The authors have no conflict of interest
Metabolic Acidosis Stimulates RANKL RNA Expression in Bone Through a Cyclo-oxygenase-Dependent Mechanism†
Article first published online: 1 JUL 2003
Copyright © 2003 ASBMR
Journal of Bone and Mineral Research
Volume 18, Issue 7, pages 1317–1325, July 2003
How to Cite
Frick, K. K. and Bushinsky, D. A. (2003), Metabolic Acidosis Stimulates RANKL RNA Expression in Bone Through a Cyclo-oxygenase-Dependent Mechanism. J Bone Miner Res, 18: 1317–1325. doi: 10.1359/jbmr.2003.18.7.1317
- Issue published online: 2 DEC 2009
- Article first published online: 1 JUL 2003
- Manuscript Accepted: 24 JAN 2003
- Manuscript Revised: 6 DEC 2002
- Manuscript Received: 1 OCT 2002
Metabolic acidosis inhibits osteoblastic bone formation and stimulates osteoclastic resorption. To determine whether acidosis alters expression of RNA for the osteoclastic differentiation factor RANKL, mouse calvariae were incubated in neutral or physiologically acidic media. Acidosis resulted in a significant cyclo-oxygenase-dependent increase in RANKL RNA levels, which would be expected to induce the associated increase in bone resorption.
Introduction: Metabolic acidosis increases net calcium efflux from bone, initially through physicochemical mechanisms and later through predominantly cell-mediated mechanisms. Acidosis decreases osteoblastic bone formation and increases osteoclastic resorption. The growth and maturation of osteoclasts, derived from hematopoietic precursors in the monocyte/macrophage lineage, are dependent on the interplay of a number of factors. Commitment of pre-osteoclasts to osteoclasts is induced by the interaction of the osteoclastic cell-surface receptor RANK with a ligand expressed by osteoblasts, RANKL. The RANK/RANKL interaction not only initiates a differentiation cascade that culminates in mature bone-resorbing osteoclasts but also increases osteoclastic resorptive capacity and survival.
Methods: To test the hypothesis that metabolic acidosis increases expression of RANKL, we cultured neonatal mouse calvariae in acidic (initial medium pH ∼7.1 and [HCO3−] ∼11 mM) or neutral (initial medium pH ∼7.5 and [HCO3−] ∼25 mM) medium for 24 and 48 h. We determined the relative expression of RANKL RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitated the expression by Northern analysis.
Results: In this model of metabolic acidosis, there was significantly increased expression of RANKL RNA at both 24 (2-fold) and 48 h (5-fold) compared with respective controls. Net calcium efflux from bone was also increased in acidic medium compared with control medium. At 48 h, net calcium efflux correlated directly with RANKL expression (r = 0.77, n = 15, p < 0.001). Inhibition of prostaglandin synthesis with indomethacin blocked the acid-induced increase in RANKL RNA as well as the increased calcium efflux.
Conclusions: Metabolic acidosis induces osteoblastic prostaglandin synthesis, followed by autocrine or paracrine induction of RANKL. This increase in RANKL would be expected to augment osteoclastic bone resorption and help explain the increase in cell-mediated net calcium efflux.
Metabolic acidosis increases urine calcium excretion(1–3) without a parallel increase in intestinal calcium absorption,(4,5) resulting in a net loss of calcium from the body.(3,5) Because the vast majority of body calcium resides within the skeleton, bone has been implicated as the source of this additional urinary calcium.(6) Indeed, metabolic acidosis has been shown to deplete bone mineral stores.(7,8) Mild chronic metabolic acidosis in the elderly, because of an inability to excrete the endogenous acid load of a typical Western diet, has been implicated in the pathogenesis of osteoporosis.(9,10)
In vitro studies have supported the hypothesis that metabolic acidosis, modeled by a primary reduction in medium bicarbonate concentration, induces a net efflux of calcium from bone.(11–21) Metabolic acidosis stimulates calcium efflux through both early (0–24 h) physicochemical events(15,18,20) and later (>24 h) predominantly cell-mediated(11–14,16,17,), 19 mechanisms. Using cultured neonatal mouse calvariae, we have demonstrated that the cell-mediated calcium release observed in response to a model of metabolic acidosis results from both an inhibition of osteoblastic activity and a stimulation of osteoclastic activity.(17,19) Compared with calvariae incubated in neutral medium, osteoblastic collagen synthesis and alkaline phosphatase activity were both decreased after incubation in acidic medium.(17,19) In cells isolated from calvariae, RNA expression for the immediate early response gene egr-1 was inhibited by acidosis,(15) as was RNA expression for the bone matrix genes, matrix gla protein and osteopontin.(13,14) Levels of osteoclastic β-glucuronidase, an enzyme whose release correlates with osteoclast-mediated bone resorption, were increased during culture of calvariae in acidic medium.(17,19) The acid-induced cell-mediated increase in net calcium flux seems to be caused, in large part, by an increase in osteoblastic prostaglandin E2 (PGE2) release, which is known to stimulate osteoclastic resorption.(11,12)
