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Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06520-8020
Address all correspondence and requests for reprints to: Dr. Masiukiewicz , Section of Endocrinology, Yale University School of Medicine, P.O. Box 208020, 333 Cedar Street, FMP 109, New Haven, Connecticut 06520-8020. E-mail: urszula.masiukiewicz{at}yale.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increasing evidence suggests that IL-6 may be one of the key cytokines mediating PTH’s proresorptive effect in bone. Thus, in vitro studies have shown that IL-6 is produced by stromal/osteoblastic cells in response to PTH (3, 4, 5, 6, 7, 8, 9). In addition, PTH-induced bone resorption can be attenuated in a rat osteoblast/osteoclast coculture system by using a neutralizing antibody to the IL-6 receptor (10). We have recently reported that IL-6 plays an important role in PTH-induced bone resorption in vivo. First, we observed that, in patients with primary and secondary hyperparathyroidism circulating levels of IL-6 are markedly elevated and return to normal after correction of hyperparathyroidism (11, 12). Second, infusion of PTH in rodents and humans results in elevations in circulating levels of IL-6. The rise in circulating levels of IL-6 correlates with elevations in bone resorption markers. Systemic administration of IL-6 neutralizing antisera blocks PTH-induced bone resorption in mice, and the ability of PTH to induce resorption in mice with targeted disruption of the IL-6 gene is markedly attenuated (13).
Interleukin-6 has also been shown to be important in mediating the
increase in bone remodeling that follows sex steroid withdrawal. It has
been shown that blockade of IL-6 prevents the increase in
osteoclastogenesis seen in estrogen-deficient mice (14). In keeping
with this observation, IL-6 knock-out mice do not lose trabecular bone
following ovariectomy (15). In vitro, estrogen has been
shown to suppress cytokine induced IL-6 production. Specifically,
estrogen inhibits tumor necrosis factor (TNF), and IL-1 stimulated IL-6
gene transcription via binding of the estrogen receptor-
ligand complex to nuclear factor (NF)
B and CAAT
enhancer-binding protein (C/EBPß) transcription factors and
presumably preventing binding to the IL-6 promoter (16, 17, 18, 19, 20, 21).
In the present study, we investigated whether estrogen modulates PTH-induced IL-6 production in vitro in osteosarcoma Saos-2 cells and in primary human osteoblasts, and in vivo in mice. We demonstrate that, following estrogen withdrawal, PTH-induced IL-6 production is augmented in vitro and in vivo and that the augmented effect of PTH on circulating levels of IL-6 in vivo can be prevented by estrogen replacement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of cytokines, markers of bone turnover and serum
estradiol
Murine IL-6, serum collagen cross-links, and urine collagen
cross-links were measured as previously reported (13). The sensitivity
and intra and interassay coefficients of variation (CV) for these three
assays in our laboratory are IL-6: 3.9 pg/ml, 3.2% and 4.1%; serum
collagen cross-links: 0.5 µg/liter, 2.8% and 3.6%; and urine
collagen cross-links: 25 µg/mmol creatinine, 3.8% and 4.9%.
Human IL-6, IL-11, and CSF-1 in cell culture conditioned media, were measured using ELISA kits (R&D Systems, Minneapolis, MN). For the measurement of IL-11, antibody incubation times were adjusted to increase the sensitivity of the assay, which in our laboratory is 1.6 pg/ml. The intraassay and interassay CVs for this assay in our laboratory are 3.9% and 5.1%, respectively. For human IL-6, the sensitivity in our laboratory is 0.1 pg/ml and the intraassay, and interassay CVs are 4.4% and 5.4% respectively. For CSF-1, the sensitivity is 6 pg/ml and intraassay, and interassay CVs are 3.1% and 4.3% respectively.
Serum estradiol levels were measured using an estradiol RIA kit from Diagnostics Systems Laboratories, Inc., Webster, TX. The sensitivity of the assay in our laboratory is 4.7 pg/ml. The intraassay and interassay CVs for this assay are 3.8% and 4.8%, respectively.
