Effects of electromagnetic stimulation on the functional responsiveness of isolated rat osteoclasts

We report the effects of pulsed electromagnetic fields (PEMFs) on the responsiveness of osteoclasts to cellular, hormonal, and ionic signals. Osteoclasts isolated from neonatal rat long bones were dispersed onto either slices of devitalised cortical bone (for the measurement of resorptive activity) or glass coverslips (for the determination of the cytosolic free Ca²⁺ concentration, [Ca²⁺]). Osteoclasts were also cocultured on bone with osteoblastlike, UMR-106 cells. Bone resorption was quantitated by scanning electron microscopy and computer-assisted morphometry. PEMF application to osteoblast–osteoclast cocultures for 18 hr resulted in a twofold stimulation of bone resorption. In contrast, resorption by isolated osteoclasts remained unchanged in the presence of PEMFs, suggesting that osteoblasts were necessary for the PEMF-induced resorption simulation seen in osteoblast–osteoclast cocultures. Furthermore, the potent inhibitory action of the hormone calcitonin on bone resorption was unaffected by PEMF application. However, PEMFs completely reversed another quite distinct action of calcitonin on the osteoclast: its potent inhibitory effect on the activation of the divalent cation-sensing (or Ca²⁺) receptor. For these experiments, we made fura 2-based measurements of cytosolic [Ca²⁺] in single osteoclasts in response to the application of a known Ca²⁺ receptor agonist, Ni²⁺. We first confirmed that activation of the osteoclast Ca²⁺ receptor by Ni²⁺ (5 mM) resulted in a characteristic monophasic elevation of cytosolic [Ca²⁺]. As shown previously, this response was attenuated strongly by calcitonin at concentrations between 0.03 and 3 nM but remained intact in response to PEMFs. PEMF application, however, prevented the inhibitory effect of calcitonin on Ni²⁺-induced cytosolic Ca²⁺ elevation. This suggested that the fields disrupted the interaction between the calcitonin and Ca²⁺ receptor systems. In conclusion, we have shown that electromagnetic fields stimulate bone resorption through an action on the osteoblast and, by abolishing the inhibitory effects of calcitonin, also restore the responsiveness of osteoclasts to divalent cations. J. Cell. Physiol. 176:537–544, 1998. © 1998 Wiley-Liss, Inc.     

Effects of extracellular calcium and protons on osteoclast potassium currents

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca²⁺ and H⁺. We examined the effects of these cations on two types of K⁺ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca²⁺, pH 7.4), the voltage-gated, outwardly rectifying K⁺ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V _0.5). Increasing [Ca²⁺]_out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca²⁺]_out, respectively. This effect of elevating [Ca²⁺]_out was due to a positive shift of the K⁺ channel voltage activation range. Zn²⁺ or Ni²⁺ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K⁺ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K⁺ current, the potency sequence was Zn²⁺ > Ni²⁺ > Ca²⁺. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K⁺ current, changes in the concentration of extracellular H⁺ or Ca²⁺ did not shift the voltage activation range of the inwardly rectifying K⁺ current. These findings are consistent with Ca²⁺ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K⁺ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council.     

Capacitative calcium entry and proliferation of human osteoblast-like MG-63 cells

Abstract. Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca²⁺) control in numerous cellular functions; however, involvement of capacitative Ca²⁺ entry (CCE) in proliferation of bone cells is less well investigated. Objectives: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast‐like MG‐63 cells. Meterials and Methods: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. Results: Intracellular Ca²⁺ store depletion by thapsigargin induced CCE in MG‐63 cells; this was characterized by a rapid transient increase of intracellular Ca²⁺ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet‐derived growth factor. Inhibitors of store‐operated Ca²⁺ channels (2‐APB and SKF‐96365) prevented CCE, while voltage‐dependent Ca²⁺ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage‐dependent Ca²⁺ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2‐APB and SKF‐96395, inhibited it. Cell cycle analysis showed that 2‐APB and SKF‐96395 lengthen the S and G₂/M phases, which would account for the reduction in cell proliferation. Conclusions: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca²⁺ influx involved in osteoblast proliferation.     

Calcium signaling in osteoclasts

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca²⁺) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca²⁺ oscillations occur during RANKL-mediated osteoclastogenesis. Ca²⁺ oscillations provide a digital Ca²⁺ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca²⁺/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca²⁺ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca²⁺ oscillations, and we discuss possible molecular players in this specialized Ca²⁺ response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Localized calcium signaling in multinucleated osteoclasts

Localized intracellular Ca²⁺ ([Ca²⁺]_i) pulses, fluctuations, and repetitive spikes were detected in multinucleated rabbit osteoclasts in the presence of serum and in response to calcitonin using the fluorescent calcium indicator fluo-3 and a laser scanning microscope. We observed that these [Ca²⁺]_i changes were often restricted within a region of the cell body or propagated from the initial region of occurrence to other parts of the cell body but not to all parts. These observations suggest the existence of significant barriers to Ca²⁺ transport between different cytoplasmic regions of the osteoclast. To further investigate this phenomenon, we mechanically perturbed different cellular regions by touching locally with a micropipette. This usually induced a local increase in cytosolic and nuclear free [Ca²⁺]_i. In some cases there was propagation of the [Ca²⁺]_i increase to other regions but with part of the cell body not affected. Those regions of the cell body to which the [Ca²⁺]_i increase did not propagate had a [Ca²⁺]_i response to a direct mechanical perturbation. Our data show that osteoclasts can have different [Ca²⁺]_i activities in apparently equivalent cellular regions, no matter how generated. This suggests that there can be a number of spatially separate Ca²⁺ regulatory systems within an osteoclast cell body. © 1996 Wiley-Liss, Inc.     