Osteoclasts are derived from hemopoietic precursors in the monocyte/macrophage lineage.(22–25) Growth of these precursors is dependent on macrophage colony-stimulating factor (M-CSF; also known as CSF-1), a growth factor that is produced by osteoblasts.(26) Commitment of pre-osteoclasts to osteoclasts is induced by the interaction of the osteoclastic cell-surface receptor RANK with a molecule expressed on the surface of osteoblasts, RANKL.(22–25) The RANK/RANKL interaction activates the DNA binding protein NFκB to initiate a differentiation cascade that leads to functional multinuclear osteoclasts capable of resorbing bone. In addition to promoting the maturation of pre-osteoclasts, RANKL also increases the bone-resorbing activity of mature osteoclasts(27–29) and decreases osteoclastic apoptosis.(30) Osteoblasts also produce osteoprotegerin (OPG), which acts as a decoy receptor.(22–25) The binding of RANKL to OPG prevents the RANK/RANKL interaction and limits osteoclastic activity. Thus, the equilibrium between levels of OPG and RANKL is critical in determining the extent of osteoclastic bone resorption.
In this study we tested the hypothesis that the expression of RANKL or one of the other mediators of osteoclastic activity would be altered by metabolic acidosis. We compared the expression of these mediators by reverse transcriptase-polymerase chain reaction (RT-PCR), and when the data suggested an acidosis-induced increase in RANKL, we quantitated its expression by Northern analysis. We found that metabolic acidosis significantly increased the expression of RANKL RNA at both 24 and 48 h, which was significantly correlated with calcium efflux from bone at the latter time period. Indomethacin blocked the acidosis-induced increase in RANKL RNA, indicating that prostaglandin synthesis mediates this effect. The acidosis-induced increase in RANKL would be expected to result in increased osteoclastic bone resorption and help explain the mechanism by which acidosis induces the cell-mediated increase in net calcium efflux.
MATERIALS AND METHODS
Organ culture of bone
Exactly 2.8 ml of Dulbecco's modified Eagle's medium (DMEM), containing 15% heat-inactivated horse serum, was preincubated at a partial pressure of carbon dioxide (PCO2) of 40 mm Hg, at 37°C for at least 3 h in 35-mm dishes.(11–21) Calvariae were dissected from 4- to 6-day-old mice, and immediately before adding two bones per dish, 1 ml of medium was removed to determine preincubation pH, PCO2, and calcium concentration. Medium pH and PCO2 were determined with a blood-gas analyzer (ABL5; Radiometer, Copenhagen, Denmark), and calcium was measured by an ion-selective electrode (10; Nova Biomedical, Waltham, MA, USA). At the conclusion of each 24-h incubation period, medium was removed and analyzed for pH, PCO2, and calcium. The concentration of bicarbonate ([HCO3−]) was calculated from pH and PCO2 as described previously.(21) Net calcium flux was calculated as Vm × ([Ca]f − [Ca]i), where Vm is the medium volume (1.8 ml) and [Ca]f and [Ca]i are the final and initial medium calcium concentrations, respectively.
Calvariae were divided into two groups. Some were incubated in medium at neutral pH (∼7.5, CTL) and others at acidic pH (∼7.1, MET), produced by a primary decrease in [HCO3−], as a model of metabolic acidosis. PCO2 was held at 40 mm Hg throughout all experiments. To closely replicate physiological conditions, only the HCO3−/CO2 buffer system was used.(31) To produce the acid medium for MET, an aliquot of 2.4 M HCl was added to the DMEM + horse serum (10 μl/ml of medium), resulting in a reduction of [HCO3−] and thus pH. To produce the neutral pH medium for CTL, an aliquot of 2.4 M HCl was added to the DMEM + horse serum (4 μl/ml of medium), resulting in physiologically normal [HCO3−] and thus pH. Some calvariae were incubated for only 24 h and harvested for RNA. Others were incubated for a total of 48 h, with transfer to similar, preincubated fresh medium at 24 h, before harvesting for RNA at the completion of the experiment. To study the role of prostaglandin biosynthesis in RANKL expression, 0.56 μM indomethacin was added to some CTL or MET cultures; this is a concentration that we have previously shown to inhibit prostaglandin synthesis in similar bone organ cultures.(12)
The two calvariae from each dish were pooled for isolation of total RNA using an RNeasy kit (Qiagen, Valencia, CA, USA. In brief, 600 μl buffer RLT (proprietary) containing 1% β-mercaptoethanol was added to the two bones, and the mixture vortexed vigorously (10 s × 6). The solution and bones were pipetted into a QIAshredder and centrifuged 2 minutes at 13,000 rpm. Six hundred microliters of 70% ethanol was added to the filtrate and mixed. The extract was loaded as two aliquots onto an RNeasy minispin column, and RNA was loaded onto the column by centrifugation each time for 15 s (13,000 rpm). The column was washed by centrifugation sequentially with 700 μl buffer RW1 (proprietary) and twice with 500 μl buffer RPE (proprietary), each time for 15 s (13,000 rpm). The column was dried by centrifugation for 1 minute at 13,000 rpm. RNA was eluted from the column with two 30-μl aliquots of RNase-free water by centrifugation for 1 minute at 13,000 rpm. RNA was quantitated by ultraviolet spectrophotometry.
