Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.     

1,25(OH)2D3 and calcipotriol (MC903) have similar effects on the induction of osteoclast-like cell formation in human bone marrow cultures

MC903 is a novel analogue of 1,25(OH)2D3 which exhibits similar inhibitory effects on cell proliferation and like, 1,25(OH)2D3, stimulates synthesis of osteoblast specific proteins by osteoblast-like cells in vitro. It is less active than 1,25(OH)2D3 in causing hypercalcemia in vivo. Since 1,25(OH)2D3 is known to stimulate bone resorption and increase the number of osteoclasts in several systems (in vivo and in vitro) we examined the effects of MC903 on the formation of osteoclast-like cells in vitro. As reported previously 1,25(OH)2D3 promoted the formation of multinucleated cells with phenotypic and functional characteristics of osteoclasts from adult human bone-marrow cultures at concentrations between 10(-8)M to 10(-12)M. Higher doses consistently suppressed multinucleated cell formation to values seen in the absence of 1,25(OH)2D3. Cells cultured in the presence of MC903 or for three weeks consistently induced the formation of multinucleated cells at concentrations 10(-8)M to 10(-12)M. As seen with 1,25(OH)2D3, MC903 also inhibited multinucleated cell formation at very high concentrations (10(-6)M). In two separate experiments MC903 appeared to be more potent than 1,25(OH)2D3 at lower concentrations (10(-10)M - 10(-12)M). From this study we conclude that MC903 is at least as potent as 1,25(OH)2D3 in inducing the formation human osteoclast-like cells in vitro. The decreased ability of MC903 to induce hypercalcemia in vivo is not therefore a result of a less marked effect than 1,25(OH)2D3 on the regulation of osteoclast formation.     

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.     

Characterization of zinc effect to inhibit osteoclast-like cell formation in mouse marrow culture: Interaction with dexamethasone

The inhibitory effect of zinc compounds on osteoclast-like cell formation in mouse marrow culture in vitro was characterized. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing agent, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or prostaglandin E2 (PGE2). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1,25(OH)2D3 (10-8 M) or PGE2 (10-6 M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were enhanced by the presence of dexamethasone (10-9 to 10-6 M). The dexamethasone (10-7 M)-enhanced osteoclast-like cell formation was not inhibited by the presence of zinc sulfate (10-6 M) or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; 10-6 M), although the zinc compounds had an inhibitory effect on osteoclastic formation in the absence of the steroid. The effect of dexamethasone was not seen, when the steroid was added at the later stage of culture with bone-resorbing agents. In this case, the inhibitory effect of zinc compounds was clearly revealed. This effect of zinc compounds disappeared in the presence of Ca2+-chelating agent (0.5 mM EGTA). The present study suggests that zinc compounds have an inhibitory effect at the stage of differentiation of preosteoclastic cells in bone marrow cell culture system. (Mol Cell Biochem 166: 145-151, 1997)     

The effect of low molecular weight chitosan on bone resorption in vitro and in vivo.

We studied the effect of low molecular weight chitosan (LMWC) on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. LMWC at 440 microg/ml inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha,25(OH)2D3. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of LMWC on pit formation by OCLs was examined using dentin slices, and LMWC inhibited pit formation at 440 microg/ml. Oral administration of the LMWC to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the body and uterus weights. These results suggested that LMWC prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.     

1,25-Dihydroxyvitamin D3 decreases alkaline phosphatase activity in cultures of embryonic chick tibiae

The influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the alkaline phosphatase (AlPase) activity in cultures of chick embryo tibiae was determined. A dose-related, decreased release (30-47%) of AlPase from the bones was seen with the metabolite at 0.05-0.5 ng/ml of medium with a similar effect on the bone content of enzyme. The highest dose (1 ng/ml) decreased the bone content by 38% without further effect on AlPase release. Combining a low level of 1,25(OH)2D3 (0.05 ng/ml) with parathyroid hormone (PTH, 1 U/ml) reduced release of enzyme additively, but caused no greater decrease in bone content of activity than PTH alone. No effects of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3, 0.5 ng/ml] on release or bone content of AlPase were found when this metabolite was added alone or in combination with PTH; however, 24,25(OH)2D3 did prevent the inhibition of release of AlPase when added with 1,25(OH)2D3. After a 1-day exposure to 1,25(OH)2D3, continued incubation in metabolite-free medium resulted in an 89% increase in bone content of AlPase. The results suggest that 1,25(OH)2D3, as well as PTH, may have regulatory roles in bone growth through their effects on AlPase.     

Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells

The predominant cell responsible for bone resorption, the multinucleated osteoclast, has been difficult to study because of inaccessibility. When feline marrow-derived mononuclear cells are established in long-term culture, multinucleated cells form within 48 h, reaching maximum numbers at 16 d. We have observed that these cultured cells have many of the features of osteoclasts. Morphologically, they are multinucleated, contain large numbers of branched mitochondria, have a peripheral cytoplasm lacking organelles (a clear zone), and have extensive cell-surface processes. In addition to these ultrastructural features, the cells contain a tartrate- resistant acid phosphatase, the activity of which is increased by parathyroid hormone (PTH) and inhibited by calcitonin. PTH, prostaglandin E2, and 1,25(OH)2 vitamin D3 increased multinucleated cell formation, while calcitonin inhibited the stimulatory effects of PTH. Time-lapse cinemicrographic and autoradiographic studies indicated that the multinucleated cells formed by fusion of the mononuclear progenitors. The multinucleated cells were phagocytic and stained with nonspecific esterase, consistent with their being derived from immature monocytes. Further, cell populations enriched for multinucleated cells release 45Ca from devitalized bone. Density-gradient centrifugation on Percoll was used to enrich and characterize the mononuclear progenitors of these multinucleated cells. The progenitor cells were found predominantly in Percoll density layers of 1.065 to 1.08 g/ml and were enriched up to 30-fold as compared to unfractionated cells. The bone marrow mononuclear cells that formed the multinucleated cells were initially nonadherent to plastic, stained heavily with nonspecific esterase, and appeared to be immature monocytes histologically. These data indicate that the multinucleated osteoclast-like cells in our cultures are derived from nonadherent monocytic progenitor cells that are responsive to osteotropic hormones. The ability to grow and characterize these cells in vitro should facilitate studies to elucidate the role these cells play in normal and pathologic states of bone resorption.     

A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells

Summary A “sequential culture step” system was devised to study osteoclast differentiation from newborn porcine bone marrow cells. Nonadherent cells were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in low-serum medium supplemented with L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adherent cells mainly composed of M-CSF-dependent macrophage/osteoclast progenitors, but devoid of stromal-like cells, were further cultured in medium supplemented with L9-CM and CM derived from serum-free cultures of fetal rat calvarial bones. This phase was characterized by a rapid induction of mono- and multinucleated (pre)osteoclast-like cells, positive for cytochemical TRAP activity, but negative for nonspecific esterase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] stimulated osteoclast generation, whereas calcitonin treatment significantly inhibited this process. The osteoclastic nature of the cells was confirmed by the occurrence of extensive, characteristic bone resorption on dentin slices, which was associated with release of type I collagen N-telopeptides from the bone matrix into the culture medium. The presence of a DNA synthesis inhibitor (HU) during the first 3 d of culture completely inhibited osteoclast formation, whereas HU treatment during the last phase did not affect production of multinucleated osteoclast-like cells. Likewise, a specific antibody directed against M-CSF during the first preculture period, completely abolished osteoclast formation. Adding the antibody during the last phase of the culture, however, strongly inhibited multinucleated osteoclast formation, accompanied by a significant increase in a mononuclear TRAP-positive, NSE-positive (osteoclast precursor) cell fraction. These results indicate that M-CSF is essential for progenitor proliferation as well as for (pre)osteoclast maturation and/or fusion into multinucleated cells, but also suggest that additional soluble (bone-derived) factors are involved as cofactors in the differentiation process to committed mononuclear osteoclast precursors. The porcine marrow culture approach provides a suitable model system to investigate specific soluble osteoclast-inducing factors affecting different stages of osteoclast development.     

