1 Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York 10092
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ABSTRACT |
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Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of ~0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.
cytokines, hepatocytes, hepatic endothelial cells, Kupffer cells, bone
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INTRODUCTION |
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PARATHYROID HORMONE (PTH) plays a critical role in calcium homeostasis, defending against hypocalcemia by acting on its "classical" target organs, bone and kidney, to stimulate bone resorption, promote renal conservation of calcium, and induce production of 1,25-dihydroxyvitamin D, which in turn enhances intestinal calcium absorption (5). These effects are thought to be mediated by the type 1 PTH/PTH-related protein (PTHRP) receptor, which is highly expressed in both bone and kidney (43). However, the receptor is also expressed in a number of other tissues (41, 43), suggesting that PTH may be exerting effects at sites other than those generally recognized as targets.
The cytokine interleukin-6 (IL-6), whose bioactivity is enhanced in the presence of its soluble receptor (IL-6sR; see Refs. 34, 40), stimulates osteoclastogenesis and bone resorption (36). We recently reported that IL-6 plays an important role in PTH-induced bone resorption in vivo (15). Circulating levels of both IL-6 and IL-6 soluble receptor (IL-6sR) are increased in humans and rodents in response to chronic PTH excess and short-term low-dose PTH infusion (15, 16). The tissue source(s) of the systemic IL-6 and IL-6sR released in response to PTH is uncertain. Bone may contribute, since bone marrow stromal/osteoblastic cells produce IL-6 after treatment with PTH in vitro (10, 14, 19, 22, 26, 27, 32, 38), but other organs may also play a role. The liver expresses high levels of IL-6 (42), IL-6sR (1, 8), and the PTH/PTHRP receptor (41, 43), and PTH has previously been shown to be active in hepatic tissue (6, 18, 21, 23, 24, 29, 30, 31). We therefore investigated the possibility that the liver contributes to PTH-induced upregulation of systemic levels of IL-6 and IL-6sR. Our results demonstrate that PTH potently induces hepatic production of bioactive IL-6 and its soluble receptor by the rat liver ex vivo and that hepatocytes, liver endothelial cells, and Kupffer cells each produce IL-6 and IL-6sR in response to PTH in vitro.
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MATERIALS AND METHODS |
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Reagents. Human PTH-(1-84) and bovine PTH-(7-34) were purchased from Bachem (King of Prussia, PA). Human calcitonin, type IV placental collagen, and FBS were obtained from Sigma (St. Louis, MO). Monoclonal rat anti-mouse IL-6, polyclonal goat anti-mouse/rat IL-6 antibody, and recombinant murine IL-6 protein were purchased from R&D Systems (Minneapolis, MN). Reagents for staining liver endothelial cells for uptake of di-I-acetylated low-density lipoprotein (LDL) were obtained from Biomedical Technologies (Stoughton, MA).
Perfusion of the isolated rat liver.
Perfusion of the isolated rat liver was performed as previously
described (24). In brief, mature (250-350 g) male
Sprague-Dawley rats (Taconic, Germantown, NY) were anesthetized with
pentobarbital sodium (50 mg/kg body wt) administered by intraperitoneal
injection. The abdomen was opened, and the pancreaticoduodenal branch
of the portal vein was ligated. A ligature was placed around the inferior vena cava (IVC) above the renal veins, and the IVC was injected with 500 units of heparin. The portal vein was cannulated with
a 14-gauge Teflon catheter, and the liver was perfused at 37°C at a
rate of 30 ml/min with Krebs solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 1.2 mM
MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3,
5.5 mM glucose, and 200 U/100 ml heparin), oxygenated with 95%
O2-5% CO2. After cannulation of the
supradiaphragmatic IVC and ligation of the IVC above the renal veins,
the liver was transferred to a temperature-controlled perfusion chamber
and continuously perfused via the portal vein in a single-pass fashion at a rate of 40 ml/min with buffer containing no heparin. The buffer
was continuously gassed with the 95% O2-5%
CO2 mixture, and the temperature was monitored with a
thermistor probe inserted between the lobes of the liver. Test peptides
or vehicle control was added to the perfusate by means of a syringe
pump programmed to deliver 400 µl/min of a 100× stock solution.
Two-milliliter aliquots of the hepatic effluent were collected at 10 to
20-min intervals for 130 min from the catheter placed in the IVC. The effluent was stored at 70°C until analyzed for IL-6 or IL-6sR immunoreactivity or IL-6 bioactivity. The viability of each perfusion was assessed by measurement of perfusion pressure and oxygen
consumption. Oxygen consumption was measured using a
Lex-O2-Con instrument (Cavitron, Anaheim, CA) at timed
intervals. Adequacy of perfusion was assessed in each experiment by
trypan blue distribution within the liver.
