Osteoclast Responses to Lipopolysaccharide, Parathyroid Hormone and Bisphosphonates in Neonatal Murine Calvaria Analyzed by Laser Scanning Confocal Microscopy
Department of Pharmacology, School of Dentistry, Showa University, Tokyo, Japan (KS,SY); Division of Pharmacology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, Sendai, Japan (ST,TK,HS); and CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada (JS)
Correspondence to: Keiko Suzuki, PhD, Department of Pharmacology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan. E-mail: suzukik{at}dent.showa-u.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: calvarial cells bone remodeling bisphosphonates osteopontin vß3 integrin confocal microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bone resorption initially involves the differentiation of large multinucleated tartrate-resistant acid phosphatase (TRACP)-positive osteoclasts from the fusion of TRACP-negative monocytic precursors, stimulated by receptor activator of nuclear factor B-ligand (RANK-L) and macrophage colony-stimulating factor (M-CSF) (Lacey et al. 1998; Yasuda et al. 1998
). Mature osteoclasts are highly motile cells that form a tight attachment to the mineralized bone surface to be resorbed through a sealing zone, which involves the rearrangement of the cytoskeleton so that actin forms a dense belt-like structure, the "actin ring" (Lakkakorpi et al. 1989
). The part of the plasma membrane enclosed by the actin enlarges into the highly convoluted ruffled border through which protons and liposome proteases such as cathepsin K, a cysteine protease highly active in digesting native collagen fibers, are transported (Vaananen et al. 2000
). Formation of the ruffled border and the intravesicular transport of degradation products are modulated by TRACP (Hollberg et al. 2002
), whereas the attachment of osteoclasts to the bone surface involves ligation of osteoclast integrins with extracellular matrix proteins within the bone matrix.
Results of numerous studies of osteoclasts and osteoblasts have been obtained from cells cultured on artificial substrates, such as plastic or glass. However, two-dimensional monolayer cultures are limited by distortions introduced by the cells having to adapt to artificial flat and rigid surfaces (Themistocleous et al. 2004). Studies using three-dimensional culture systems, which have the potential to simulate cellcell interactions that take place in tissues under physiological and pathophysiological conditions, have been used successfully in the investigation of complex biological processes such as angiogenesis, wound healing, tumor invasion, and metastasis (Edelman and Keefer 2005
). Consequently, the importance of studying cells within their natural environment is being increasingly recognized.
Organ cultures of fetal and neonatal bones, in which the patency of local interactions between osteoblastic and osteoclastic cells is retained within the context of a three-dimensional bone matrix (Stern and Krieger 1983), are frequently used for the biochemical analysis of bone remodeling. However, few studies have examined the cellular responses to factors that modulate bone cell activity. Therefore, we have used laser scanning confocal microscopy to study the effects on bone cells of PTH and LPS, which stimulate bone resorption, and bisphosphonates (BPs) that suppress bone resorption in cultured neonatal mouse calvaria. Responses were determined by fluorescence staining of calvaria for TRACP activity, F-actin, ß3 integrin, osteopontin (OPN), alkaline phosphatase (ALP) activity, collagen type I, bone sialoprotein (BSP), and bone mineral.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fluorescent Staining of Calvaria
Calvaria were cut into four pieces along the sutures producing one frontal, two parietal, and one occipital bone. Only parietal bones were used in these experiments. Bone pieces were washed twice with PBS, blocked with 2% BSA-0.01% Triton X-100 in PBS (BSA buffer) for 1 hr, and incubated with primary antibodies (diluted 1:100 in BSA buffer) overnight at 4C. After washing with BSA buffer for 2 hr, bones were incubated with secondary antibodies (diluted 1:200 in BSA buffer) for 2 hr, washed with BSA buffer for 2 hr, and mounted on glass slides using a PermaFluor (ThermoShandon; Pittsburgh, PA) aqueous mounting medium. Rabbit anti-human OPN (LF123), which cross-reacts with murine OPN, was generously provided by Dr. L.W. Fisher (National Institutes of Health; Bethesda, MD). Hamster anti-mouse ß3 integrin monoclonal antibody (clone 2C9.G2), diluted 1:100, was from BD Biosciences (San Jose, CA). Alexa Fluor 488- or rhodamine-conjugated phalloidin was from Molecular Probes (Eugene, OR). Rabbit anti-rat type I collagen, which cross-reacts with murine type I collagen, was from LSL Co. Ltd. (Tokyo, Japan). Expression of BSP was determined using an affinity-purified antibody raised in rabbits against the N-terminal peptide of rat BSP. This antibody is specific for bone cells and cross-reacts with mouse BSP.
