&-Microglobulin from cultured SHARON Nephrology
induces calcium efflux neonatal mouse calvariae
M. MOE AND STUART Program,
Pritzker
M. SPRAGUE
School of Medicine, University of Chicago, Chicago, Illinois 60637
Moe, Sharon M., and Stuart M. Sprague. &Microglobulin induces calcium efflux from cultured neonatal mousecalvariae. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F540-F545, 1992.~&Microglobulin (&M) polymerizes to form amyloid fibrils that deposit and cause destructive bone lesionsin patients on chronic dialytic therapy. &M is mitogenic to osteoblasts;however, its effect on bone mineralization is unknown. To determine whether P,M causesbone demineralization, neonatalmousecalvariae wereincubated with and without ,&M, and net calcium flux was calculated. Following a 48-h but not 3- or 24-h incubation, ,&M ( 10-8-10-6 M) induced a net calcium efflux. The efflux was similar to that observed with lo-lo M parathyroid hormone (PTH) but less than that observedwith lo-” M PTH. Devitalizing the calvariae resulted in a net calcium influx that wasunaffected by the addition of&M, indicating a cell-mediatedphenomenon.The releaseof ,&glucuronidase,an osteoclastenzyme, increasedafter a 48-h but not a 24-h incubation with ,&M. Calcitonin, an osteoclastinhibitor, blocked the &M-induced calcium efflux and ,&glucuronidase release,suggestingosteoclastinvolvement. Thus ,&M inducesa dose- and time-dependent, cell-mediated calcium efflux from neonatal mousecalvariae that involves osteoclaststimulation. osteoblast; osteoclast; ,&-microglobulin amyloidosis; bone resorption
THE PROTEIN ,&-microglobulin &M) has a molecular mass of 11.8 kDa and is found on the surface of all
nucleated cells noncovalently associated with the light chain component of the class I major histocompatibility antigens (MHC I) (13). Serum concentrations in humans with normal renal function range from 1-2 x lOA M; however, concentrations 5-10 times greater are found in patients undergoing chronic dialytic therapy (25). ,&M frequently deposits as amyloid in patients with chronic renal failure, resulting in skeletal and periarticular disease, including the development of bone cysts and destructive osteoarthropathies (13). Data from a preliminary study in which &M was injected subcutaneously over the parietal bones of young mice suggested that ,&M induced bone resorption (17). Evidence that ,&M has a physiological function in bone metabolism is supported by the finding that bonederived growth factor isolated from rat calvariae is homologous to &M (6). Subsequent investigations have demonstrated that ,&M is an osteoblast mitogen (6, 7, 9). Some investigators believe that ,&M is not a typical growth factor but, instead, a regulator of other growth factors (5, 9). &M enhances both receptor number and gene transcription of insulin-like growth factor I (IGF-I) receptors (7) and may therefore represent a signal link in the complex regulatory system of bone remodeling involving both systemic and local factors. To determine whether &M induces net calcium efflux from bone, we incubated neonatal mouse calvariae with and without graded concentrations of &M. Cultured neonatal mouse calvariae have functioning osteoblasts and osteoclasts, respond to hormones and protons as F540
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$2.00
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does human bone in vivo, and thus provides an appropriate model for the in vitro study of bone mineral dissolution (l-4, 23). METHODS Organ
Culture
of
Bone
Neonatal (4-6 days old) CD-l mice (Charles River, Portage, MI) were killed by cervical dislocation, and their calvariae were removed by dissectionas previously described(l-4, 16, 18, 23, 24). Three milliliters of Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 15% (vol:vol) heat-inactivated (56”C, 1 h) horse serum and 2.8 mM L-glutamine (all from Whittaker Bioproducts, Walkersville, MD), heparin sodium(10 U/ml), and potassiumpenicillin (100 U/ml) were preincubated at 5% CO, and 37°C for 3 h in 35-mm Petri dishes.One milliliter was then removed for initial measurements,and two calvariae were placed in each dish on a stainlesssteel wire grid. Calvariae were incubated for 3, 24, or 48 h, at which time a secondsampleof mediumwasremoved for analysis.Experimental and control cultures were performed in parallel and random order, and the data presentedin Figs. 1-7 and Table 1 represent a minimum of three independent experiments to achieve the number of pairs of calvariae in each group. Experimental
Conditions
Calvariae were cultured with and without ,&M at concentrations ranging from lo-l1 to 10e6M. Varying concentrations of human P,M (purified by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis;SigmaChemical, St. Louis, MO) wereadded to the mediumin a constant volume to achieve the desiredfinal concentrations. Additionally, some calvariae were incubated with or without bovine parathyroid hormone [PTH-( 1- 34); IO-lo and IO- 8 M] or with or without 3 x 10eg M salmon calcitonin (both from Bachem, Torrance, CA). These concentrations of PTH and calcitonin have previously been shownto increase(2, 19, 23) and inhibit (8, 14, 23) calcium releasefrom cultured calvariae, respectively. P,M, PTH, and calcitonin were addedprior to preincubating the medium.In someexperiments, the bone cells in calvariae were killed by three cycles of rapid freezing (-70°C) followed by rapid thawing (+23”C) before incubation (2, 23). DNA
Synthesis
DNA synthesis was determined by measuringthe uptake of [3H]thymidine into calvariae. Paired calvariae were incubated for 48 h with either low5 M phorbol 12-myristate 13-acetate (PMA) or &M (lO-g-lO-7 M). The calvariae were then removed from each culture dish and incubated for an additional hour in serum-freeDMEM containing 1.0mM thymidine, 5 &i [3H]thymidine, and the appropriate concentration of &M or PMA. The calvariae were then digestedwith 5% trichloroacetic acid, rinsed with acetoneand ether, weighed,solubilized for 18 h (Solvable; New England Nuclear ResearchProducts, Boston, MA), and placed in scintillation fluid for counting. DNA synthesis is expressedas counts per minute per microgram dry weight.
0 1992 the American
Physiological
Society
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,&-MICROGLOBULIN-INDUCED Enzyme
Flux Calculations
Analysis
Regressionswere calculated by least square analysis, and group meanswere compared with analysis of variance using conventional statistical programs (Instat, San Diego, CA) written for a personalcomputer. All valuesare meanst SE; P < 0.05 was consideredsignificant. RESULTS
Effect of P,M on Net Calcium Flux
The addition
of ,&M in concentrations from 1O-11 to induced a dose-dependent net calcium efflux from bone after a 48-h incubation (Fig. 1). The peak response to &M was observed at a concentration of 10msM (P,M 581.5 t 59.2 vs. control 207.3 t 26.9 nmol bone w-148 h-l; P < 0.001). Greater concentrations of &M led to a similar efflux of calcium. The data presented in Fig. 1 suggest that the slope of the doseresponse curve is maximal between low9 and 10es M &M. To test whether this phenomenon was time dependent, calvariae were incubated for 3 and 24 h in control medium 10e6 M to the culture medium
l
l
16”
l$’
lo-’ lo-” If7
&Microglobulin
lo-” PTH
(M)
Fig. 1. Effect of increasing concentrations of µglobulin (P2M) on net calcium flux in calvariae incubated for 48 h. Values are means t SE. Twenty-one pairs of calvariae were incubated in control (Ctl) medium (open bar), and 6-9 pairs were incubated at each concentration of &M (solid bars). Compared with control, pzM induced a net calcium efflux at concentrations ranging from lops to 10m6 M, although this was less than that observed with 10e8 M parathyroid hormone (PTH; hatched bar). Symbols indicate significant difference (P < 0.05): * compared with Ctl; T compared with lo-l1 M P2M; o compared with lo-lo M P2M; and $ compared with 10Mg M ,&M.