Tissue culture.
Hepatocytes were isolated from livers of Sprague-Dawley rats by
low-speed centrifugation after in situ collagenase perfusion, as
previously reported (3). The hepatocyte fraction was
resuspended in Lebovitz media containing 10% FBS and seeded in
six-well plates at a density of 7.5 × 105 cells/well.
After 1 h, the cultures were treated for either 2 or 4 h with
108 M human PTH-(1-84) or its vehicle. At the end of
the treatment period, the conditioned media were collected and stored
at
20°C until assayed for IL-6 or IL-6sR. Cell viability, as
assessed by trypan blue exclusion, was at least 95% at the conclusion
of the treatment period.
IL-6 assays. Immunoreactive IL-6 was measured in liver effluent and tissue culture media using a murine solid-phase enzyme-linked immunosorbent assay (R&D Systems). Antibody incubation times were adjusted to increase the sensitivity of the assay. In our laboratory, the detection limit is 3.9 pg/ml. Any sample in which the level was undetectable was assigned a value of 1 pg/ml. The intra-assay and interassay coefficients of variation (CV) for this assay in our laboratory are 3.2 and 4.1%, respectively.
IL-6 bioactivity in the liver effluent or conditioned media from liver cell cultures was determined by the proliferation of the factor-dependent cell line B13.29 (B9), as previously described (32). Briefly, 104 B9 cells were cultured with various dilutions of hepatic effluent or cell-conditioned media in 96-well flat-bottomed tissue culture plates (Costar, Cambridge, MA) for 72 h at 37°C in RPMI 1640 medium containing 2% FBS. Cells cultured in media alone served as the negative control. Proliferation was measured by the incorporation of [3H]thymidine (1 µCi/well, 5-8 Ci/mmol) added during the last 16 h of culture. The cultures were harvested on an automated sample harvester (Cambridge Technologies, Cambridge, MA) using glass fiber filters. Radioactivity was measured with a scintillation spectrometer (Packard Instrument, Meriden, CT). Data are presented as means ± SE of triplicate cultures. To confirm that the bioactivity noted in the liver effluent was due solely to the presence of IL-6, hepatic effluent was preincubated for 30 min with a polyclonal goat anti-mouse/rat IL-6 neutralizing antibody at a concentration of 2 µg/ml before its addition to the B9 cell cultures. As a control in these neutralization experiments, a monoclonal neutralizing antibody to mouse IL-6 was used at a concentration of 0.1 µg/ml.IL-6sR assay. IL-6sR was measured in hepatic effluent and conditioned media as follows. Rat (monoclonal) anti-mouse IL-6 receptor (Sigma) was diluted to a working concentration of 2 µg/ml in PBS without carrier protein. Ninety-six-well microplates (Costar) were immediately coated with 100 µl/well of the diluted antibody. The plate was sealed and incubated overnight at room temperature. Each well was then aspirated and washed with 400 µl of wash buffer (0.05% Tween 20 in PBS, pH 7.4). The process was repeated for a total of three washes using a Bio-Tek autowasher. The wells were blocked with 300 µl of block buffer (1% BSA, 5% sucrose in PBS with 0.05% sodium azide) for a minimum of 1 h at room temperature. The aspiration/wash cycle was then repeated as described above.
A seven-point standard curve using twofold serial dilutions of mouse IL-6sR (Diaclone, Besançon, France) was diluted in 0.1% BSA and 0.05% Tween 20 in Tris-buffered saline (20 mM Trizma base and 150 mM NaCl), pH 7.3. The standards range from 15 to 1,000 pg/ml. Tris-saline buffer (100 µl) was added to each well, followed by the addition of 100 µl of either standard or sample. After a 2-h incubation at room temperature, the plate was washed as before. Two-hundred micoliters of polyclonal antibody (200 ng/ml) against IL-6sR conjugated to horseradish peroxidase (R&D) were then added followed by a 2-h incubation at room temperature. The aspiration/wash cycle was repeated as above followed by the addition of 100 µl of equal volumes of hydrogen peroxide and tetramethylbenzidine (R&D). After a 30-min incubation at room temperature, the reaction was stopped with 50 µl of 2 N sulfuric acid. The optical density of each well was determined by using a microplate reader set to 450 nm and to 540 nm for wavelength correction. Calculations were done using a computer-generated 4PL curve fit. The sensitivity of the assay is 7 pg/ml. Any sample in which the level was undetectable was assigned a value of 4 pg/ml. The intra-assay CV was 2.9% and the interassay CV was 3.8%.Detection of IL-6 transcripts in hepatocytes. Total RNA was isolated from rat hepatocytes using Trizol reagent (GIBCO-BRL). IL-6 mRNA was detected using the GeneAmp RNA PCR kit (Perkin-Elmer). Total RNA (2 µg) was reverse transcribed with random hexamers. A DNA fragment of 426 bp was amplified by PCR using the following primers: 5'-GAGTTGTGCAATGGCAATTCTG-3' (sense) and 5'-CCGAGTAGACCTCATAGTGAC-3' (antisense). As a control for the amount of total RNA reverse transcribed, RNA was also used to amplify transcripts for glyceraldehyde-3'-phosphate dehydrogenase (GAPDH).