TRACP activity was detected as described previously (Mostafa et al. 1982) but using naphthol AS-MX phosphate as a substrate, and ALP activity was detected by an ELF 97 endogenous phosphatase detection kit (Molecular Probes). Mineral was stained by incubation with Alizarin Red S diluted at 10 mg/ml in 0.25% ammonia solution for 10 min (Burdi 1965
).
Osteoclast Culture and Fluorescent Staining
Bone marrow cells obtained from tibias and femurs of 5- to 7-week-old ddY mice were treated with 0.83% NH4Cl in 10 mM of Tris-HCl buffer (pH 7.4) for 20 min on ice to hemolyze red blood cells. The remaining cells were suspended in 0.51.0 ml of -minimal essential medium (
-MEM) with 10% FCS adjusted to pH 7.0 after adding FCS and applied onto a Sephadex G-10 (Amersham Pharmacia Biotech; Uppsala, Sweden) column (bed volume 510 ml) and incubated for 45 min at 37C to remove macrophages and stromal cells. Non-adherent cells were collected by eluting with culture media and plated onto Lab-Tek chamber slides (eight-well glass slide; Nalge Nunc International, Naperville, IL) at a density of 5 x 105 cells/cm2 and then cultured in
-MEM with 10% FCS for 3 days in the absence or presence of 25 µM clodronate or 2.5 µM risedronate. The Lab-Tek chamber slides were precoated by adding 10 µl/well of mixture of recombinant human M-CSF (10 ng/ml of culture media; Pepro Tech EC Ltd., London, UK) and recombinant human soluble receptor activator of nuclear factor
B (NF-
B) ligand (sRANKL, 50 ng/ml of culture media; Pepro Tech EC Ltd.), which was air dried for 2 hr.
For fluorescent staining, cells were fixed with 2% paraformaldehyde for 15 min at 4C. After washing with PBS and treating with 0.1% Triton X-100 in PBS for 5 min, cells were incubated with Alexa Fluor 488-conjugated phalloidin (diluted 1:100 in PBS; Molecular Probes) for 30 min.
Laser Scanning Confocal Microscopy and Image Processing
Stained calvaria and osteoclast cultures were examined under a laser scanning confocal microscope equipped with an optical laser unit and a scanning unit (Inverted System Microscope IX70; Olympus Optical Co., Tokyo, Japan). Images were obtained with a UPLAPOx20 [numerical aperture (N.A.): 0.70] or a UPLAPOx40 (N.A.: 0.85) objective lens at various magnifications (1 to 10x) obtained using the microscope software. Alexa Fluor 488 and ELF 97 alcohol precipitate, a product generated by the enzymatic cleavage of ELF 97 phosphatase substrate, were excited with the 488-nm line of an air-cooled argon ion laser and detected with a 510/540 band-pass emission filter (FVX-BA510/540). Alexa Fluor 594, Texas Red, rhodamine, Alizarin Red, and TRACP staining were excited with the 568-nm line of an air-cooled krypton ion laser and detected with a 585-nm long-pass barrier filter (FVX-BA585IF) to avoid the carryover between green and red fluorescence. Z-stacks of serial optical sections of XY images taken at the indicated intervals were processed to create a composite image by estimating the regions of best focus using image-processing and analysis software (MetaMorph; Universal Imaging Co., West Chester, PA).