Cell culture
Human osteoblast-like osteosarcoma cells, Saos-2 cells, were
obtained from ATCC (Rockville, MD). Cells were maintained
in RPMI-1640 medium supplemented with 10% FBS and 1%
penicillin/streptomycin. For dose response experiments, cells were
plated at an initial density of 1.2 x 106
cells per well (9.6 cm2). At this plating
density, cells were confluent after 24 h of culture. Media were
changed daily and cells were maintained in culture for 72 h before
PTH treatment. After 72 h of culture, cells were treated with
(1–84) h PTH at the indicated concentrations for 24 h. Treatment
media were harvested 24 h post PTH treatment and assayed for IL-6.
For estrogen withdrawal experiments, Saos-2 cells were plated in 6-well
plates, at the initial density indicated above, and grown to confluence
in RPMI-1640 cell culture media, supplemented with 10% FBS and 1%
penicillin/streptomycin. At confluence (24 h post plating), treatment
media were changed to phenol red-free RPMI-1640 supplemented with 10%
charcol/dextran-treated FBS with or without the addition of 17ß
estradiol at a final concentration of 10 -9
M. The cells were stabilized for 48 h under these new
culture conditions with a media change at 24 h. After 48 h
(time = 0), cells were treated with 10-8
M (1–84) h PTH for 24 h, at which time media were
harvested and assayed for IL-6, IL-11, and CSF-1. At the end of PTH
treatment in both dose-response and estrogen-withdrawal experiments,
cell number was determined and viability assessed by trypan blue
exclusion. In all experiments the mean cell number/well were not
different and averaged 3.6 ± 0.1 x
106.
Primary human osteoblasts were kindly provided by Dr. Mark C. Horowitz
(Cell Core, Yale Core Center for Musculoskeletal Disorders) and
isolated and cultured as previously described (22). Isolated cells were
grown to confluence for 2 weeks in 
-MEM supplemented with 10% FBS
and 1% penicillin/streptomycin, at which point treatment media were
changed to phenol red-free RPMI-1640 supplemented with 10%
charcol/dextran-treated FBS with or without the addition of 17ß
estradiol at a final concentration of 10-9
M. The cells were stabilized for 48 h under these new
culture conditions with a media change at 24 h. After 48 h
(time = 0) cells were treated with PTH and IL-6 measured in the
conditioned media as described above. In all experiments, the mean cell
number/well were not different and averaged 4.9 ± 0.1 x
105.
Animal studies
PTH infusions were carried out as previously described (13). In
brief, 4-week-old CD-1 mice were ovariectomized or sham-ovariectomized.
Two weeks following surgery, animals underwent sc implantation of
pellets containing either 17ß-estradiol or placebo. Two weeks
following pellet implantation, interscapular sc miniosmotic pumps were
implanted; the pumps were loaded with (1–84) h PTH to deliver hormone
at a rate of 4.3 pmol/h for 5 days. At the end of 5 days, animals were
killed and serum collected and assayed for IL-6, collagen cross-links
and estradiol and urine collected and assayed for collagen cross-links.
The results of urine collagen cross-links were corrected for urinary
creatinine, which was measured by a colorimetric method using alkaline
picrate solution. These studies were approved by the Yale Animal Care
and Use Committee.
Statistical analyses
All values are expressed as mean ± SEM.
Comparisons between IL-6 production in Saos-2 cells and primary human
osteoblasts cultured under estrogen-replete and deficient conditions
and comparisons between groups of PTH-treated animals were made using
Student’s t test for unpaired samples. Statistical analysis
of the Saos-2 cells IL-6 dose-response to PTH stimulation was performed
by one-way ANOVA. Comparisons of the effect of PTH treatment on serum
IL-6 and urine and serum collagen cross-links in sham-operated and
ovariectomized mice were made using two-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment of Saos-2 cells, with (1–84) h PTH resulted in a
dose-dependent increase in the release of immunoreactive IL-6 measured
in the conditioned media at 24 h (Fig. 1
). Thus, PTH at a concentration of 5
x10-10 M, induced a 4.4 ±
0.5-fold increase in IL-6 release compared with vehicle-treated cells
and at the highest concentration tested (10-8
M) stimulated a 10.0 ± 0.3 fold increase over
vehicle-treated cells. The mean concentration of immunoreactive IL-6 in
the media from PTH-treated (10 -8 M)
cells, was 9.3 ± 0.2 pg/ml compared with 0.9 ± 0.18 pg/ml
in the media from vehicle-treated cells (n = 3, P
= 0.003).