Statistical analyses. Statistical analyses were performed using the SAS statistical package, version 6.11 (SAS Institute, Durham, NC). Comparisons between the effects of the various peptides on cytokine immunoreactivity and bioactivity in effluents from isolated perfused rat livers were made using repeated-measures ANOVA. Comparisons between the effects of PTH and vehicle on production of IL-6 or IL-6sR by cultured liver cells were made using two-way ANOVA.
The study was approved by the Yale University Animal Care and Use Committee. ![]() |
RESULTS |
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PTH stimulates production of immunoreactive IL-6 by the isolated
rat liver.
Figure 1A summarizes levels of
immunoreactive IL-6 in effluents collected at 10- to 20-min intervals
from ex vivo rat livers. In preparations perfused with vehicle or 100 pM human calcitonin, IL-6 levels remained below 6 pg/ml throughout the
130-min procedure. In contrast, perfusion with 100 pM PTH-(1-84)
induced a substantial increase in IL-6 production that became
detectable after 40 min of the study and increased thereafter in a
linear fashion to reach a mean value of 30.7 ± 2.7 pg/ml at 130 min (P < 0.0001 vs. vehicle or calcitonin).
PTH-(7-34) stimulated production of IL-6 that was intermediate in
magnitude between that induced by PTH-(1-84) and either vehicle or
calcitonin. The mean IL-6 level in hepatic effluent after 130 min of
perfusion with PTH-(7-34) was 10.8 ± 0.8 pg/ml [P < 0.0001 vs. vehicle, calcitonin, and PTH-(1-84)].
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PTH stimulates hepatic IL-6 production at physiological concentrations. Perfusion of the isolated rat liver with PTH-(1-84) induced IL-6 production in a dose-dependent manner. Thus, as shown in Fig. 1B, 1 pM concentrations of PTH-(1-84) in the perfusate (equivalent to 2.4 pmol/h) resulted in IL-6 levels of 14.8 ± 0.4 pg/ml at the end of the perfusion (P < 0.001 vs. vehicle). Given a perfusion rate of 40 ml/min, near- physiological concentrations of PTH are therefore capable of inducing hepatic IL-6 production at a rate of 0.6 ng/min in this ex vivo system.
PTH stimulates hepatic production of the IL-6sR.
PTH-(1-84) stimulated production of the IL-6sR by the isolated rat
liver in a dose-dependent fashion. As demonstrated in Fig. 2, levels of the IL-6sR in hepatic
effluent collected after 10 min of perfusion with either vehicle or PTH
were undetectable. Thereafter, IL-6sR levels in effluent from livers
perfused with vehicle remained constant at a value of ~50 pg/ml
throughout the study. Each concentration of PTH-(1-84) studied (1, 10, and 100 pM) induced hepatic production of IL-6sR that was
significantly greater than that induced by perfusion with vehicle
(P < 0.001). However, the time course of the increase
in hepatic IL-6sR production in response to PTH was different from that
observed for IL-6. Thus levels of the IL-6sR increased initially to
reach a peak at 60 min and fell gradually thereafter, although they
remained above control values at the conclusion of the 130-min
perfusion period.
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PTH-induced hepatic IL-6 is bioactive.
To determine whether the immunoreactive IL-6 produced by the isolated
rat liver in response to perfusion with PTH was biologically active, we
tested the ability of effluent from isolated rat livers to induce
proliferation of the B9 cell line, as described in MATERIALS AND
METHODS. As shown in Fig.