RNA samples from each individual experiment were simultaneously analyzed for RANKL, OPG, M-CSF, and β-actin. Total RNA (1 μg) was mixed with oligo dT to serve as primer and heated to 70°C for 5 minutes in reverse transcription buffer (20 mM Tris HCl, pH = 8.4, 50 mM KCl, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP). After cooling to 42°C, MgCl2 (5 mM final concentration), DTT (10 mM), RNaseOut (40 U), and RNnase H− M-MLV reverse transcriptase (50 U, Superscript II; GIBCO/Invitrogen, Carlsbad, CA, USA) were added, and the mixture was incubated at 42°C for 60 minutes. The reaction was stopped by heating to 75°C for 10 minutes, and then cooled to 37°C. RNase H (2 U) was added to digest RNA hybridized to DNA, and the mixture was incubated at 37°C for 20 minutes. Aliquots (1/10 volume) were subsequently amplified using gene-specific primers for β-actin, M-CSF,(26) OPG,(32) and RANKL(32) (Table 1) and TaqDNA polymerase (Platinum Taq; GIBCO/Invitrogen). MgCl2 was added to 1 mM (final concentration) for M-CSF amplification, 2 mM for OPG and β-actin amplification and 3 mM for RANKL amplification. For PCR, samples were heated to 94°C for 3 minutes and subjected to 35 cycles of denaturation, 94°C, 45 s; annealing, cool to 50°C over 60 s; hold at 50°C for 30 s; elongation, warm to 68°C over 30 s; hold at 68°C for 60 s. At the end of cycling, the samples were incubated at 72°C for 15 minutes. Aliquots (10 μl) of the reactions were electrophoresed on 1.5% agarose (standard agarose:Agarose-1000; [GIBCO/Invitrogen], 1:1; containing 1 μg/ml ethidium bromide) in TBE (89 mM Tris-borate, 2 mM EDTA, pH = 8.0). Gels were photographed on an ultraviolet transilluminator using 667 film (Polaroid, Cambridge, MA, USA).
Northern blotting and hybridization conditions
RNA samples (15 μg) were electrophoresed on 1.5% agarose containing 1.9% formaldehyde and transferred to charged nylon membranes (GeneScreen Plus; NEN, Boston, MA, USA) by capillary transfer as previously described.(15) For detection of RANKL mRNA, the product from RANKL PCR reactions was cloned into the vector pCR4 by topoisomerase 1-mediated ligation (Invitrogen), purified plasmid DNA was digested with EcoR1, and insert DNA was purified after agarose gel electrophoresis. The RANKL fragment was labeled with α-32P-dCTP using a random primer labeling system (Ambion, Austin, TX, USA; or Invitrogen). Hybridization conditions were as described by Ikeda et al.(33) In brief, filters were prehybridized for 15–20 minutes in Rapid Hybridization buffer (Amersham Pharmacia, Piscataway, NJ, USA) at 65°C, and then probe was added and hybridization was continued for 3 h at 65°C. The filter was washed once in 2× SSC (SSC = 0.15 mM NaCl, 0.015 mM sodium citrate), 0.2% SDS at 65°C for 20 minutes, and then twice in 0.2× SSC and 0.2% SDS at 65°C for 45 minutes each. Filters were exposed either to X-ray film (Kodak, Rochester, NY, USA) at −70°C with intensifying screens or for the indomethacin experiments in a PhosphorImager cassette (Molecular Dynamics, Amersham Pharmacia). Bands of hybridization were quantitated using either a densitometer (Molecular Dynamics) for autoradiograms or the PhosphorImager. To normalize for RNA loading, filters were stripped and reprobed for GAPDH as previously described.(15)
All experiments using animals were conducted with protocols approved by the University of Rochester Committee on Animal Research.