Statistical Analyses
The area and volume of resorption lacunae, the area of regions stained with Alizarin Red S or fluorescent-conjugated phalloidin, and the percent of resorption were averaged from a minimum of three values in replicate experiments from which the mean ± SD (mean ± SEM in area and volume of resorption lacunae) were calculated. Levene's test was used to determine homogeneity of variance, and Bonferroni's procedure was used in the endpoint adjustment of p values for multiplicity, using Statistical Package for Social Science (SPSS) for Windows 11.0.1J (SPSS Japan Inc.; Tokyo, Japan).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
Actin (Figure 4B) also colocalized with OPN (Figure 4C), as observed in resorbing osteoclasts stimulated by 10 µg/ml LPS. The colocalization was observed at the periphery of the bone-resorbing osteoclasts (Figure 4D) and coincided with the edges of resorption lacunae (Figure 4A), as seen with the actin and ß3 integrin. In cultures treated with 2.5 µM risedronate in combination with LPS (Figures 4E4J), the resorption lacunae were reduced in size and depth (Figure 4E), and the staining of actin (Figure 4F) and especially the OPN (Figure 4G) were markedly reduced and their peripheral colocalization lost (Figure 4I: merged image of Figures 4E and 4H). The loss of F-actin and OPN, which colocalized in multinucleated osteoclasts, was more clearly evident at higher magnification (Figure 4J).
XZ (Depth) Analyses of Double-stained Calvaria by Using a Laser Scanning Confocal Microscope
To further demonstrate the spatial organization of actin and ß3 integrin, images of osteoclasts double stained for actin and ß3 integrin were taken at a focal plane beginning 16.1 µm above (Figures 5A5D) the level of the sealing zone (Figures 3B3D) in LPS-treated calvaria. Serial optical sections through to 35.7 µm above the resorption lacunae revealed cells with morphological features of osteoblasts. To determine whether these actin-staining cells were osteoblasts, calvaria were double stained for actin and ALP activity (Figures 6A6K), for collagen type I and mineral (Figures 7A7H), or for BSP and mineral (Figures 7I and 7J). The ALP and actin appear to be coexpressed (Figures 6H and 6K, yellow) as expected in osteoblastic cells, which fill the resorbed area and proliferate on top of the resorbed area of bone (Figures 6F6K). That the osteoblastic cells were newly formed was confirmed by experiments in which bromodeoxyuridine added to the culture media was incorporated into the cell nuclei (data not shown). In actin-stained areas lacking ALP activity at the periphery of the resorption area (indicated as blue and pink lines in Figures 6H and 6K, respectively), the actin appears localized to the sealing zone of bone-resorbing osteoclasts. The osteoblastic cells stained strongly for collagen type I (Figures 7F and 7H), whereas little staining for collagen is observed in control calvaria (Figures 7B and 7D). Similarly, in the LPS-treated cultures the putative osteoblastic cells were concentrated at resorption sites, as indicated by the diminished staining with Alizarin Red, and stained strongly for BSP (Figure 7J), which is a marker of mature osteoblasts (Ganss et al. 1999). In contrast, much weaker staining for BSP was observed in the cells associated with the shallow resorption lacunae in control calvaria (Figure 7I).
From XZ (depth)-scans of the calvaria double stained for actin and mineral after culturing under various conditions (Figure 8), it was evident from the actin staining that cell proliferation had been stimulated in LPS-treated calvaria (Figure 8J). In control (Figure 8B) and clodronate-treated calvaria (Figure 8F), little accumulation of cells was observed, indicating that the proliferation of bone-forming cells occurs in response to accelerated bone resorption induced by LPS. The area of the remaining bone mineral and proliferation of bone-forming cells were measured from the mineral staining in XZ images of calvaria stained with Alizarin Red S and actin staining in XZ images of calvaria stained with Alexa Fluor 488-conjugated phalloidin, respectively, assuming that the ALPase-positive and collagen-producing cells were bone-forming cells. The area of the remaining bone mineral in control and LPS was 12.6 ± 2.6 (mean ± SD; n=40) and 7.0 ± 1.8 µm2 (mean ± SD; n=49), respectively. The areas of proliferation of bone-forming cells were 0.2 ± 0.3 (mean ± SD; n=40) and 12.7 ± 6.0 µm2 (mean ± SD; n=49), respectively. There was a statistically significant correlation between these parameters in the LPS group (r = 0.699, p=0.002, n=49). Furthermore, it was evident from the Alizarin Red staining of mineral (Figures 8O and 8S) and the immunostaining of actin (Figures 8N and 8R), that LPS-induced resorption was decreased in the presence of BPs whereas the accumulation of osteoblastic cells was still evident, albeit not as marked as in calvaria treated with LPS only (Figure 8J).