3A, the IL-6 bioactivity in
samples collected from livers perfused with PTH-(1-84), PTH-(7-34),
calcitonin (all at 100 pM), or vehicle reflected the pattern of IL-6
immunoreactivity measured in the same samples. Neither calcitonin nor
vehicle perfusion induced any change in IL-6 bioactivity over the
130-min experiment. In effluent from livers perfused with 100 pM
PTH-(1-84), IL-6 bioactivity began to increase after 40 min and
thereafter increased in a linear fashion to reach a level at 130 min
that was five times greater than that detected in response to perfusion
with either vehicle or calcitonin (P < 0.0001). As was
true of IL-6 immunoreactivity, PTH-(7-34) induced IL-6 bioactivity
that was intermediate between that observed in response to PTH-(1-84)
and that induced by perfusion with either calcitonin or vehicle. Thus PTH-(7-34) induced 2.2- and 2.1-fold greater IL-6 bioactivities compared with vehicle or an equimolar concentration of calcitonin, respectively (P < 0.001).
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Liver cells produce IL-6 and IL-6sR in response to PTH in vitro.
Immunoreactive IL-6 and IL-6sR were measured in conditioned media from
rat liver cells, isolated and cultured as described in MATERIALS
AND METHODS, and treated with 10 nM PTH-(1-84) or its vehicle
for 2 or 4 h. In vitro data were obtained at this pharmacological
PTH concentration only. Mean ± SE levels of IL-6 and IL-6sR in
the cell-conditioned media of each hepatic cell type at each time point
are shown in Tables 1 and
2, respectively. IL-6 was not
detectable in Kupffer cell-conditioned medium from 9 of 20 wells
treated with vehicle, whereas IL-6sR was not detectable in Kupffer
cell-conditioned medium from 15 of 22 wells treated with vehicle and 1 of 22 wells treated with PTH.
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Hepatocytes express mRNA for IL-6.
To determine if the increased expression of IL-6 protein by hepatocytes
was accompanied by changes in the level of IL-6 mRNA, semiquantitative
RT-PCR was performed using total RNA isolated from hepatocytes at 1, 2, and 3 h after treatment with PTH. As shown in Fig.
6, PTH induced a time-dependent increase
in IL-6 transcript expression, whereas there was no change in levels of GAPDH mRNA. It should be noted that, although IL-6 mRNA was always detected, upregulation of transcript expression was not seen in all
hepatocyte preparations examined, probably due to the low levels of
IL-6 transcript expression in these cells and the modest degree of
increase in protein levels (2.0-fold).
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DISCUSSION |
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PTH regulates the circulating levels of IL-6 in humans and rodents such that serum levels of IL-6 are increased in the face of chronic PTH excess and short-term PTH infusion and decreased in hypoparathyroidism (15, 16). Changes in circulating levels of the IL-6sR also occur in response to PTH in the same direction as those observed for IL-6 (16). The current study demonstrates that perfusion of the isolated rat liver with near physiological concentrations of PTH-(1-84) stimulates production of both bioactive IL-6 and the IL-6sR. Mean levels of IL-6 in effluent from ex vivo rat livers perfused with 2.4 pmol/h PTH were 14.8 pg/ml, strikingly similar to systemic levels of IL-6 (14.4 pg/ml) observed in intact rats infused with 12.9 pmol/h PTH (15). These data strongly suggest that hepatic production could contribute substantially to PTH-induced increases in systemic levels of IL-6 and its soluble receptor. We find that three of the four major liver cell types (hepatocytes, endothelial cells, and Kupffer cells) produce both IL-6 and IL-6sR in response to PTH in vitro. Given that hepatocytes constitute, by far, the majority of the cellular mass of the liver, it is likely that they are responsible for most of the PTH-induced hepatic production of IL-6 and IL-6sR.
The role of PTH in the regulation of calcium homeostasis is in part dependent on its ability to stimulate osteoclastic bone resorption. IL-6 promotes osteoclastogenesis (36) and plays an important role in mediating the bone-resorbing actions of PTH in vivo (15). PTH stimulates production of IL-6 in both skeletal (10, 14, 19, 22, 26, 27, 32, 38) and hepatic (current study) tissue, and we can not yet determine whether the dependence of PTH-induced bone resorption on IL-6 is attributable to cytokine produced at either site or both. However, our current findings raise the possibility that PTH-induced, liver-derived IL-6 may act in an endocrine fashion and may have as a potential site of action, bone. That IL-6 can act in this fashion is suggested by recent evidence demonstrating that its systemic administration influences the hypothalamic-pituitary-adrenal axis (33). An endocrine action of liver-derived IL-6 on bone would potentially be augmented by the increased circulating levels of the IL-6sR induced by PTH, since osteoclastogenesis induced by IL-6 in vitro is enhanced in the presence of its soluble receptor (40). The IL-6sR also prolongs the half-life of circulating IL-6 in vivo, thereby augmenting further the biological actions of IL-6 (34).