All values were expressed as mean ± SE. Tests of significance were calculated using the Student t-test and regression analysis using conventional programs (BMDP; Statistical Solutions, Saugus, MA, USA) on a personal computer. p < 0.05 was considered significant.
Medium pH, PCO2, [HCO3−], and calcium flux
To determine the effect of metabolic acidosis on RANKL, OPG, and M-CSF expression, we incubated calvariae for 24 or 48 h in neutral (CTL) or reduced (MET) pH medium. By design, in the 24-h experiments and in both 24-h periods of the 48-h experiments, the initial medium pH in MET was significantly lower than in CTL (Table 2) because of a reduction in the medium [HCO3−]. There was no difference in PCO2 between CTL and MET in any time period. At the conclusion of the 24-h experiments, and in each 24-h time period of the 48-h experiments, compared with their respective controls, incubation in MET led to a significant increase in net calcium efflux from bone (Fig. 1). There was no difference in the initial medium pH, PCO2, [HCO3−], or in net calcium efflux in the incubations subsequently used for RT-PCR compared with those subsequently used for Northern analysis.
To determine whether the expression of RANKL, OPG, and M-CSF were altered (relative to β-actin as a control) by a reduction of medium pH, RNA was extracted from calvariae, before incubation (PRE) and after 24-h and 48-h incubations in either CTL or MET, and subjected to RT-PCR. Compared with PRE and CTL, incubation in acidic medium (MET) stimulated expression of RANKL at both 24 and 48 h (Fig. 2). Compared with PRE and CTL, incubation in MET appeared not to alter expression of OPG, M-CSF, or β-actin at either time period. Figure 2 represents data from one of three separate experiments; in each experiment, all cultures were incubated and analyzed in parallel, and in each, the results were similar.
While RT-PCR is useful for determining the presence of a specific RNA, it is not optimal to quantitate the abundance of that RNA. To determine the amount of RANKL RNA, without the possible artifacts induced by PCR amplification, RNA samples from calvariae were quantitated by Northern analysis. Incubation in acidic medium (MET) led to greater RANKL RNA accumulation compared with calvariae before incubation (PRE) or calvariae incubated in neutral medium (CTL) at 24 and 48 h (Fig. 3, representative autoradiogram). GAPDH RNA accumulation was similar in all groups. Compilation of the four separate experiments at 24 h demonstrated an approximately 2-fold greater accumulation of RANKL RNA in MET (n = 11 pairs of calvariae) compared with CTL (n = 9; p < 0.05; Fig. 4). Compilation of the four separate experiments at 48 h demonstrated an approximately 5-fold greater accumulation in MET (n = 7 pairs of calvariae) compared with CTL (n = 8; p < 0.05).
There was a strong direct correlation between net calcium flux during the second 24 h of the 48-h experiment and RANKL RNA levels (r = 0.771, n = 15, p < 0.001; Fig. 5) as well as cumulative net calcium flux during the entire 48-h experiment and RANKL RNA levels (r = 0.763, n = 15, p < 0.001; data not shown). There was no correlation between net calcium flux during the 24-h experiment and RANKL RNA levels (r = 0.349, n = 20, p = not significant; data not shown), consistent with acid-induced calcium efflux from bone being primarily caused by physicochemical dissolution over this initial 24-h period.
Inhibition of cyclo-oxygenase with indomethacin
Metabolic acidosis increases secretion of PGE2, and cyclo-oxygenase inhibition with indomethacin abolishes this cell-mediated calcium release.(11,12) To determine the effect of inhibiting prostaglandin synthesis on RANKL expression, indomethacin was added to parallel cultures of calvariae in CTL or MET medium and calcium efflux, RANKL, and GAPDH RNA content was determined. Initial culture conditions were not different from those shown in Table 2.
At 48 h, compared with CTL, metabolic acidosis caused a significant increase in calcium efflux, and this increase was significantly inhibited by indomethacin (Fig. 6). Compared with bones incubated in acidic medium (n = 11 pairs of calvariae), RANKL RNA content was significantly reduced by indomethacin (n = 10; Fig. 7, representative northern blot; Fig. 8, quantitation of all cultures). Indomethacin did not alter GAPDH RNA content. Similar results were obtained at 24 h (data not shown).