Together these observations suggest that the cells occupying areas where bone resorption has taken place are ALP-positive, collagen type I-, and BSP-producing osteoblastic cells, and that there is an induction of bone formation coupled with LPS-stimulated bone resorption. Notably, similar results were obtained when 108 M PTH was used to stimulate bone resorption (Suzuki et al. 2003).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Regulation of osteoclastic activity by BPs is used extensively in the treatment of metabolic bone diseases associated with increased bone resorption. However, the precise mechanism of action on bone metabolism of BPs is still unclear. BPs cause osteoclast retraction, condensation, cellular fragmentation, and induce apoptosis, recognized by morphological changes (Schenk et al. 1973; Flanagan and Chambers 1989
; Hughes et al. 1995
). BPs also inhibit osteoclast recruitment and differentiation (Lowik et al. 1988
; Hughes et al. 1989
; Cecchini and Fleisch 1990
), the attachment of osteoclasts to the bone surface (Colucci et al. 1998
), and ruffled border formation (Sato et al. 1991
), which are essential for bone resorption. Notably, these effects may be a consequence of BPs interfering with the remodeling of the actin cytoskeleton (Selander et al. 1994
; Murakami et al. 1995
) and microtubule formation, which are required for the formation of cell processes and osteoclast fusion (Figure 2). However, BPs also affect osteoblasts, causing the release of a factor that inhibits osteoclast activity or formation (Sahni et al. 1993
; Vitte et al. 1996
). Although it is unclear which of these processes of bone resorption is most sensitive to inhibition by BPs, it has been suggested that the most likely route by which BPs inhibit bone resorption is through a direct effect on resorbing osteoclasts (Rogers et al. 2000
).
Although there have been many investigations on the effect of BPs on osteoclast differentiation and formation, the results remain controversial. In our studies, BP caused vacuolarization (Figures 2B2D, 2G2I) and the retraction of pseudopods (Figures 2B2D, 2F2I) in some osteoclasts. Although cytoplasmic vacuolarization could reflect toxic effects of BPs, whether it is associated with apoptosis or causes the suppression of bone resorption activity is not clear at present. As observed previously (Gravel et al. 1994; Suzuki et al. 2002a
), reduced pseudopod formation is suggestive of an impaired ability of preosteoclasts to fuse, an important step in osteoclastic differentiation. Although BPs may inhibit the resorption-stimulating activity by osteoblasts, it is evident from our studies on TRACP-positive mononuclear cells derived from bone marrow that BPs inhibit M-CSF and RANKL-induced fusion of osteoclast precursors in the absence of osteoblasts (Figures 2F and 2G).
That cell adhesion molecules play an important role in skeletal growth, development, and homeostasis is well established. Cell attachment molecules such as OPN and its vß3 integrin receptor are involved in osteoclast differentiation, migration to sites of resorption, fusion of postmitotic osteoclast precursors, cellular polarization, and tight sealing zone formation required for bone resorption (Reinholt et al. 1990
; Yamate et al. 1997
; Nakamura et al. 1999
; Duong et al. 2000
; McHugh et al. 2000
; Chellaiah and Hruska 2003
). The cessation of resorption by detachment of osteoclasts from the bone matrix also involves adhesion molecules (Ek-Rylander et al. 1994
; Katayama et al. 1998
), which are important for osteoclast survival (Horton et al. 2002
). In addition to mediating cell adhesion, cell attachment molecules signal through receptors and affect cytoskeletal remodeling and gene transcription.