750
Net calcium flux was calculated as V,(C, - Ci) where V, is the mediumvolume (2 ml), and C, and Ci arethe final and initial calcium concentrations, respectively, in nanomolesper milliliter (l-4,23). A positive flux (efflux) indicates movement of calcium from bone into medium, and a negative flux (influx) indicates the movement from medium into bone. Statistical
ctl
Determination
Total calcium was determined by automatic fluorometric titration (Calcette; Precision Systems, Sudbury, MA) (l-4, 23). The closeagreementof bone culture medium calcium concentration measuredby fluorometric titration with that measured by atomic absorption spectrophotometry has been previously demonstratedin our laboratory (3). Calcium
F541
Determination
Phosphatase actiuity. Release of acid and alkaline phosphataseare indicative of osteoclastand osteoblastactivity, respectively. Specific phosphataseactivities in medium were determined by a modification (23) of the method describedby Dziak et al. (22). The medium was incubated with 30 mM p-nitrophenolphosphate and either an acid (citrate, pH 4.8, 1 h; for acid phosphatase)or alkaline (glycine, pH 10.3, 30 min; for alkaline phosphatase)buffer at 37°C. The reaction was terminated by adding cold 0.1 N NaOH, and the amount of p-nitrophenol releasedwasmeasuredspectrophotometrically at 410nm (23). Acid and alkaline phosphataseactivity is expressedas nanomolesof p-nitrophenol releasedper bone per hour. ,&Glucuronidase. ,&Glucuronidasereleaseis indicative of osteoclast bone resorption and correlates with calcium efflux (8, 23). ,&Glucuronidaseactivity wasdetermined using a modification of the assay described by Fishman et al. (Sigma) (12). Immediately following the experimental culture period, an aliquot of mediumwas incubated (1 h, 56°C) with 0.03 M phenolphthalein mono-@-glucuronicacid and 0.2 M acetate buffer. The reaction was terminated by adding 0.1 M 2-amino-2-methyl-lpropanol buffer, and the amount of phenolphthalein released was measured spectrophotometrically at 550 nm. ,&Glucuronidaseactivity wascalculated asmicrogramsof phenolphthalein releasedper bone per hour and expressedas percent of control activity. Calcium
CALCI JM EFFLUX
X
i
600-
3 E
0
Control
m
4 x 10-8M
eZa
4 x 10-7M
*+o T
F4500
c-l
0 I-150-
3
24 Time (hr)
48
Fig. 2. Effect of ,&M on net calcium flux in calvariae incubated for 3,24, and 48 h. Values are means t SE with at least 6 pairs of calvariae per group. During a 3- or 24-h incubation, fi,M did not alter net calcium flux relative to control incubations. However, after a 48-h incubation, ,&M induced a net calcium efflux that was greater than control and greater than that observed when calvariae were incubated with similar concentrations of P2M for 3 or 24 h. * Compared with control at same time period, P < 0.001. 7 Compared with same pzM concentration after 3 h, P < 0.01. o Compared with same ,&M concentration after 24 h, P < 0.001.
and medium supplemented with 4 x 10Vs and 4 x low7 M &-M. In contrast to a 48-h incubation, &M did not induce a significant calcium efflux relative to control during the shorter incubation periods (Fig. 2). However, the calcium efflux observed at these concentrations of &M during a 48-h incubation was greater than that seen with equivalent ,&M concentrations incubated for 3 or 24 h (P < 0.001; Fig. 2).
To determine whether the magnitude of the net calcium efflux from bone was comparable to that of PTH, a known physiological stimulator of calcium release, the calcium efflux induced by 10m7 M &M was compared with that induced by PTH with and without ,&M. Calvariae incubated with &M had a calcium efflux similar to
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F542
,&-MICROGLOBULIN-INDUCED
that observed from calvariae incubated with a submaxima1 concentration of PTH (lOs10 M). However, calvariae incubated with lo- 8 M PTH exhibited a significantly greater calcium efflux than calvariae incubated with ,&M alone or lo- lo M PTH alone (P < 0.01; Fig. 3). The combination of either concentration of PTH and &M did not further increase the net calcium efflux observed with PTH alone. Mechanism of &M-Induced
Calcium Efflux
To determine whether the &M-induced net calcium efflux was ceil mediated or due to an alteration in the physicochemical driving forces for mineralization, calvariae were killed by successive freeze-thaw cycles, a maneuver previously shown to cause net calcium influx into bone (2, 23). After a 48-h incubation, ,&M (4 x 10v7 M) induced a net calcium efflux from live calvariae that was greater than that observed in live calvariae incubated in unsupplemented medium (P < 0.02; Fig. 4). Killing the calvariae prior to incubation resulted in a net calcium influx that was not altered by the addition of ,&M to the medium (Fig. 4). A 3-h incubation was performed because previous studies have demonstrated that alterations in the physicochemical driving forces for bone mineralization occur during this short time period (2); however, ,&M did not alter the net calcium flux in either live or dead calvariae after a 3-h incubation (Fig. 4). Compared with control, the release of the osteoclastic lysosomal enzyme ,&glucuronidase was significantly increased by the addition of &M at concentrations greater than lo-” M (Fig. 5). The maximal release of ,LI-glucuronidase was observed at 10e7 M ,&M. The ,&glucuronidase release stimulated by 10m7 and 10V6 M ,&M was similar to that induced by 10m8 M PTH, a known osteoclast stimulator (10 -7 M /!&M, 145.7 t 11.7%; lO-6 M ,&M, 136.7 t 11.4%; and PTH, 154.9 t 8.7%; all P < 0.001 vs. control, Fig. 5). The release of acid phosphatase, also an osteoclast enzyme, from calvariae incubated with lo-’ M &M for 48 h
CALCIUM
EFFLUX
600 I
Control
-
400 X
2 A 200LL g E< O. .“J z -2~ --Orw -400 u c-l
-600
-
-800
Live
Live
Dead
3 hr
Dead
48 hr-
Fig. 4. Effect of P,M on net calcium flux in live and dead calvariae. Values are means rfl SE for 6-8 pairs of calvariae per group. Calvariae were incubated for 3 and 48 h. A positive flux represents calcium efflux from bone into medium; a negative flux represents calcium influx from medium into bone. After a 3-h incubation in control medium, there was a small calcium influx in both live and dead calvariae that was not altered by addition of ,&M (4 x lob7 M). After a 48-h incubation, addition of ,&M resulted in a net calcium efflux. Killing the calvariae resulted in a calcium influx that was not altered by addition of &M. * Different from control, same duration, P < 0.02. t Different from live, same treatment, P < 0.001.
was greater than control incubations and similar to that observed with 10m8 M PTH (Table 1). Alkaline phosphatase, a marker of osteoblast activity, was not affected by a 48-h incubation with &M (Table 1). Neither acid nor alkaline phosphatase was stimulated by a 24-h incubation with &M. Calcitonin (3 X 10 -g M), an osteoclast inhibitor, induced a net calcium influx into bone incubated in control medium (P < 0.01; Fig. 6). The addition of calcitonin to medium supplemented with &M (4 x 10m8 M) blocked both the &M-induced calcium efflux (Fig. 6) and release of ,&glucuronidase (Fig. 7).