Isolation and characterization of a cDNA clone encoding a novel peptide (OSF) that enhances osteoclast formation and bone resorption

Using an expression cloning approach, we identified and cloned a novel intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption, termed OSF. Conditioned media from 293 cells transiently transfected with the 0.9 kb OSF cDNA clone stimulated osteoclast-like cell formation in both human and murine marrow cultures in the presence or absence 10(-9) M 1,25-dihydroxyvitamin D3. In addition, conditioned media from 293 cells transfected with the OSF cDNA clone enhanced the stimulatory effects of 1,25-(OH)2D3 on bone resorption in the fetal rat long bone assay. In situ hybridization studies using antisense oligomers showed expression of OSF mRNA in highly purified osteoclast-like cells from human giant cell tumors of the bone. Northern blot analysis demonstrated ubiquitous expression of a 1.3 kb mRNA that encodes OSF in multiple human tissues. Sequence analysis showed the OSF cDNA encoded a 28 kD peptide that contains a c-Src homology 3 domain (SH3) and ankyrin repeats, suggesting that it was not a secreted protein, but that it was potentially involved in cell signaling. Consistent with these data, immunoblot analysis using rabbit antisera against recombinant OSF demonstrated OSF expression in cell lysates but not in the culture media. Furthermore, recombinant OSF had a high affinity for c-Src, an important regulator of osteoclast activity. Taken together, these data suggest that OSF is a novel intracellular protein that indirectly enhances osteoclast formation and osteoclastic bone resorption through the cellular signal transduction cascade, possibly through its interactions with c-Src or other Src-related proteins.     

1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

1,25(OH)2D3 acts as a bone-forming agent in the hormone-independent senescence-accelerated mouse (SAM-P/6)

Recent studies suggest that vitamin D signaling regulates bone formation. However, the overall effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone turnover in vivo is still unclear. In this study, our aim was to examine the effect of 1,25(OH)2D3 on bone turnover in SAM-P/6, a hormone-independent mouse model of senile osteoporosis characterized by a decrease in bone formation. Male and female 4-mo-old SAM-P/6 mice were treated with 1,25(OH)2D3 (18 pmol/24 h) or vehicle for a period of 6 wk, and a group of age- and sex-matched nonosteoporotic animals was used as control. Bone mineral density (BMD) at the lumbar spine increased rapidly by >30 +/- 5% (P < 0.001) in 1,25(OH)2D3-treated SAM-P/6 animals, whereas BMD decreased significantly by 18 +/- 2% (P < 0.01) in vehicle-treated SAM-P/6 animals and remained stable in control animals during the same period. Static and dynamic bone histomorphometry indicated that 1,25(OH)2D3 significantly increased bone volume and other parameters of bone quality as well as subperiosteal bone formation rate compared with vehicle-treated SAM-P/6 mice. However, no effect on trabecular bone formation was observed. This was accompanied by a marked decrease in the number of osteoclasts and eroded surfaces. A significant increase in circulating bone formation markers and a decrease in bone resorption markers was also observed. Finally, bone marrow cells, obtained from 1,25(OH)2D3-treated animals and cultured in the absence of 1,25(OH)2D3, differentiated more intensely into osteoblasts compared with those derived from vehicle-treated mice cultured in the same conditions. Taken together, these findings demonstrate that 1,25(OH)2D3 acts simultaneously on bone formation and resorption to prevent the development of senile osteoporosis.     