Compared with culture in neutral medium, incubation of neonatal mouse calvariae in medium acidified by a decrease in the concentration of bicarbonate, a model of metabolic acidosis, results in a cell-mediated increase in net calcium efflux. In this model of acidosis, we have observed a reciprocal suppression of osteoblastic activity and stimulation of osteoclastic activity.(11,16,19) Multiple factors expressed by osteoblasts could mediate osteoclastic activity. Acidosis increases osteoblastic secretion of PGE2, which is known to stimulate osteoclastic bone resorption.(11,12,34) Other factors produced by osteoblasts, such as RANKL, M-CSF, and OPG, are also known to modulate osteoclastic differentiation and subsequent activity. A TNF-related protein, RANKL, is expressed on the osteoblastic cell surface and interacts with a receptor, RANK, on the surface of pre-osteoclasts and osteoclasts.(22–25) The interaction of RANKL with RANK stimulates differentiation of pre-osteoclasts into functionally mature, bone-resorbing osteoclasts,(27) increases the resorptive activity of mature osteoclasts,(27–29) and decreases apoptosis of osteoclasts.(30) The principal findings of this study are that culture of calvariae in acidic medium increases RANKL RNA and that this increase is dependent on cyclo-oxygenase activity. Increased RANKL would be expected to stimulate osteoclastic bone resorption and would help to explain the mechanism for the cell-mediated augmentation of net calcium efflux induced by acidosis. The acidosis-induced increase in RANKL RNA is specific and not a generalized upregulation of RNA synthesis; while there was an increase in RANKL, there was no apparent change in RNA levels for OPG, M-CSF, β-actin, or GAPDH.
The stimulation of RANKL by acidosis could be mediated through an autocrine or a paracrine mechanism. Metabolic acidosis inhibits osteoblastic collagen synthesis(11,16,19) but increases osteoblastic PGE2 secretion both in organ culture and in primary cultures of calvarial cells.(11,12,34) PGE2 increased RANKL in pre-osteoblastic marrow stromal cells.(35,36) Addition of PGE2 to osteoblastic cells caused a 2-fold increase in RANKL RNA, whereas addition of PGE2 to osteoblasts from mice deficient in EP2 PGE2 receptors had little effect.(37) In this study, inhibition of cyclo-oxygenase activity with indomethacin suppressed both acid-induced calcium efflux and the increase in RANKL RNA. These results suggest that acidosis triggers increased cyclo-oxygenase activity, resulting in the release of prostaglandins, including PGE2, which then bind to their respective receptors and cause the observed heightened levels of RANKL RNA. Prostaglandins, especially PGE2, directly stimulate bone resorption in organ culture(38,39) and mediate resorption of mouse calvariae in response to a variety of cytokines and growth factors including epidermal growth factor,(40) platelet-derived growth factor,(41) TNF-α,(42) transforming growth factor-β,(43,44) and metabolic, but not respiratory, acidosis.(11,12) Osteoclast formation in marrow cultures in mice deficient in PGHS-2 (COX-2) was reduced by 60–70% relative to wild-type controls, but could be restored to normal levels by adding exogenous PGE2.(45) Spleen cells from mice deficient in the EP2 PGE2 receptor did not respond to PGE2 with an increase in the number of osteoclasts as measured by TRACP+ multinuclear cells (MNC).(37) An acidosis-induced increase in osteoblastic PGE2, leading to an increase in RANKL and enhanced osteoclastic resorption, may serve as a coupling factor in the coordinated response of osteoblasts and osteoclasts to acidosis.(11,12,16,), 19 While metabolic acidosis leads to increased net calcium efflux from bone, stimulated osteoclastic activity, suppressed osteoblastic collagen synthesis and increased osteoblastic PGE2 production, in contrast respiratory acidosis (a fall in medium pH brought about by an increase in the partial pressure of carbon dioxide) has little effect on any of these parameters.(17,19,46) Further studies will also be necessary to determine if there is an alteration in RANKL RNA during models of respiratory acidosis.
In addition to promoting the maturation of pre-osteoclasts into resorbing bone cells,(27) RANKL has been shown to simulate the activity of mature osteoclasts.(27–29) RANKL treatment of TRACP+ MNCs increases pseudopodial motility(28) and the appearance of actin rings, which are cytoskeletal structures located at regions of contact between the osteoclast and bone.(29) When TRACP+ MNCs are placed on bone mineral, treatment with RANKL increases the number of osteoclastic resorption pits and the total area resorbed,(27–29) apparently by decreasing the probability that a resorbing osteoclast will dissociate from bone.(29) RANKL also can act as an osteoclastic survival factor; it decreased apoptosis of adherent rabbit osteoclasts by 40–60%.(30) In the current study, it is unclear which of these mechanisms, alone or in combination, were responsible for the acidosis-induced increase in bone resorption.