Lyn inhibits osteoclast differentiation by interfering with PLCγ1-mediated Ca signaling

Osteoclasts differentiate from macrophage-lineage cells to become specialized for bone resorption function. By a proteomics approach, we found that Lyn was down-regulated by the osteoclast differentiation factor, receptor activator of NF-κB ligand (RANKL). The forced reduction of Lyn caused a striking increase in the RANKL-induced PLCγ1, Ca²⁺, and NFATc1 responses during differentiation. These data suggest that Lyn plays a negative role in osteoclastogenesis by interfering with the PLCγ1-mediated Ca²⁺ signaling that leads to NFATc1 activation. Consistent with the in vitro results, in vivo injection of Lyn specific siRNA into mice calvariae provoked a fulminant bone resorption. Our study provides the first evidence of the involvement of Lyn in the negative regulation of osteoclastogenesis by RANKL.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

Response of the electrochromic dye,merocyanine 540, to membrane potential in rat liver mitochondria

Summary We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca^2–] _a -dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca^2–-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca²⁺-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K^–] _a , nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba²⁺] _a . All these data are consistent with the concept that PTX may act on Ca^2– channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca²⁺ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca²⁺ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca²⁺ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca²⁺ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca²⁺-channel conductance (6 nS/cell at 2.6mm [Ca²⁺] _a ). PTX evoked the activation of a new class of Ca²⁺-selective channels (5 pS in 25mm [Ca²⁺]_pipet), which are rather insensitive to membrane potential. We have termed theseG-type calcium channels. These data suggest that treatment with PTX not only increases the probability of L-type Ca²⁺-channel activation at more negative potentials, but also increases the probability of opening of an entirely new, voltage-independent, Ca²⁺ channel. These actions of PTX should promote Ca²⁺ entry and might explain the stimulation by the toxin of CA secretion from medullary chromaffin cells in culture.     

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca²⁺. Here, we show that extracellular Ca²⁺ (Ca²⁺_o) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca²⁺_o compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca²⁺_o in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cβ (PLCβ) led to an abolition of the Ca²⁺_o-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca²⁺_o-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca²⁺_o concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca²⁺-rich environment.     

Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium

The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca²⁺]_e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca²⁺]_i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca²⁺]_i increased when [Ca²⁺]_e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca²⁺]_e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca²⁺]_e (up to 20 mM [Ca²⁺]_e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca²⁺]_e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca²⁺]_e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca²⁺]_i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca²⁺]_i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C. © 1996 Wiley-Liss, Inc.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: A patch-clamp study

There is evidence for a role of increased cytoplasmic Ca²⁺ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate—the universality of Ca²⁺ as a component of the signaling chain, and the source of the increased Ca²⁺, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca²⁺, and of internal Ca²⁺ buffers to control levels of cytoplasmic Ca²⁺. We show that ABA-induced reduction of the K⁺ inward rectifier can occur in the absence of external Ca²⁺, but is abolished when Ca²⁺ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca²⁺ is a necessary component of the signaling chain by which ABA decreases the K⁺ inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca²⁺ from internal stores is capable of mediating the response, in the absence of any Ca²⁺ influx from the extracellular medium. The work also shows that enhancement of the K⁺ outward rectifier by ABA is Ca²⁺ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish (Proc. Natl. Acad. Sci. USA 89:1790–1794, 1990) and M.R. Blatt (J. Gen. Physiol. 99:615–644, 1992), is an attractive working hypothesis.     

Structural Basis for the Biological Effects of Pr(III) Ions: Alteration of Cell Membrane Permeability

The biological effects of rare-earth ions on the organism have been studied using Pr³⁺ as a probe ion and Escherichia coli cell as a target. Atomic force microscopy (AFM) observation of the surface of E. coli cells shows that the presence of Pr³⁺ substantially changes the structure of the outer membrane. By induced coupled plasma-mass spectrometry (ICP-MS), more Cu²⁺ was found in the cells grown in the presence of Pr³⁺, indicating changes of cell permeability. Using energy dispersive X-ray spectroscopy (EDX), Ca²⁺ is found on the outer surface of the original cell. It is proposed that Pr³⁺ can replace Ca²⁺ from the binding sites because of their close ionic radii and similar ligand speciality.     

Role of Ca2+ in the activity of rhicadhesin from Rhizobium leguminosarum biovar viciae,which mediates the first step in attachment of Rhizobiaceae cells to plant root hair tips

The first step in attachment of Rhizobiaceae cells to plant root hair tips is mediated by a Ca²⁺-dependent, Ca²⁺-binding protein, rhicadhesin. The possible role of Ca²⁺ in synthesis, anchoring and activity of rhicadhesin was investigated. Growth of Rhizobium leguminosarum biovar viciae cells under Ca²⁺-limitation was found to result in loss of attachment ability. Under these conditions, rhicadhesin could not be usolated from the bacterial cell surface, but was found to be excreted in the growth medium. Divalent ions appeared to be essential for the ability of purified rhicadhesin to inhibit attachment of R. leguminosarum biovar viciae cells to pea root hair tips. Calcium ions were found not to be involved in binding of rhicadhesin to the plant surface, but appeared to be involved in anchoring of the adhesin to the bacterial cell surface. A model for the role of Ca²⁺ in activity of rhicadhesin is presented.     

Magnesium deficiency results in an increased formation of osteoclasts

Magnesium (Mg²⁺) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg²⁺ deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg²⁺ restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg²⁺ (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg²⁺ deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg²⁺-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP⁺) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg²⁺ deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg²⁺ deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg²⁺ deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg²⁺. In conclusion, Mg²⁺ deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg²⁺ deficient patients.