1000 i
Q 0
Ctl Ctl Basal
&M Medium
Ctl
&M
PTH (lo-“M)
1 d”
I 0-l’
I 6’
I o-a
&Microglobulin
I o-’
I 0-’
PTH
(M)
Ctl PTH (10-8M)
Fig. 3. Effect of ,&M and PTH on net calcium flux in calvariae incubated for 48 h. Values are means t SE for 8-18 pairs of calvariae per group. Compared with control (Ctl; open bars), ,&M (10s7 M; solid bars) induced a net calcium efflux which was similar to that observed with lo-lo M PTH b u t 1ess than that observed with lob8 M PTH. Addition of ,&M to medium containing PTH did not alter the PTH-induced calcium efflux. * Different from Ctl, same PTH concentration, P < 0.01. t Different from basal medium, same ,&M treatment, P < 0.05. 0 Different from lo-lo M PTH, P < 0.01.
Fig. 5. Effect of increasing concentrations of ,&M on ,&glucuronidase release in calvariae incubated for 48 h. Values are means k SE for 7-30 pairs of calvariae per group. ,&Glucuronidase activity was calculated as pg phenolphthalein released. bone- lhl and is expressed as % of control activity. Compared with control (Ctl, open bar) ,&M (solid bars) significantly increased release of ,&glucuronidase at concentrations ranging from low8 to lop6 M. Release of ,&glucuronidase induced by low7 and lo-” M ,&M was similar to that observed with 10ms M PTH (hatched bar). Symbols indicate significant difference (P < 0.05): * compared with Ctl; t compared with lo-l1 M ,&M; o compared with lo-lo M P2M; $ compared with 10vg M ,&M.
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&MICROGLOBULIN-INDUCED
DNA synthesis, as measured by [3H]thymidine uptake after a 48-h incubation, was not stimulated by the addition of ,&M (10 -‘-10e7 M), compared with calvariae incubated in control medium (lo-’ M ,&M, 5.85 t 0.88; lo+ M &M, 6.67 t 0.93; 1O-7 M ,&M, 6.47 t 1.01; control, 5.14 t 0.61 countsmin-l l pg dry wt-‘; not significant). However, the addition of 10m5 M PMA, a known mitogen in calvarial cultures (1 l), stimulated [3H] thymidine uptake (9.14 t 1.38 counts min-l pg dry wt-l; P < 0.01 compared with control). l
l
DISCUSSION
,&M induces a dose-dependent net calcium efflux from cultured neonatal mouse calvariae at concentrations ranging from 10 -8 to 10m6 M. The calcium efflux occurs after incubation of live but not dead calvariae, indicating that &M induces a cell-mediated release of calcium. The observation that the ,&M-induced calcium efflux occurred after a 48-h but not 24-h incubation, as did the release of the osteoclast enzymes ,&glucuronidase and acid phosphatase, is consistent with a cell-mediated phe-
-200 -300
Ctl
PM 2
Calc
&M 8c Calc
Fig. 6. Effect of &M and calcitonin (Calc) on net calcium flux in calvariae incubated for 48 h. Values are means t SE for 8-13 pairs of calvariae per group. Compared with control (Ctl), ,&M (4 x 10es M) induced a net calcium efflux. Calcitonin (3 x 10Bg M) induced a net calcium influx and completely blocked efflux induced by ,&M. * Compared with Ctl, P < 0.005. “f Compared with &M, P < 0.001. 175 CI>
I-
150
k!i -u -125 l
-
-
Ctl
PM 2
Calc
&M 8c Calc
Fig. 7. Effect of ,&M and calcitonin (Calc) on P-glucuronidase release in calvariae incubated for 48 h. Values are means rt SE for 3-9 pairs of calvariae per group. P-Glucuronidase activity was calculated as pg phenolphthalein released bone-l. h-l and is expressed as % of control activity. Compared with control (Ctl), ,&M (4 x low8 M) significantly increased, whereas calcitonin (3 x 10Bg M) inhibited, release of @-glucuronidase. Addition of calcitonin to calvariae incubated with ,&M blocked ,&M-induced release of P-glucuronidase. * Compared with Ctl, P c 0.05. t Compared with P2M, P < 0.001. l
CALCIUM
F543
EFFLUX
Table 1. Effect of &M on calvarial enzyme release
Control P2M
,6-Glucuronidase, % of control (n = 7-30)
Acid Phosphatase, nmol.bone-l-48 (n = 9-23)
100 127.5&11.4* 154.9+8.7*-t
228.5t17.6 294.8t34.4* 356.0t25.8*
h-l
nmol
Alkaline Phosphatase, bone-l 48 h-1 ;n = 9-26)
952.8258.5 1,038.9+73.3 1,012.8+83.1
PTH Values are means t SE; n, no. of pairs of calvariae. Calvariae were incubated for 48 h in unaltered control medium, medium supplemented with 10m8 M fi2-microglobulin (P2M), or medium supplemented with 10v8 M parathyroid hormone (PTH). ,&Glucuronidase activity was measured as pg phenolphthalein bone-l 48 h-l and is expressed as % of control; acid and alkaline phosphatase activity are expressed as nmol p-nitrophenol released bone-l. 48 h-l. * Different from control and + different from P2M (P < 0.05). l
l
l
nomenon. It is possible that our measurements were not sufficiently sensitive to detect small differences over a shorter time period; however, these data suggest a delayed stimulation of ,&M on bone mineral dissolution. This significant time delay supports the hypothesis that p2M acts via the activation of other growth factors in the complex regulation of bone mineralization (5, 9). The &M-induced net calcium efflux from bone could represent either increased bone resorption or decreased bone mineralization. Bone resorption and formation are integrated processes regulated by the interaction between osteoclasts and osteoblasts. p2M significantly increased the release of ,&glucuronidase and acid phosphatase, lysosomal enzymes that correlate with increased osteoclast activity, suggesting ,&M stimulates osteoclastic bone resorption. Further supporting a role for increased osteoelastic bone resorption was the observation that calcitonin, a specific osteoclast inhibitor (10, 14), blocked the &M-induced calcium efflux and ,&glucuronidase release. The mechanisms by which osteoclasts are stimulated to resorb bone are complex and may be mediated by the activation of osteoblasts through a variety of agents (21, 26). Whether P2M directly stimulated osteoclasts, uncoupled bone formation from resorption, or decreased osteoblast bone mineralization could not be determined from these studies. P2M not only affects osteoclast function but has been shown to stimulate cell proliferation and protein synthesis in isolated osteoblast cultures (6, 7, 9). In previous studies variable concentrations of ,&M have been required to stimulate osteoblast proliferation as measured by [3H]thymidine uptake (6, 7, 9). Similar to findings of Canalis et al. (6), we were unable to demonstrate an effect of P2M (1O-g-1O-7 M) on DNA synthesis in calvarial cultures. The incongruity observed in [3H] thymidine incorporation between isolated osteoblast and calvarial cultures suggests that additional components of bone may also be affected by P2M, complicating the interpretation of this experimental measurement. In further support of such complexity, Evans et al. (9) demonstrated that, although osteoblasts respond to and produce &M, &M did not affect the production of alkaline phosphatase or osteocalcin by isolated osteoblasts. Similarly, in the present study we were also unable to demonstrate an effect of P,M on alkaline phosphatase release. These data suggest that,