OPN, which interacts with the vß3 integrin through an RGD sequence (Ross et al. 1993
; Grano et al. 1994
) and mediates the attachment of osteoclasts to the bone surface (Reinholt et al. 1990
; Flores et al. 1992
), has a prominent role in bone resorption. OPN is localized to the basolateral surfaces in the clear zone and in ruffled border membranes (Andersson and Johansson 1996
; Chellaiah et al. 2003
) and is deposited in the resorption pits during bone resorption. Although OPN in the lamina limitans coating the bone surface is produced by osteoblasts following bone formation (McKee and Nanci 1995
), osteoclasts also produce OPN (Tezuka et al. 1992
; Tong et al. 1994
), which is found in resorption lacunae formed during the remodeling of intramembranous and endochondral bone (Maeda et al. 1994
) and in developing osteophytes (Dodds et al. 1995
). The presence of the OPN is likely important for the chemotaxis and attachment of lining cells that migrate into the resorption lacunae and initiate bone formation (Everts et al. 2002
). In addition to mediating attachment, OPN stimulates the resorptive activity of osteoclasts (Horton et al. 1995
; Chellaiah and Hruska 2003
), reflecting its cytokine properties that also appear to be important in the migration and fusion of osteoclast precursors (Suzuki et al. 2002a
).
OPN secreted from the basolateral surfaces of osteoclasts during bone resorption (Chellaiah et al. 2003) acts as an autocrine factor by binding to the
vß3 integrin (Chellaiah and Hruska 2003
). In previous studies we have shown that OPN colocalizes with the ß3 integrin exclusively at the cell periphery and in the cell processes of prefusion osteoclasts cultured with M-CSF and RANKL (Suzuki et al. 2002b
). However, colocalization of OPN and ß3 integrin is not evident in the sealing zone of multinuclear osteoclasts grown on glass, even though an actin ring was formed around the cell periphery (Suzuki et al. 2003
). In contrast, in calvarial organ cultures we have confirmed that both OPN and ß3 integrin colocalize with actin along the edge of resorbing osteoclasts (Figure 3 and Figure 4). These results indicate that the cytoskeletal reorganization involved in the formation of a sealing zone around resorbing osteoclasts is strictly regulated by the substrata on which the cell locates and emphasizes the importance of studying these cells in their natural environment. Although our results do not directly demonstrate the importance of OPN in cytoskeletal organization, they support previous studies that have demonstrated this relationship (Katayama et al. 1998
; Suzuki et al. 2002a
).
There have been few reports regarding the effects of BPs on the expression of OPN and vß3 integrin in bone cells in spite of their functional importance in bone metabolism. Although BPs have been reported to downregulate OPN mRNA expression by normal osteoblasts in culture (Sodek et al. 1995
) and by an osteoblastic cell line (Mackie et al. 2001
), which can impact on the recruitment and bone-resorptive activity of osteoclasts, there are no reports on the effect of BPs on OPN expression in osteoclasts. However, BPs have been shown to inhibit the adhesion of human osteoclast-like cells onto coverslips coated with BSP, but not fibronectin, indicative of the interference with receptors such as
vß3 integrins that specifically recognize bone matrix proteins (Colucci et al. 1998
) including OPN. Although it is not known whether BPs have a direct effect on OPN expression by osteoclasts, our studies clearly show that BP treatment of calvaria greatly diminishes the production of OPN, which appears to impact on the colocalization of OPN with actin and the formation of the sealing zone of resorbing osteoclasts (Figure 4).