Decreased extracellular pH is also known to directly stimulate osteoclasts. Teti et al. demonstrated that a reduction in extracellular pH acidifies osteoclasts, reduces cytosolic calcium, and promotes expression of cell-matrix attachment structures.(47) These structures, termed podosomes, are located in the area where the osteoclast contacts the bone during resorption.(47) Activation of NFκB, involving its translocation from the cytosol of pre-osteoclasts to the nucleus, is important for their maturation into bone-resorbing osteoclasts. Acidosis activates NFκB in human ovarian carcinoma cells(48) and pancreatic adenocarcinoma cells.(49) It is possible that a reduction in pH also directly stimulates the translocation of NFκB in pre-osteoclasts and/or in mature osteoclasts. Given the relative paucity of osteoclasts in the neonatal bone used in these studies,(50) we expect that it would be difficult to determine if NFκB is directly stimulated by metabolic acidosis. The activity of RANKL may also be pH dependent.
Osteoblasts express both the ligand RANKL and the “decoy” receptor OPG. OPG binds to RANKL, and this interaction prevents RANKL from occupying its receptor, RANK. The greater the ratio of RANKL to OPG, the greater the probability that RANKL will bind to RANK and result in increased maturation of pre-osteoclasts to functional bone-resorbing osteoclasts and in increased osteoclastic activity and survival.(27–29) A coordinate decrease in OPG with an increase in RANKL expression has been shown for some bone-resorbing agents such as parathyroid hormone (PTH),(51) 1,25(OH)2D3,(52) glucocorticoids,(53,54) and interleukin (IL)-11.(52) This reciprocal regulation would be expected to markedly stimulate bone resorption. In contrast, certain antiresorptive agents such as estradiol(55) and vasoactive intestinal peptide(56) increase OPG levels and thus decrease osteoclastic formation and bone resorption. In the current study, the increase in RANKL was not associated with an apparent alteration in the amount of OPG RNA as determined by RT-PCR (Fig. 2). We cannot exclude the possibility that acidosis induced small differences in the amount of OPG RNA, which were not detected by RT-PCR. However these changes, if any, would be small compared with the readily apparent changes in RANKL detected by this method. This increase in RANKL, without an apparent decrease in OPG, would be expected to increase the probability of a RANKL/RANK interaction and stimulate bone resorption, although perhaps not as robust an increase as if OPG had also fallen. In addition to RANKL and OPG, increases in M-CSF or in RANK would also be expected to increase bone resorption.(22–25) However, we did not observe a change in M-CSF RNA with acidosis. Osteoblasts also produce other cytokines reported to act synergistically with RANKL to promote osteoclast activity, including TNF-α(57) and TGF-β.(58) Further studies will be necessary to determine the importance, if any, of these cofactors in acid-induced increases in calcium efflux.
In addition to osteoblasts, other cell types including fibroblasts,(59–61) microvascular endothelial cells,(62) and activated T-cells(63) have been shown to express RANKL and induce osteoclastogenesis. While the neonatal bones used in these studies are rich in osteoblasts and osteoblast precursors, future studies will be necessary to determine the precise cells expressing acid-induced RANKL RNA. It is also possible that not all of the active RANKL is in the cell-bound form; human osteosarcoma cell lines have been reported to secrete a soluble form of RANKL.(64)
Previously we have shown that during the initial 3 h of exposure to acidic medium, physicochemical mineral dissolution is responsible for the increase in net calcium flux from bone.(15,18,20) Over longer time periods, greater than 24 h, acidosis induces cell-mediated bone resorption.(11–14,16,17,), 19 The precise time at which cell-mediated bone resorption predominates over physiochemical dissolution is difficult to determine. The results of this study, showing an increase in RANKL at 24 h, which is a cell-mediated effect, are consistent with the previously observed lag in initiation of cell-mediated resorption. The biological effect of RANKL, that of stimulating production and function of mature osteoclasts, would not be evident until some later time period. Burgess et al. reported that after injection of RANKL into mice, an increase in blood ionized calcium could be detected 1 h later; this increase was attributed to activation of mature osteoclasts.(29) In this study, the 5-fold increase in RANKL RNA expression at 48 h was numerically far greater than the 2-fold increase at 24 h, suggesting that the longer duration of acidosis led to greater stimulation of RANKL RNA. Further studies on the kinetics of calcium efflux after the addition of RANKL to calvarial cultures will be necessary to assess the relative roles of precursor maturation, mature osteoclast activation, and survival in this system.