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F544
,&-MICROGLOBULIN-INDUCED
in addition to osteoblast proliferation, other growth factors or cell types may be required to stimulate bone cell enzyme production and subsequent mineralization. The precise mechanism by which ,&M induces osteoclast-mediated bone resorption and the role of osteoblasts and other growth factors in this process requires further study. Bone remodeling is controlled by hormonal and local factors in a complex regulatory system, and ,&M may act via either of these pathways. Using both a submaximal and pharmacological concentration of PTH, ,&M did not augment calcium efflux from calvariae incubated with both these agents. Similarly, previous studies were unable to demonstrate an effect of PTH on ,&M transcription (20) or an effect of 1,25dihydroxyvitamin D3 on ,&M immunoreactivity (9) in osteoblast cultures. These preliminary data suggest &M may not interact with these calciotropic hormones. Conversely, ,&M does interact with other growth factors in the local regulation of bone remodeling. Centrella et al. (7) demonstrated that ,&M and IGF-I have additive effects on osteoblast proliferation. &M enhances IGF-I receptor number and increases IGF-I transcripts and polypeptide levels in osteoblast-enriched cultures (7). These stimulatory effects of &M appear to be selective, because it does not enhance transforming growth factor-p receptors or transcripts (7). ,&M induces bone mineral dissolution in the mouse calvariae model after a significant time delay; whether this delay is due to a multi-step process involving IGF-I and/or other cytokines remains to be determined. In conclusion, these studies demonstrate that ,&M stimulates calcium efflux and ,&glucuronidase release from cultured neonatal mouse calvariae in a dose- and time-dependent manner. The mechanism of ,&M-induced bone mineral dissolution is cell mediated and involves osteoclast stimulation either directly or by inducing osteoblasts and/or bone cytokines. Further study delineating the role of&M in the interaction between osteoblasts and osteoclasts as well as its possible role in the regulation of cytokine production in bone remodeling is required. We thank S. A. Barrett for expert technical assistance. This study was supported by the Baxter Health Care Extramural Grant Program and by a grant-in-aid from the National Kidney Foundation of Illinois. S. M. Moe was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Training Grant 5T32DK-07510-01. These data were presented, in part, to the 24th Annual Meeting of The American Society of Nephrology, Baltimore, MD, November 1720. Present address of S. M. Moe: Dept. of Medicine, Indiana Univ. Med. School, 108 Sessler Hall, 1120 South Dr., Indianapolis, IN 46202. Address for reprint requests: S. M. Sprague, Dept. of Medicine, Box 28, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Received 18 September 1991; accepted in final form 23 March 1992. REFERENCES 1. Bushinsky, D. A. Effects of parathyroid hormone on net proton flux from neonatal mouse calvariae. Am. J. Physiol. 252 (Renal FZuid Electrolyte Physiol. 21): F585-F589, 2987. 2. Bushinsky, D. A., J. M. Goldring, and F. L. Coe. Cellular contribution to pH-mediated calcium flux in neonatal mouse calvariae. Am. J. Phvsiol. 248 (Renal Fluid Electrolyte Physiol. 17):
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EFFLUX
F785-F789, 1985. Bushinsky, D. A., N. S. Krieger, D. I. Geisser, E. B. Grossman, and F. L. Coe. Effects of pH on bone calcium and proton fluxes in vitro. Am. J. Physiol. 245 (Renal Fluid Electrolyte Physiol. 14): F204-F209, 1983. 4. Bushinsky, D. A., and R. J. Lechleider. Mechanism of proton-induced bone calcium release: calcium carbonate dissolution. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F998F1005, 1987. 5. Canalis, E., T. McCarthy, and M. Centrella. Growth factors and the regulation of bone remodeling. J. Clin. Invest. 81: 277-281, 1988. and M. Centrella. A bone-derived 6. Canalis, E., T. McCarthy, growth factor isolated from rat is µglobulin. Endocrinology 121: 1198-1200, 1987. 7. Centrella, M., T. L. McCarthy, and E. Canalis. ,&-microglobulin enhances insulin-like growth factor I receptor levels and synthesis in bone cell cultures. J. Biol. Chem. 264: 18268-18271, 1989. 8. Eilon, G., and L. G. Raisz. Comparison of the effects of stimulators and inhibitors of resorption on the release of lysosomal enzymes and radioactive calcium from fetal bone in organ cultures. Endocrinology 103: 1969-1975, 1978. 9. Evans, D. B., M, Thavarajah, and J. A. Kanis, Immunoreactivity and proliferative actions of ,&-microglobulin on human bone-derived cells in vitro. Biochem. Biophys. Res. Commun. 175: 795-803, 1991. 10. Farly, J. R., N. M. Tarbaux, S. L. Hall, T. A. Linkhart, and D. J. Baylink. The anti-bone-resorptive agent calcitonin also acts in vitro to directly increase bone formation and bone cell proliferation. Endocrinology 123: 159-167, 1988. 11. Feyen, J. H. M., D. N. Petersen, and B. E. Kream. Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate. J. Bone Miner. Res. 3: 173-179, 1988. W. H., K. Kato, C. L. Anstiss, and S. Green. 12. Fishman, Human serum ,&glucuronidase; its measurement and some of its properties. Clin. Chim. Acta 15: 435-447, 1967. 13. Gorevic, P. D., T. T. Casey, W. J. Stone, C. R. Diraimondo, F. C. Prelli, and B. Frangione. ,&-Microglobulin is an amyloidogenic protein in man. J. Chin. Invest. 76: 2425-2429, 1985. M. E., L. G. Raisz, and H. A. Simmons. The effects 14. Holtrop, of parathyroid hormone, colchicine, and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture. J. Cell Biol. 60: 346-355, 1974. 15. Hurst, N. P., R. Van Den Berg, A. Disney, M, Alcock, L. Albertyn, M. Green, and V. Pascoe. Dialysis related arthropathy. Ann. Rheum. Dis. 48: 409-420, 1989. 16. Ivey, J. L., D. R. Wright, and A. H. Tashijan. Bone resorption in organ culture. J. Clin. Invest. 58: 1327-1338, 1976. 17. Kang, M. S., C. C. Li, and J. Petersen. In vivo effect of ,&-microglobulin on bone resorption in mice (Abstract). Kidney Int. 37: 304, 1990. G., and G. Vaes. Collagenase, procollagenase 18 Lenaers-Claeys, and bone resorption effects of heparin, parathyroid hormone and calcitonin. Biochim. Biophys. Acta 584: 375-388, 1979. 19# Lerner, U. H. Modifications of the mouse calvarial technique improve the responsiveness to stimulators of bone resorption. J. 3.