In this study, we also assessed bone formation using the phalloidin-stained actin in combination with ALP, collagen type I and BSP as an indicator of the proliferation and activity of bone-forming cells (Figure 5Figure 8). Notably, bone formation activity was increased in association with bone resorptive activity induced by LPS and PTH (Suzuki et al. 2003), consistent with the concept that the activities of osteoblasts and osteoclasts are coupled. Although it was not possible to quantify bone resorption by measuring differences in the thickness of the calvaria before and after inducing bone resorption by morphometry, the inclusion of biochemical analyses (Tatrai et al. 1992
) in future studies should circumvent these problems.
In conclusion, examination of fluorescent-stained calvaria by laser scanning confocal microscope provides a valuable approach for studying cellular mechanisms of bone remodeling and evaluating the effects of biological agents on bone cells, maintained in their natural environment for a relatively short and controlled experimental time period. Using this approach we have shown the relationship between cell attachment molecules involved in osteoclast activity and how these relationships are influenced by LPS and PTH that stimulate osteoclast activity and BPs that suppress osteoclast activity.
![]() |
Acknowledgments |
---|
We are grateful to Prof. Yasushi Sakai (Division of Physiology, School of Nursing and Rehabilitation Science, Showa University) for providing the confocal microscope facilities and for helpful suggestions for the experiments.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andersson G, Johansson EK (1996) Adhesion of human myelomonocytic (HL-60) cells induced by 1,25-dihydroxyvitamin D3 and phorbol myristate acetate is dependent on osteopontin synthesis and the alpha v beta 3 integrin. Connect Tissue Res 35:163171[Medline]
Burdi AR (1965) Toluidine Blue-Alizarin Red S staining of cartilage and bone in whole-mount skeletons in vitro. Stain Technol 40:4548[Medline]
Cecchini MG, Fleisch H (1990) Bisphosphonates in vitro specifically inhibit, among the hematopoietic series, the development of the mouse mononuclear phagocyte lineage. J Bone Miner Res 5:10191027[Medline]
Chellaiah MA, Hruska KA (2003) The integrin alpha(v)beta(3) and CD44 regulate the actions of osteopontin on osteoclast motility. Calcif Tissue Int 72:197205[CrossRef][Medline]
Chellaiah MA, Kizer N, Biswas R, Alvarez U, Strauss-Schoenberger J, Rifas L, Rittling SR, et al. (2003) Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Mol Biol Cell 14:173189
Colucci S, Minielli V, Zambonin G, Cirulli N, Mori G, Serra M, Patella V, et al. (1998) Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces. Calcif Tissue Int 63:230235[CrossRef][Medline]
Dodds RA, Connor JR, James IE, Rykaczewski EL, Appelbaum E, Dul E, Gowen M (1995) Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: an in vitro and ex vivo study of remodeling bone. J Bone Miner Res 10:16661680[Medline]
Duong LT, Lakkakorpi P, Nakamura I, Rodan GA (2000) Integrins and signaling in osteoclast function. Matrix Biol 19:97105[CrossRef][Medline]
Edelman DB, Keefer EW (2005) A cultural renaissance: in vitro cell biology embraces three-dimensional context. Exp Neurol 192:16[CrossRef][Medline]
Ek-Rylander B, Flores M, Wendel M, Heinegard D, Andersson G (1994) Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro. J Biol Chem 269:1485314856
Everts V, Delaisse JM, Korper W, Jansen DC, Tigchelaar-Gutter W, Saftig P, Beertsen W (2002) The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation. J Bone Miner Res 17:7790[Medline]
Flanagan AM, Chambers TJ (1989) Dichloromethylenebisphosphonate (Cl2MBP) inhibits bone resorption through injury to osteoclasts that resorb Cl2MBP-coated bone. Bone Miner 6:3343[CrossRef][Medline]