Synthesis in vitro of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by interferon-gamma-stimulated normal human bone marrow and alveolar macrophages

Cultured human macrophages from normal donors were examined for their capability to metabolize 25-hydroxyvitamin D3 (25-(OH)D3). Upon exposure to recombinant human interferon-gamma (IFN-gamma) both bone marrow-derived macrophages (BMM) and pulmonary alveolar macrophages (PAM) produced a polar 25-(OH)D3 metabolite which was purified from conditioned media and unequivocally identified as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by UV-absorbance spectrophotometry and mass spectrometry. The BMM and PAM also synthesized a second 25-(OH)D3 metabolite which was structurally identified as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). The time course of 25-(OH)D3 metabolism by macrophages suggested that the production of 24,25-(OH)2D3 was stimulated by high intracellular levels of 1,25-(OH)2D3 and not by IFN-gamma. The 1,25-(OH)2D3 obtained from BMM and PAM promoted macrophage-like differentiation of promyelocytic HL-60 leukemia cells and inhibited IFN-gamma production by normal human lymphocytes. Our data suggest that locally high levels of 1,25-(OH)2D3 in the microenvironment of IFN-gamma-stimulated BMM and PAM may modulate the function of hormone-responsive cells.     

Mitochondrial alkaline phosphatase as an intracellular signal in the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in LLC-PK1 cells

In previous works we have found a mitochondrial alkaline phosphatase (AP) activity in LLC-PK1. The aim of this work has been to study the possible involvement of mitochondrial AP activity in the synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) from the substrate 25(OH)D3. Renal phenotype LLC-PK1 cells were incubated with 25(OH)D3 as substrate and treated with or without 1,25(OH)2D3, forskolin, 12-myristate-13-acetate (PMA) and 1,25(OH)2D3 in conjunction with PMA. Incubation of LLC-PK1 cells with forskolin (adenylate cyclase activator) not only stimulated the 1-hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial AP activity. The addition of 1,25(OH)2D3, the main activator of 24-hydroxylase, produced a decrease of mitochondrial AP activity, a decrease of 1,25(OH)2D3 synthesis and an increase of the 24,25(OH)2D3 synthesis. Incubation with PMA, a potent activator of protein kinase C, did not produce any changes in mitochondrial AP activity, but an inhibition of 1,25(OH)2D3 and an activation of 24,25(OH)2D3 synthesis were found. Moreover, incubation of LLC-PK1 cells with PMA in conjunction with 1,25(OH)2D3 produced an additive effect in the decrease of 1,25(OH)2D3 and an increase of 24,25(OH)2D3 synthesis remaining mitochondrial AP activity as cells treated only with 1,25(OH)2D3. Our results suggest that mitochondrial AP activity could be involved as an intracellular signal in the regulation of 25(OH)D3 metabolism to the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in renal phenotype LLC-PK1 cells through cAMP protein kinase system.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis

We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.     

Generation of osteoclasts in cultures of rabbit bone marrow and spleen cells

The primary and specific function of the osteoclast is the resorption of bone. We have applied this criterion, and a monoclonal antibody that binds specifically to osteoclasts, to cultures of tissues that may contain osteoclastic precursors. Bone marrow and spleen cells were incubated for up to 4 weeks in the presence or absence of parathyroid hormone, interleukin 1, or 1,25(OH)2 vitamin D3, on plastic coverslips or slices of devitalised bone. Osteoclasts (as judged by the presence of resorption cavities and the appearance of monoclonal antibody-positive cells) did not develop in cultures incubated without added hormones, nor in cultures containing parathyroid hormone or interleukin 1, but were regularly observed when bone marrow cells were incubated with 1,25(OH)2 vitamin D3. Although multinucleate giant cells were common after incubation, especially in the presence 1,25(OH)2 vitamin D3, monoclonal antibody bound not to these cells but to a minor and distinctive population of mononuclear cells and cells of low multinuclearity. We found no excavations and no monoclonal antibody-positive cells after incubation of peritoneal macrophages with 1,25(OH)2D3. These results provide direct evidence of osteoclastic function arising in cultures of haemopoietic tissues.