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McCarthy, T. L., M. Centrella, and E. Canalis. Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone. Endocrinology 124: 1247-1253, 1989. 21. Rodan, G. A., and T. J. Martin. Role of osteoblasts in hormonal control of bone resorption-a hypothesis. C&if Tissue Int. 33: 349-351, 1981. 22. Shlossman, M., M. Brown, E. Shapiro, and R. Dziak. Calcitonin effects on isolated bone cells. Calcif. Tissue Int. 34: 19020.
196, 1982. 23.
Sprague, S. M., and D. A, Bushinsky. Mechanism of aluminum-induced calcium efflux from cultured neonatal mouse calvariae. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F583-F588, 1990.
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,&-MICROGLOBULIN-INDUCED 24.
Stern, P. H., and N. S. Krieger. bones and neonatal mouse mone and 1,25dihydroxyvitamin 176, 1983.
25.
calvaria:
Ullian, M. E., W. S. Hammond, and B. A. Molitoris. ,&-Microglobulin
CALCIUM
Comparison of fetal rat limb effects of parathyroid horD3. Calcif. Tissue Int. 35: 172-
A. C. Alfrey, associated
A. Schultz, amyloidosis
in
EFFLUX
F545
chronic hemodialysis patients with carpel tunnel syndrome. Medicine 68: 107-115, 1989. 26. Vaes, G. Cellular biology and biochemical mechanism of bone resorption: a review of recent developments on the formation, activation, and mode of action of osteoclasts. CZin. Orthop. Relat. Res. 231: 239-271, 1988.
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induces calcium efflux neonatal mouse calvariae
M. MOE AND STUART Program,
Pritzker
M. SPRAGUE
School of Medicine, University of Chicago, Chicago, Illinois 60637
Moe, Sharon M., and Stuart M. Sprague. &Microglobulin induces calcium efflux from cultured neonatal mousecalvariae. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F540-F545, 1992.~&Microglobulin (&M) polymerizes to form amyloid fibrils that deposit and cause destructive bone lesionsin patients on chronic dialytic therapy. &M is mitogenic to osteoblasts;however, its effect on bone mineralization is unknown. To determine whether P,M causesbone demineralization, neonatalmousecalvariae wereincubated with and without ,&M, and net calcium flux was calculated. Following a 48-h but not 3- or 24-h incubation, ,&M ( 10-8-10-6 M) induced a net calcium efflux. The efflux was similar to that observed with lo-lo M parathyroid hormone (PTH) but less than that observedwith lo-” M PTH. Devitalizing the calvariae resulted in a net calcium influx that wasunaffected by the addition of&M, indicating a cell-mediatedphenomenon.The releaseof ,&glucuronidase,an osteoclastenzyme, increasedafter a 48-h but not a 24-h incubation with ,&M. Calcitonin, an osteoclastinhibitor, blocked the &M-induced calcium efflux and ,&glucuronidase release,suggestingosteoclastinvolvement. Thus ,&M inducesa dose- and time-dependent, cell-mediated calcium efflux from neonatal mousecalvariae that involves osteoclaststimulation. osteoblast; osteoclast; ,&-microglobulin amyloidosis; bone resorption
THE PROTEIN ,&-microglobulin &M) has a molecular mass of 11.8 kDa and is found on the surface of all
nucleated cells noncovalently associated with the light chain component of the class I major histocompatibility antigens (MHC I) (13). Serum concentrations in humans with normal renal function range from 1-2 x lOA M; however, concentrations 5-10 times greater are found in patients undergoing chronic dialytic therapy (25). ,&M frequently deposits as amyloid in patients with chronic renal failure, resulting in skeletal and periarticular disease, including the development of bone cysts and destructive osteoarthropathies (13). Data from a preliminary study in which &M was injected subcutaneously over the parietal bones of young mice suggested that ,&M induced bone resorption (17). Evidence that ,&M has a physiological function in bone metabolism is supported by the finding that bonederived growth factor isolated from rat calvariae is homologous to &M (6). Subsequent investigations have demonstrated that ,&M is an osteoblast mitogen (6, 7, 9). Some investigators believe that ,&M is not a typical growth factor but, instead, a regulator of other growth factors (5, 9). &M enhances both receptor number and gene transcription of insulin-like growth factor I (IGF-I) receptors (7) and may therefore represent a signal link in the complex regulatory system of bone remodeling involving both systemic and local factors. To determine whether &M induces net calcium efflux from bone, we incubated neonatal mouse calvariae with and without graded concentrations of &M. Cultured neonatal mouse calvariae have functioning osteoblasts and osteoclasts, respond to hormones and protons as F540
0363-6127/92
$2.00
Copyright
does human bone in vivo, and thus provides an appropriate model for the in vitro study of bone mineral dissolution (l-4, 23). METHODS Organ
Culture
of
Bone
Neonatal (4-6 days old) CD-l mice (Charles River, Portage, MI) were killed by cervical dislocation, and their calvariae were removed by dissectionas previously described(l-4, 16, 18, 23, 24). Three milliliters of Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 15% (vol:vol) heat-inactivated (56”C, 1 h) horse serum and 2.8 mM L-glutamine (all from Whittaker Bioproducts, Walkersville, MD), heparin sodium(10 U/ml), and potassiumpenicillin (100 U/ml) were preincubated at 5% CO, and 37°C for 3 h in 35-mm Petri dishes.One milliliter was then removed for initial measurements,and two calvariae were placed in each dish on a stainlesssteel wire grid. Calvariae were incubated for 3, 24, or 48 h, at which time a secondsampleof mediumwasremoved for analysis.Experimental and control cultures were performed in parallel and random order, and the data presentedin Figs. 1-7 and Table 1 represent a minimum of three independent experiments to achieve the number of pairs of calvariae in each group. Experimental
Conditions
Calvariae were cultured with and without ,&M at concentrations ranging from lo-l1 to 10e6M. Varying concentrations of human P,M (purified by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis;SigmaChemical, St. Louis, MO) wereadded to the mediumin a constant volume to achieve the desiredfinal concentrations. Additionally, some calvariae were incubated with or without bovine parathyroid hormone [PTH-( 1- 34); IO-lo and IO- 8 M] or with or without 3 x 10eg M salmon calcitonin (both from Bachem, Torrance, CA). These concentrations of PTH and calcitonin have previously been shownto increase(2, 19, 23) and inhibit (8, 14, 23) calcium releasefrom cultured calvariae, respectively. P,M, PTH, and calcitonin were addedprior to preincubating the medium.In someexperiments, the bone cells in calvariae were killed by three cycles of rapid freezing (-70°C) followed by rapid thawing (+23”C) before incubation (2, 23). DNA
Synthesis
DNA synthesis was determined by measuringthe uptake of [3H]thymidine into calvariae. Paired calvariae were incubated for 48 h with either low5 M phorbol 12-myristate 13-acetate (PMA) or &M (lO-g-lO-7 M). The calvariae were then removed from each culture dish and incubated for an additional hour in serum-freeDMEM containing 1.0mM thymidine, 5 &i [3H]thymidine, and the appropriate concentration of &M or PMA. The calvariae were then digestedwith 5% trichloroacetic acid, rinsed with acetoneand ether, weighed,solubilized for 18 h (Solvable; New England Nuclear ResearchProducts, Boston, MA), and placed in scintillation fluid for counting. DNA synthesis is expressedas counts per minute per microgram dry weight.