Flores ME, Norgard M, Heinegard D, Reinholt FP, Andersson G (1992) RGD-directed attachment of isolated rat osteoclasts to osteopontin, bone sialoprotein, and fibronectin. Exp Cell Res 201:526530[CrossRef][Medline]
Ganss B, Kim RH, Sodek J (1999) Bone sialoprotein. Crit Rev Oral Biol Med 10:7998
Grano M, Zigrino P, Colucci S, Zambonin G, Trusolino L, Serra M, Baldini N, et al. (1994) Adhesion properties and integrin expression of cultured human osteoclast-like cells. Exp Cell Res 212:209218.[CrossRef][Medline]
Gravel MR, Zheng ZG, Sims SM, Dixon SJ (1994) Platelet-activating factor induces pseudopod formation in calcitonin-treated rabbit osteoclasts. J Bone Miner Res 9:17691776[Medline]
Hollberg K, Hultenby K, Hayman A, Cox T, Andersson G (2002) Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport. Exp Cell Res 279:227238[CrossRef][Medline]
Horton MA, Nesbit SA, Bennett JH, Stenbeck G (2002) Integrins and other cell surface attachment molecules of bone cells. In Bilezikian JP, Raisz LG, Rodan GA, eds. Principles of Bone Biology. 2nd ed. San Diego, Academic Press, 265286
Horton MA, Nesbit MA, Helfrich MH (1995) Interaction of osteopontin with osteoclast integrins. Ann NY Acad Sci 760:190200[Abstract]
Hughes DE, MacDonald BR, Russell RG, Gowen M (1989) Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. J Clin Invest 83:19301935[Medline]
Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, Mundy GR, et al. (1995) Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 10:14781487[Medline]
Katayama Y, House CM, Udagawa N, Kazama JJ, McFarland RJ, Martin TJ, Findlay DM (1998) Casein kinase 2 phosphorylation of recombinant rat osteopontin enhances adhesion of osteoclasts but not osteoblasts. J Cell Physiol 176:179187[CrossRef][Medline]
Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, et al. (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165176[CrossRef][Medline]
Lakkakorpi P, Tuukkanen J, Hentunen T, Jarvelin K, Vaananen K (1989) Organization of osteoclast microfilaments during the attachment to bone surface in vitro. J Bone Miner Res 4:817825[Medline]
Lowik CW, van der Pluijm G, van der Wee-Pals LJ, van Treslong-De Groot HB, Bijvoet OL (1988) Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate. J Bone Miner Res 3:185192[Medline]
Mackie PS, Fisher JL, Zhou H, Choong PF (2001) Bisphosphonates regulate cell growth and gene expression in the UMR 10601 clonal rat osteosarcoma cell line. Br J Cancer 84:951958[CrossRef][Medline]
Maeda H, Kukita T, Akamine A, Kukita A, Iijima T (1994) Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin. Histochemistry 102:247254[CrossRef][Medline]
McHugh KP, Hodivala-Dilke K, Zheng MH, Namba N, Lam J, Novack D, Feng X, et al. (2000) Mice lacking ß3 integrins are osteosclerotic due to dysfunctional osteoclasts. J Clin Invest 104:433440
McKee MD, Nanci A (1995) Osteopontin and the bone remodeling sequence. Colloidal-gold immunocytochemistry of an interfacial extracellular matrix protein. Ann NY Acad Sci 760:177189[Abstract]
Mostafa YA, Meyer RA Jr, Latorraca R (1982) A simple and rapid method for osteoclast identification using a histochemical method for acid phosphatase. Histochem J 14:409413[CrossRef][Medline]
Mulari MT, Qu Q, Harkonen PL, Vaananen HK (2004) Osteoblast-like cells complete osteoclastic bone resorption and form new mineralized bone matrix in vitro. Calcif Tissue Int 75:253261[CrossRef][Medline]
Murakami H, Takahashi N, Sasaki T, Udagawa N, Tanaka S, Nakamura I, Zhang D, et al. (1995) A possible mechanism of the specific action of bisphosphonates on osteoclasts: tiludronate preferentially affects polarized osteoclasts having ruffled borders. Bone 17:137144[CrossRef][Medline]
Nakamura I, Pilkington MF, Lakkakorpi PT, Lipfert L, Sims SM, Dixon SJ, Rodan GA, et al. (1999) Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone. J Cell Sci 112:39853993
