis. These results provide new insights for understanding excessive bone loss in periodontitis. J. Cell. Biochem. 112: 1890–1897, 2011.

Inhibitory regulation of osteoclast bone resorption by signal regulatory protein α

Osteoclasts mediate bone resorption, which is critical for bone development, maintenance, and repair. Proper control of osteoclast development and function is important and deregulation of these processes may lead to bone disease, such as osteoporosis. Previous studies have shown that the cytosolic protein tyrosine phosphatase SHP‐1 acts as a suppressor of osteoclast differentiation and function, but putative inhibitory receptors that mediate recruitment and activation of SHP‐1 in osteoclasts have remained unknown. In the present study, we identify the SHP‐1‐recruiting inhibitory immunoreceptor signal regulatory protein (SIRP) α as a negative regulator of osteoclast activity. SIRPα is expressed by osteoclasts, and osteoclasts from mice lacking the SIRPα cytoplasmic tail and signaling capacity display enhanced bone resorption in vitro. Consequently, SIRPα‐mutant mice have a significantly reduced cortical bone mass. Fur‐thermore, osteoclasts from SIRPα‐mutant mice show an enhanced formation of actin rings, known to be instrumental in bone resorption. SIRPα mutation did not significantly affect osteoclast formation, implying that the role of SIRPα was limited to the regulation of mature osteoclast function. This identifies SIRPα as a bona fide inhibitory receptor that regulates the bone‐resorption activity and supports a concept in which osteoclast function is balanced by the signaling activities of activating and inhibitory immunoreceptors.—Van Beek, E. M., de Vries, T. J., Mulder, L., Schoenmaker, T., Hoeben, K. A., Matozaki, T., Langenbach, G. E. J., Kraal, G., Everts, V., van den Berg, T. K. Inhibitory regulation of osteoclast bone resorption by signal regulatory protein α. FASEB J. 23, 4081‐4090 (2009). www.fasebj.org

Increased osteoclast formation and activity by peripheral blood mononuclear cells in chronic liver disease patients with osteopenia.

Osteoporosis is a common complication of chronic liver disease, and the underlying mechanisms are not understood. We aimed to determine if osteoclasts develop from osteoclast precursors in peripheral blood mononuclear cells (PBMCs) of chronic liver disease patients with osteopenia compared with controls. PBMCs were isolated and fluorescence‐activated cell sorting was performed to quantify the activated T lymphocyte population and receptor activator of nuclear factor κβ ligand (RANKL) expression. The activated T lymphocyte populations were comparable for all 3 groups, and RANKL was not detectable. The percentage of CD14+CD11b+ cells containing osteoclast precursors was comparable between the 3 groups. To assess the formation and functional activity of osteoclasts formed from circulating mononuclear cells, PBMCs were cultured (1) without addition of cytokines, (2) with macrophage colony‐stimulating factor (M‐CSF), (3) with M‐CSF and osteoprotegerin, and (4) with M‐CSF and RANKL. The number of tartrate‐resistant acid phosphatase‐positive multinucleated cells and bone resorption was assessed. PBMCs from chronic liver disease patients with osteopenia formed more osteoclast‐like cells, which, when cultured in the presence of M‐CSF and RANKL resorbed more bone than controls. The number of osteoclast‐like cells and the amount of bone resorption correlated with lumbar bone densities. Addition of M‐CSF increased numbers of osteoclast‐like cells formed in healthy controls; however, this was not observed in either of the chronic liver disease groups. Plasma levels of M‐CSF were elevated in both patient groups compared with healthy controls. Conclusion: Circulating mononuclear cells from chronic liver disease patients with osteopenia have a higher capacity to become osteoclasts than healthy controls or chronic liver disease patients without osteopenia. This could partially be due to priming with higher levels of M‐CSF in the circulation. (HEPATOLOGY 2007.)