0 1992 the American
Physiological
Society
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,&-MICROGLOBULIN-INDUCED Enzyme
Flux Calculations
Analysis
Regressionswere calculated by least square analysis, and group meanswere compared with analysis of variance using conventional statistical programs (Instat, San Diego, CA) written for a personalcomputer. All valuesare meanst SE; P < 0.05 was consideredsignificant. RESULTS
Effect of P,M on Net Calcium Flux
The addition
of ,&M in concentrations from 1O-11 to induced a dose-dependent net calcium efflux from bone after a 48-h incubation (Fig. 1). The peak response to &M was observed at a concentration of 10msM (P,M 581.5 t 59.2 vs. control 207.3 t 26.9 nmol bone w-148 h-l; P < 0.001). Greater concentrations of &M led to a similar efflux of calcium. The data presented in Fig. 1 suggest that the slope of the doseresponse curve is maximal between low9 and 10es M &M. To test whether this phenomenon was time dependent, calvariae were incubated for 3 and 24 h in control medium 10e6 M to the culture medium
l
l
16”
l$’
lo-’ lo-” If7
&Microglobulin
lo-” PTH
(M)
Fig. 1. Effect of increasing concentrations of µglobulin (P2M) on net calcium flux in calvariae incubated for 48 h. Values are means t SE. Twenty-one pairs of calvariae were incubated in control (Ctl) medium (open bar), and 6-9 pairs were incubated at each concentration of &M (solid bars). Compared with control, pzM induced a net calcium efflux at concentrations ranging from lops to 10m6 M, although this was less than that observed with 10e8 M parathyroid hormone (PTH; hatched bar). Symbols indicate significant difference (P < 0.05): * compared with Ctl; T compared with lo-l1 M P2M; o compared with lo-lo M P2M; and $ compared with 10Mg M ,&M.
750
Net calcium flux was calculated as V,(C, - Ci) where V, is the mediumvolume (2 ml), and C, and Ci arethe final and initial calcium concentrations, respectively, in nanomolesper milliliter (l-4,23). A positive flux (efflux) indicates movement of calcium from bone into medium, and a negative flux (influx) indicates the movement from medium into bone. Statistical
ctl
Determination
Total calcium was determined by automatic fluorometric titration (Calcette; Precision Systems, Sudbury, MA) (l-4, 23). The closeagreementof bone culture medium calcium concentration measuredby fluorometric titration with that measured by atomic absorption spectrophotometry has been previously demonstratedin our laboratory (3). Calcium
F541
Determination
Phosphatase actiuity. Release of acid and alkaline phosphataseare indicative of osteoclastand osteoblastactivity, respectively. Specific phosphataseactivities in medium were determined by a modification (23) of the method describedby Dziak et al. (22). The medium was incubated with 30 mM p-nitrophenolphosphate and either an acid (citrate, pH 4.8, 1 h; for acid phosphatase)or alkaline (glycine, pH 10.3, 30 min; for alkaline phosphatase)buffer at 37°C. The reaction was terminated by adding cold 0.1 N NaOH, and the amount of p-nitrophenol releasedwasmeasuredspectrophotometrically at 410nm (23). Acid and alkaline phosphataseactivity is expressedas nanomolesof p-nitrophenol releasedper bone per hour. ,&Glucuronidase. ,&Glucuronidasereleaseis indicative of osteoclast bone resorption and correlates with calcium efflux (8, 23). ,&Glucuronidaseactivity wasdetermined using a modification of the assay described by Fishman et al. (Sigma) (12). Immediately following the experimental culture period, an aliquot of mediumwas incubated (1 h, 56°C) with 0.03 M phenolphthalein mono-@-glucuronicacid and 0.2 M acetate buffer. The reaction was terminated by adding 0.1 M 2-amino-2-methyl-lpropanol buffer, and the amount of phenolphthalein released was measured spectrophotometrically at 550 nm. ,&Glucuronidaseactivity wascalculated asmicrogramsof phenolphthalein releasedper bone per hour and expressedas percent of control activity. Calcium
CALCI JM EFFLUX
X
i
600-
3 E
0
Control
m
4 x 10-8M
eZa
4 x 10-7M
*+o T
F4500
c-l
0 I-150-
3
24 Time (hr)
48
Fig. 2. Effect of ,&M on net calcium flux in calvariae incubated for 3,24, and 48 h. Values are means t SE with at least 6 pairs of calvariae per group. During a 3- or 24-h incubation, fi,M did not alter net calcium flux relative to control incubations. However, after a 48-h incubation, ,&M induced a net calcium efflux that was greater than control and greater than that observed when calvariae were incubated with similar concentrations of P2M for 3 or 24 h. * Compared with control at same time period, P < 0.001. 7 Compared with same pzM concentration after 3 h, P < 0.01. o Compared with same ,&M concentration after 24 h, P < 0.001.
and medium supplemented with 4 x 10Vs and 4 x low7 M &-M. In contrast to a 48-h incubation, &M did not induce a significant calcium efflux relative to control during the shorter incubation periods (Fig. 2). However, the calcium efflux observed at these concentrations of &M during a 48-h incubation was greater than that seen with equivalent ,&M concentrations incubated for 3 or 24 h (P < 0.001; Fig. 2).