Our data are consistent with a pathway involving induction of cyclo-oxygenase activity by metabolic acidosis, resulting in increased prostaglandin levels, which stimulate RANKL, consequently increasing the maturation of pre-osteoclasts to resorbing osteoclasts and augmenting cell-mediated bone resorption. These data further confirm that prostaglandins mediate the increased RANKL RNA expression and serve as pivotal coupling factors between osteoblasts and osteoclasts during acidosis.
We thank Susan B. Smith for expert technical assistance. This work was supported in part by National Institutes of Health Grants AR 46289, DK 57716, and DK 56788 and funds from the Renal Research Institute.
- 11984 Pathophysiology of hypercalciuria. Am J Physiol 247:F1–F13.,
- 21979 Urinary calcium excretion in human beings. N Engl J Med 301:535–541., ,
- 31982 Effects of metabolic acidosis on PTH and 1, 25(OH)2D3 response to low calcium diet. Am J Physiol 243:F570–F575., , , , ,
- 41980 Effect of metabolic acidosis in intestinal absorption of calcium and phosphorus. Am J Physiol 239:G480–G484., , , , , , ,
- 51966 The effects of chronic acid loads in normal man: Further evidence for the participation of bone mineral in the defense against chronic metabolic acidosis. J Clin Invest 45:1608–1614., ,
- 61964 Chemical composition of the body. In: ComarCL, BronnerF (eds.) Mineral Metabolism. Academic Press, Inc., New York, NY, USA, pp. 1–247.,
- 71986 The effects of metabolic acidosis on bone formation and bone resorption in the rat. Kidney Int 30:694–700., , ,
- 81995 The contribution of acidosis to renal osteodystrophy. Kidney Int 47:1816–1832.
- 91994 Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium bicarbonate. N Engl J Med 330:1776–1781., , , ,
- 101996 Effect of age on blood acid-base composition in adult humans: Role of age-related renal functional decline. Am J Physiol 271:F1114–F1122., ,
- 112001 Metabolic, but not respiratory, acidosis increases bone PGE2 levels and calcium release. Am J Physiol 281:F1058–F1066., , ,
- 122000 Prostaglandins regulate acid-induced cell-mediated bone resorption. Am J Physiol 279:F1077–F1082., , ,
- 131999 In vitro metabolic and respiratory acidosis selectively inhibit osteoblastic matrix gene expression. Am J Physiol 277:F750–F755.,
- 141998 Chronic metabolic acidosis reversibly inhibits extracellular matrix gene expression in mouse osteoblasts. Am J Physiol 275:F840–F847.,
- 151997 Acute metabolic acidosis inhibits the induction of osteoblastic egr-1 and type 1 collagen. Am J Physiol 272:C1450–C1456., ,
- 161996 Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts. Am J Physiol 271:F216–F222.
- 171995 Stimulated osteoclastic and suppressed osteoblastic activity in metabolic but not respiratory acidosis. Am J Physiol 268:C80–C88.
- 181993 Physicochemical effects of acidosis on bone calcium flux and surface ion composition. J Bone Miner Res 8:93–102., , , ,
- 191992 Acidosis inhibits osteoblastic and stimulates osteoclastic activity in vitro. Am J Physiol 262:F442–F448., ,
- 201985 Cellular contribution to pH-mediated calcium flux in neonatal mouse calvariae. Am J Physiol 248:F785–F789., ,
- 211983 Effects of pH on bone calcium and proton fluxes in vitro. Am J Physiol 245:F204–F209., , , ,
- 222000 Regulation of the differentiation and function of osteoclasts. J Pathol 192:4–13.
- 232001 Role of receptor activator of nuclear factor-Kappa B ligand and osteoprotegerin in bone cell biology. J Mol Med 79:243–253.,
- 242001 Control of osteoclastogenesis and bone resorption by members of the TNF family of receptors and ligands. Cytokine Growth Factor Rev 12:9–18., , ,
- 252000 Osteoprotegerin and its ligand: A new paradigm for regulation of osteoclastogenesis and bone resorption. Osteoporos Int 11:905–913.,
- 261996 Regulation of murine osteoblast macrophage colony-stimulating factor production by 1, 25(OH) 2D3. Calcif Tissue Int 59:291–296., , , ,
- 271998 Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176., , , , , , , , , , , , , , , , , , , , , , ,
- 281998 TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997–1001., , , ,
- 291999 The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527–538., , , , , , , , , , ,
- 302000 Cell adhesion is a prerequisite for osteoclast survival. Biochem Biophys Res Commun 270:550–556., , , ,
- 311995 Metabolic acidosis. In: JacobsonHR, StrikerGE, KlahrS (eds.) The Principles and Practice of Nephrology. Mosby, St. Louis, MO, USA, pp. 924–932.