IL-6 plays an important role in the acute phase response, acting to stimulate hepatic production of acute-phase proteins such as C-reactive protein (13, 17). IL-6 knockout mice exhibit impaired synthesis of acute-phase proteins (25), whereas hepatic overexpression of IL-6 in mice leads to exaggerated production of acute-phase proteins, an effect that is augmented by coexpression of the IL-6sR (35). The findings that PTH influences hepatic production of IL-6 and IL-6sR raise the possibility that disorders of PTH production may be associated with altered acute-phase responses. To our knowledge, there are no published data addressing this possibility. However, Funk et al. (12) have demonstrated that systemic administration of PTHRP, which signals through the type 1 PTH/PTHRP receptor, stimulates an increase in the circulating level of the acute-phase protein serum amyloid A in mice.
Our data provide further evidence that the liver is a target organ for PTH. The liver strongly expresses mRNA for the PTH/PTHRP receptor (41, 43), which probably mediates the actions of PTH or PTHRP on production of IL-6 and its soluble receptor (current study), gluconeogenesis (21), intracellular calcium mobilization (23), increased adenylate cyclase activity (6), insulin-like growth factor I production (7), and the acute-phase response (12) in liver tissue. It is unlikely that the PTH-2 receptor, which binds PTH more avidly than PTHRP, contributes to the effects of PTH on hepatic IL-6 production because, although it may be expressed in the liver, it is much less abundant there than the type 1 receptor (44).
Having determined that the liver is an important source of the circulating IL-6 generated in response to PTH, we examined the cellular basis of this phenomenon. Our findings demonstrate that each of three liver cell types (hepatocytes, hepatic endothelial cells, and Kupffer cells) produce both IL-6 and IL-6sR in response to PTH. In contrast, hepatic stellate cells did not produce either cytokine in response to PTH. The finding that hepatocytes respond to PTH stimulation in vitro is consistent with previous studies that have demonstrated that PTH binds to hepatocytes (2, 37) and stimulates mobilization of intracellular calcium (23), adenylate cyclase activity (29), and gluconeogenesis (30) in cultured hepatocytes. Because hepatocytes are the predominant cell type in the liver, we examined the effect of PTH on IL-6 transcript expression in these cells. The observed increase in message expression suggests that either transcriptional activation or message stabilization is contributing to the increased IL-6 protein expression induced by PTH in hepatocytes. Binding of PTH to Kupffer cells has also been demonstrated previously (4), and Kupffer cells have been implicated in hepatic degradation of PTH (4). In contrast, the current data are the first, to our knowledge, to demonstrate PTH responsiveness in hepatic endothelial cells.
We were surprised to observe stimulation of IL-6 production by the isolated rat liver in response to PTH-(7-34), since this peptide is generally considered to be biologically inactive. This finding suggests that PTH fragments may have biological activity in the liver and raises the possibility that there may be novel PTH receptors in this tissue. Indeed, some evidence exists to support the possibility that the PTH fragment containing amino acids 30-34 has biological activity (28).
In summary, the current study demonstrates that the ex vivo rat liver produces bioactive IL-6 and the IL-6sR in response to near-physiological concentrations of PTH. Hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, each produce IL-6 and IL-6sR in response to PTH, but hepatocytes are likely to be responsible for the majority of PTH-induced hepatic production of cytokine and receptor. These results suggest that the liver contributes substantially to the regulation by PTH of circulating levels of IL-6 and its soluble receptor in vivo and raise the possibility that liver-derived cytokines may play a role in PTH-induced bone resorption.
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ACKNOWLEDGEMENTS |
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We thank Lynn Sadler (Dept. of Obstetrics and Gynecology, University of Auckland School of Medicine, Auckland New Zealand) for statistical advice.
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FOOTNOTES |
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This work was supported, in part, by the Cell Isolation and Organ Perfusion Core of the Yale Liver Center. This work was also supported by grants from the Health Research Council of New Zealand (A. Grey), National Institutes of Health Grants DK-34889, AG-15345 (K. Insogna), K08D-K02596 (U. Masiukiewicz), and DK-37340 (S. Friedman), the Claude D. Pepper Older Americans Independence Center at Yale (K. Insogna), the Mount Sinai Dean's Office Research Incentive Fund (S. Friedman), and Yale Core Center for Musculoskeletal Disorders Grant AR-46032 (K. Insogna and M. Horowitz).
Address for reprint requests and other correspondence: K. Insogna, Section of Endocrinology, Yale University School of Medicine, PO Box 208020, 333 Cedar St., New Haven, CT 06520-8020 (E-mail: karl.insogna{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 February 2000; accepted in final form 9 November 2000.
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