To determine whether the magnitude of the net calcium efflux from bone was comparable to that of PTH, a known physiological stimulator of calcium release, the calcium efflux induced by 10m7 M &M was compared with that induced by PTH with and without ,&M. Calvariae incubated with &M had a calcium efflux similar to
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F542
,&-MICROGLOBULIN-INDUCED
that observed from calvariae incubated with a submaxima1 concentration of PTH (lOs10 M). However, calvariae incubated with lo- 8 M PTH exhibited a significantly greater calcium efflux than calvariae incubated with ,&M alone or lo- lo M PTH alone (P < 0.01; Fig. 3). The combination of either concentration of PTH and &M did not further increase the net calcium efflux observed with PTH alone. Mechanism of &M-Induced
Calcium Efflux
To determine whether the &M-induced net calcium efflux was ceil mediated or due to an alteration in the physicochemical driving forces for mineralization, calvariae were killed by successive freeze-thaw cycles, a maneuver previously shown to cause net calcium influx into bone (2, 23). After a 48-h incubation, ,&M (4 x 10v7 M) induced a net calcium efflux from live calvariae that was greater than that observed in live calvariae incubated in unsupplemented medium (P < 0.02; Fig. 4). Killing the calvariae prior to incubation resulted in a net calcium influx that was not altered by the addition of ,&M to the medium (Fig. 4). A 3-h incubation was performed because previous studies have demonstrated that alterations in the physicochemical driving forces for bone mineralization occur during this short time period (2); however, ,&M did not alter the net calcium flux in either live or dead calvariae after a 3-h incubation (Fig. 4). Compared with control, the release of the osteoclastic lysosomal enzyme ,&glucuronidase was significantly increased by the addition of &M at concentrations greater than lo-” M (Fig. 5). The maximal release of ,LI-glucuronidase was observed at 10e7 M ,&M. The ,&glucuronidase release stimulated by 10m7 and 10V6 M ,&M was similar to that induced by 10m8 M PTH, a known osteoclast stimulator (10 -7 M /!&M, 145.7 t 11.7%; lO-6 M ,&M, 136.7 t 11.4%; and PTH, 154.9 t 8.7%; all P < 0.001 vs. control, Fig. 5). The release of acid phosphatase, also an osteoclast enzyme, from calvariae incubated with lo-’ M &M for 48 h
CALCIUM
EFFLUX
600 I
Control
-
400 X
2 A 200LL g E< O. .“J z -2~ --Orw -400 u c-l
-600
-
-800
Live
Live
Dead
3 hr
Dead
48 hr-
Fig. 4. Effect of P,M on net calcium flux in live and dead calvariae. Values are means rfl SE for 6-8 pairs of calvariae per group. Calvariae were incubated for 3 and 48 h. A positive flux represents calcium efflux from bone into medium; a negative flux represents calcium influx from medium into bone. After a 3-h incubation in control medium, there was a small calcium influx in both live and dead calvariae that was not altered by addition of ,&M (4 x lob7 M). After a 48-h incubation, addition of ,&M resulted in a net calcium efflux. Killing the calvariae resulted in a calcium influx that was not altered by addition of &M. * Different from control, same duration, P < 0.02. t Different from live, same treatment, P < 0.001.
was greater than control incubations and similar to that observed with 10m8 M PTH (Table 1). Alkaline phosphatase, a marker of osteoblast activity, was not affected by a 48-h incubation with &M (Table 1). Neither acid nor alkaline phosphatase was stimulated by a 24-h incubation with &M. Calcitonin (3 X 10 -g M), an osteoclast inhibitor, induced a net calcium influx into bone incubated in control medium (P < 0.01; Fig. 6). The addition of calcitonin to medium supplemented with &M (4 x 10m8 M) blocked both the &M-induced calcium efflux (Fig. 6) and release of ,&glucuronidase (Fig. 7).
1000 i
Q 0
Ctl Ctl Basal
&M Medium
Ctl
&M
PTH (lo-“M)
1 d”
I 0-l’
I 6’
I o-a
&Microglobulin
I o-’
I 0-’
PTH
(M)
Ctl PTH (10-8M)
Fig. 3. Effect of ,&M and PTH on net calcium flux in calvariae incubated for 48 h. Values are means t SE for 8-18 pairs of calvariae per group. Compared with control (Ctl; open bars), ,&M (10s7 M; solid bars) induced a net calcium efflux which was similar to that observed with lo-lo M PTH b u t 1ess than that observed with lob8 M PTH. Addition of ,&M to medium containing PTH did not alter the PTH-induced calcium efflux. * Different from Ctl, same PTH concentration, P < 0.01. t Different from basal medium, same ,&M treatment, P < 0.05. 0 Different from lo-lo M PTH, P < 0.01.
Fig. 5. Effect of increasing concentrations of ,&M on ,&glucuronidase release in calvariae incubated for 48 h. Values are means k SE for 7-30 pairs of calvariae per group. ,&Glucuronidase activity was calculated as pg phenolphthalein released. bone- lhl and is expressed as % of control activity. Compared with control (Ctl, open bar) ,&M (solid bars) significantly increased release of ,&glucuronidase at concentrations ranging from low8 to lop6 M. Release of ,&glucuronidase induced by low7 and lo-” M ,&M was similar to that observed with 10ms M PTH (hatched bar). Symbols indicate significant difference (P < 0.05): * compared with Ctl; t compared with lo-l1 M ,&M; o compared with lo-lo M P2M; $ compared with 10vg M ,&M.