- 322000 Gene expression of osteoprotegerin ligand, osteoprotegerin, and receptor activator of NF-kappaB in giant cell tumor of bone: Possible involvement in tumor cell-induced osteoclast-like cell formation. Am J Pathol 156:761–767., , ,
- 332001 Determination of three isoforms of the receptor activator of nuclear factor-kappaB ligand and their differential expression in bone and thymus. Endocrinology 142:1419–1426., , ,
- 341990 H+-stimulated release of prostaglandin E2 and cyclic adenosine 3′, 5′-monophosphoric acid and their relationship to bone resorption in neonatal mouse calvaria cultures. Bone Miner 11:295–304., , , ,
- 352000 The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: An analysis using specific agonists for the respective EPs. Endocrinology 141:1554–1559., , , , , , , ,
- 361999 Reciprocal gene expression of osteoclastogenesis inhibitory factor and osteoclast differentiation factor regulates osteoclast formation. Biochem Biophys Res Commun 257:719–723.,
- 372000 Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro. Endocrinology 141:2054–2061., , , , ,
- 381970 Prostaglandins: Stimulation of bone resorption in tissue culture. Endocrinology 86:1436–1440.,
- 391972 Evidence that the bone resorption-stimulating factor produced by mouse fibrosarcoma cells is prostaglandin E2. J Exp Med 136:1329–1343., , ,
- 401978 Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria. Biochem Biophys Res Commun 85:966–975.,
- 411982 Platelet-derived growth factor stimulates bone resorption via a prostaglandin-mediated mechanism. Endocrinology 111:118–124., , ,
- 421987 Tumor necrosis factor-α (cachectin) stimulates bone resorption in mouse calvaria via a prostaglandin-mediated mechanism. Endocrinology 120:2029–2036., , , , ,
- 431985 Human transforming growth factor-alpha stimulates bone resorption in vitro. J Clin Invest 76:2016–2019., , , , , ,
- 441985 Alpha and beta human transforming growth factors stimulated prostaglandin production and bone resorption in cultured mouse calvaria. Proc Natl Acad Sci USA 82:4535–4538., , , , , , ,
- 452000 Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture. J Clin Invest 105:823–832., , , , , ,
- 462000 The effects of acid on bone. Curr Opin Nephrol Hypertens 9:369–379.,
- 471989 Extracellular protons acidify osteoclasts, reduce cytosolic calcium, and promote expression of cell-matrix attachment structures. J Clin Invest 84:773–780., , , , , , ,
- 482000 Acidic pH-induced elevation in interleukin 8 expression by human ovarian carcinoma cells. Cancer Res 60:4610–4616.,
- 492000 Regulation of interleukin-8 expression by cellular pH in human pancreatic adenocarcinoma cells. J Interferon Cytokine Res 20:1023–1028., , , , ,
- 501983 Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture. J Cell Biol 96:639–643., , ,
- 512001 Catabolic effects of continuous human PTH (1–38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 142:4047–4054., , , , , , , ,
- 521999 Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357., , , , ,
- 531999 Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: Potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 140:4382–4389., , , , ,
- 541994 Mechanisms of glucocorticoid action in bone cells. J Cell Biochem 56:295–302., ,
- 551999 Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 140:4367–4370., , , , ,
- 562000 The inhibitory effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide on osteoclast formation are associated with upregulation of osteoprotegerin and downregulation of RANKL and RANK. Biochem Biophys Res Commun 271:158–163., , , ,
- 572001 Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism. J Cell Biochem 83:70–83., , , ,
- 582000 Endogenous production of TGF-beta is essential for osteoclastogenesis induced by a combination of receptor activator of NF-kappa B ligand and macrophange-colony-stimulating factor. J Immunol 165:4254–4263., , , , , , , ,
- 592002 Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysis. J Orthop Res 20:169–173., , , ,
- 602002 Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells. Ann Rheum Dis 61:103–109., , , , , , , ,
- 612000 Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation. J Bone Miner Res 15:1459–1466., , , ,
- 622001 Receptor activator of NF-Kappa B and osteoprotegerin expression by human microvascular endothelial cells, regulation by inflammatory cytokines, and role in human osteoclastogenesis. J Biol Chem 276:20659–20672., , , , ,
- 632001 Activated human T cells directly induce osteoclastogenesis from human monocytes: Possible role of T cells in bone destruction in rheumatoid arthritis patients. Arthritis Rheum 44:1003–1012., , , , , , , , , , , , , , , ,
- 642002 Human osteosarcoma-derived cell lines produce soluble factor(s) that induces differentiation of blood monocytes to osteoclast-like cells. Int Immunopharmacol 2:25–38., , , , , , , , , ,