Reinholt FP, Hultenby K, Oldberg A, Heinegard D (1990) Osteopontina possible anchor of osteoclasts to bone. Proc Natl Acad Sci USA 87:44734475
Rogers MJ, Gordon S, Benford HL, Coxon FP, Luckman SP, Monkkonen J, Frith JC (2000) Cellular and molecular mechanisms of action of bisphosphonates. Cancer 88(suppl 12):29612978[CrossRef][Medline]
Ross FP, Chappel J, Alvarez JI, Sander D, Butler WT, Farach-Carson MC, Mintz KA, et al. (1993) Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption. J Biol Chem 268:99019907
Sahni M, Guenther HL, Fleisch H, Collin P, Martin TJ (1993) Bisphosphonates act on rat bone resorption through the mediation of osteoblasts. J Clin Invest 91:20042011[Medline]
Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, et al. (1991) Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 88:20952105[Medline]
Schenk R, Merz WA, Muhlbauer R, Russell RG, Fleisch H (1973) Effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2 MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res 12:196214
Selander K, Lehenkari P, Vaananen HK (1994) The effects of bisphosphonates on the resorption cycle of isolated osteoclasts. Calcif Tissue Int 55:368375[CrossRef][Medline]
Sodek J, Chen J, Nagata T, Kasugai S, Todescan R Jr, Li IW, Kim RH (1995) Regulation of osteopontin expression in osteoblasts. Ann NY Acad Sci 760:223241[Abstract]
Stern PH, Krieger NS (1983) Comparison of fetal rat limb bones and neonatal mouse calvaria: effects of parathyroid hormone and 1, 25-dihydroxy vitamin D3. Calcif Tissue Int 35:172176[Medline]
Suda K, Udagawa N, Sato N, Takami M, Itoh K, Woo JT, Takahashi N, et al. (2004) Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation. J Immunol 172:25042510
Suzuki K, Takeyama S, Kikuchi T, Sodek J, Yamada S, Shinoda H (2003) Evidence for the inhibitory effects of bisphosphonates on osteoclast differentiation and activation obtained using laser scanning confocal microscopy. Bone 32(suppl):S162
Suzuki K, Zhu B, Goldberg HA, Rittling SR, Denhardt DT, McCulloch CAG, Sodek J (2002b) Intracellular osteopontin in osteoclasts: impaired migration, cell fusion and resorption in osteoclasts from OPN/ and CD44/ mice. The Scientific World J 2:7981
Suzuki K, Zhu J, Rittling S, Denhard DT, Goldberg HA, McCulloch CAG, Sodek J (2002a) Co-localization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts. J Bone Miner Res 17:14861497[Medline]
Tatrai A, Foster S, Lakatos P, Shankar G, Stern PH (1992) Endothelin-1 actions on resorption, collagen and noncollagen protein synthesis, and phosphatidylinositol turnover in bone organ cultures. Endocrinology 131:603607[Abstract]
Tezuka K, Sato T, Kamioka H, Nijweide PJ, Tanaka K, Matsuo T, Ohta M, et al. (1992) Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 186:911917[CrossRef][Medline]
Themistocleous GS, Katopodis H, Sourla A, Lembessis P, Doillon CJ, Soucacos PN, Koutsilieris M (2004) Three-dimensional type I collagen cell culture systems for the study of bone pathophysiology. In Vivo 18:687696[Medline]
Tong HS, Sakai DD, Sims SM, Dixon SJ, Yamin M, Goldring SR, Snead ML, et al. (1994) Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping. J Bone Miner Res 9:577584[Medline]
Vaananen HK, Zhao H, Mulari M, Halleen JM (2000) The cell biology of osteoclast function. J Cell Sci 113:377381
Vitte C, Fleisch H, Guenther HL (1996) Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology 137:23242333[Abstract]
Yamate T, Mocharla H, Taguchi Y, Igietseme JU, Manolagas SC, Abe E (1997) Osteopontin expression by osteoclast and osteoblast progenitors in the murine bone marrow: demonstration of its requirement for osteoclastogenesis and its increase after ovariectomy. Endocrinology 138:30473055
Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:35973602
|