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&MICROGLOBULIN-INDUCED
DNA synthesis, as measured by [3H]thymidine uptake after a 48-h incubation, was not stimulated by the addition of ,&M (10 -‘-10e7 M), compared with calvariae incubated in control medium (lo-’ M ,&M, 5.85 t 0.88; lo+ M &M, 6.67 t 0.93; 1O-7 M ,&M, 6.47 t 1.01; control, 5.14 t 0.61 countsmin-l l pg dry wt-‘; not significant). However, the addition of 10m5 M PMA, a known mitogen in calvarial cultures (1 l), stimulated [3H] thymidine uptake (9.14 t 1.38 counts min-l pg dry wt-l; P < 0.01 compared with control). l
l
DISCUSSION
,&M induces a dose-dependent net calcium efflux from cultured neonatal mouse calvariae at concentrations ranging from 10 -8 to 10m6 M. The calcium efflux occurs after incubation of live but not dead calvariae, indicating that &M induces a cell-mediated release of calcium. The observation that the ,&M-induced calcium efflux occurred after a 48-h but not 24-h incubation, as did the release of the osteoclast enzymes ,&glucuronidase and acid phosphatase, is consistent with a cell-mediated phe-
-200 -300
Ctl
PM 2
Calc
&M 8c Calc
Fig. 6. Effect of &M and calcitonin (Calc) on net calcium flux in calvariae incubated for 48 h. Values are means t SE for 8-13 pairs of calvariae per group. Compared with control (Ctl), ,&M (4 x 10es M) induced a net calcium efflux. Calcitonin (3 x 10Bg M) induced a net calcium influx and completely blocked efflux induced by ,&M. * Compared with Ctl, P < 0.005. “f Compared with &M, P < 0.001. 175 CI>
I-
150
k!i -u -125 l
-
-
Ctl
PM 2
Calc
&M 8c Calc
Fig. 7. Effect of ,&M and calcitonin (Calc) on P-glucuronidase release in calvariae incubated for 48 h. Values are means rt SE for 3-9 pairs of calvariae per group. P-Glucuronidase activity was calculated as pg phenolphthalein released bone-l. h-l and is expressed as % of control activity. Compared with control (Ctl), ,&M (4 x low8 M) significantly increased, whereas calcitonin (3 x 10Bg M) inhibited, release of @-glucuronidase. Addition of calcitonin to calvariae incubated with ,&M blocked ,&M-induced release of P-glucuronidase. * Compared with Ctl, P c 0.05. t Compared with P2M, P < 0.001. l
CALCIUM
F543
EFFLUX
Table 1. Effect of &M on calvarial enzyme release
Control P2M
,6-Glucuronidase, % of control (n = 7-30)
Acid Phosphatase, nmol.bone-l-48 (n = 9-23)
100 127.5&11.4* 154.9+8.7*-t
228.5t17.6 294.8t34.4* 356.0t25.8*
h-l
nmol
Alkaline Phosphatase, bone-l 48 h-1 ;n = 9-26)
952.8258.5 1,038.9+73.3 1,012.8+83.1
PTH Values are means t SE; n, no. of pairs of calvariae. Calvariae were incubated for 48 h in unaltered control medium, medium supplemented with 10m8 M fi2-microglobulin (P2M), or medium supplemented with 10v8 M parathyroid hormone (PTH). ,&Glucuronidase activity was measured as pg phenolphthalein bone-l 48 h-l and is expressed as % of control; acid and alkaline phosphatase activity are expressed as nmol p-nitrophenol released bone-l. 48 h-l. * Different from control and + different from P2M (P < 0.05). l
l
l
nomenon. It is possible that our measurements were not sufficiently sensitive to detect small differences over a shorter time period; however, these data suggest a delayed stimulation of ,&M on bone mineral dissolution. This significant time delay supports the hypothesis that p2M acts via the activation of other growth factors in the complex regulation of bone mineralization (5, 9). The &M-induced net calcium efflux from bone could represent either increased bone resorption or decreased bone mineralization. Bone resorption and formation are integrated processes regulated by the interaction between osteoclasts and osteoblasts. p2M significantly increased the release of ,&glucuronidase and acid phosphatase, lysosomal enzymes that correlate with increased osteoclast activity, suggesting ,&M stimulates osteoclastic bone resorption. Further supporting a role for increased osteoelastic bone resorption was the observation that calcitonin, a specific osteoclast inhibitor (10, 14), blocked the &M-induced calcium efflux and ,&glucuronidase release. The mechanisms by which osteoclasts are stimulated to resorb bone are complex and may be mediated by the activation of osteoblasts through a variety of agents (21, 26). Whether P2M directly stimulated osteoclasts, uncoupled bone formation from resorption, or decreased osteoblast bone mineralization could not be determined from these studies. P2M not only affects osteoclast function but has been shown to stimulate cell proliferation and protein synthesis in isolated osteoblast cultures (6, 7, 9). In previous studies variable concentrations of ,&M have been required to stimulate osteoblast proliferation as measured by [3H]thymidine uptake (6, 7, 9). Similar to findings of Canalis et al. (6), we were unable to demonstrate an effect of P2M (1O-g-1O-7 M) on DNA synthesis in calvarial cultures. The incongruity observed in [3H] thymidine incorporation between isolated osteoblast and calvarial cultures suggests that additional components of bone may also be affected by P2M, complicating the interpretation of this experimental measurement. In further support of such complexity, Evans et al. (9) demonstrated that, although osteoblasts respond to and produce &M, &M did not affect the production of alkaline phosphatase or osteocalcin by isolated osteoblasts. Similarly, in the present study we were also unable to demonstrate an effect of P,M on alkaline phosphatase release. These data suggest that,
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F544
,&-MICROGLOBULIN-INDUCED
in addition to osteoblast proliferation, other growth factors or cell types may be required to stimulate bone cell enzyme production and subsequent mineralization. The precise mechanism by which ,&M induces osteoclast-mediated bone resorption and the role of osteoblasts and other growth factors in this process requires further study. Bone remodeling is controlled by hormonal and local factors in a complex regulatory system, and ,&M may act via either of these pathways. Using both a submaximal and pharmacological concentration of PTH, ,&M did not augment calcium efflux from calvariae incubated with both these agents. Similarly, previous studies were unable to demonstrate an effect of PTH on ,&M transcription (20) or an effect of 1,25dihydroxyvitamin D3 on ,&M immunoreactivity (9) in osteoblast cultures. These preliminary data suggest &M may not interact with these calciotropic hormones. Conversely, ,&M does interact with other growth factors in the local regulation of bone remodeling. Centrella et al. (7) demonstrated that ,&M and IGF-I have additive effects on osteoblast proliferation. &M enhances IGF-I receptor number and increases IGF-I transcripts and polypeptide levels in osteoblast-enriched cultures (7). These stimulatory effects of &M appear to be selective, because it does not enhance transforming growth factor-p receptors or transcripts (7). ,&M induces bone mineral dissolution in the mouse calvariae model after a significant time delay; whether this delay is due to a multi-step process involving IGF-I and/or other cytokines remains to be determined. In conclusion, these studies demonstrate that ,&M stimulates calcium efflux and ,&glucuronidase release from cultured neonatal mouse calvariae in a dose- and time-dependent manner. The mechanism of ,&M-induced bone mineral dissolution is cell mediated and involves osteoclast stimulation either directly or by inducing osteoblasts and/or bone cytokines. Further study delineating the role of&M in the interaction between osteoblasts and osteoclasts as well as its possible role in the regulation of cytokine production in bone remodeling is required. We thank S. A. Barrett for expert technical assistance. This study was supported by the Baxter Health Care Extramural Grant Program and by a grant-in-aid from the National Kidney Foundation of Illinois. S. M. Moe was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Training Grant 5T32DK-07510-01. These data were presented, in part, to the 24th Annual Meeting of The American Society of Nephrology, Baltimore, MD, November 1720. Present address of S. M. Moe: Dept. of Medicine, Indiana Univ. Med. School, 108 Sessler Hall, 1120 South Dr., Indianapolis, IN 46202. Address for reprint requests: S. M. Sprague, Dept. of Medicine, Box 28, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Received 18 September 1991; accepted in final form 23 March 1992. REFERENCES 1. Bushinsky, D. A. Effects of parathyroid hormone on net proton flux from neonatal mouse calvariae. Am. J. Physiol. 252 (Renal FZuid Electrolyte Physiol. 21): F585-F589, 2987. 2. Bushinsky, D. A., J. M. Goldring, and F. L. Coe. Cellular contribution to pH-mediated calcium flux in neonatal mouse calvariae. Am. J. Phvsiol. 248 (Renal Fluid Electrolyte Physiol. 17):
CALCIUM
EFFLUX
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