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, estrogen withdrawal
significantly augmented PTH-induced IL-6 production. Thus, under
estrogen-deficient conditions, treatment with
10-8 M (1–84) h PTH for
24 h resulted in a 30.0 ± 2.9-fold increase in
immunoreactive IL-6 when expressed as fold stimulation over values in
vehicle-treated cells. Adding back estrogen to the treatment media at a
final concentration of 10-9
M, decreased PTH induced IL-6 production to
15 ± 1.1-fold over basal (n = 5, P = 0.005
vs. PTH-treated estrogen-deficient cells). The mean absolute
IL-6 values after PTH-treatment were 21.8 ± 1.3 pg/ml in
estrogen-deficient cells vs. 11.0 ± 0.7 pg/ml in
estrogen replete cells. To examine the specificity of the effect of
estrogen withdrawal on PTH-induced IL-6 production, we measured the
concentration of two other cytokines, IL-11 and CSF-1, in the
conditioned media from treated and untreated cells. There was no
statistically significant effect of estrogen on PTH-induced production
of these two cytokines. The mean absolute values (estrogen-deficient
vs. estrogen replete) for CSF-1 were: PTH treatment 624
± 42 vs. 898 ± 127 pg/ml (P = 0.08);
vehicle treatment 238 ± 25 vs. 235 ± 51 pg/ml
(P = 0.9) and for IL-11: PTH treatment 248 ± 25
vs. 242 ± 33 pg/ml (P = 0.9); vehicle
treatment 10.3 ± 1.7 vs. 10.4 ± 1.3 pg/ml
(P = 1.0).
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Estrogen modulates the PTH-induced rise in circulating levels of
IL-6 and markers of bone resorption in vivo
We have previously reported that PTH infusion into CD-1 mice
results in a rise in circulating levels of IL-6 and that this rise
correlates with an increase in markers of bone resorption (13). Having
shown that estrogen withdrawal augments PTH-induced IL-6 production
in vitro, the effects of estrogen deficiency on PTH-induced
IL-6 production in vivo were next examined in ovariectomized
(ovx) and sham/ovariectomized (sham/ovx) CD-1 mice. As part of this
experiment, a group of ovx animals were implanted with slow release
estrogen pellets before the PTH infusion as outlined in Materials
and Methods. The mean circulating level of serum estradiol was
281 ± 56 pg/ml in estrogen pellet-treated ovx animals
vs. 10 ± 1 pg/ml in placebo pellet-treated ovx
animals. Measurement of baseline levels of circulating IL-6
demonstrated no differences between sham/ovx and ovx animals, 3.4
± 0.3 pg/ml vs. 3.4 ± 0.4 pg/ml, respectively. The
mean serum IL-6 level on day 5 of infusion was 60.1 ± 4.9 pg/ml
in the PTH-treated ovx animals vs. 16.9 ± 1.0 pg/ml in
the PTH-treated sham/ovx animals (P < 0.0001). Because
baseline values were the same in both groups, the mean increase in
circulating levels of serum Il-6 was greater following PTH treatment in
ovx animals compared with sham/ovx animals, (56.7 ± 4.9 pg/ml
vs. 13.5 ± 1.1 pg/ml; P < 0.001)
(Fig. 3A). Animals implanted with slow
release estrogen pellets demonstrated PTH-induced rises in circulating
levels of IL-6 comparable to those seen in PTH-treated sham/ovx animals
(mean increment of 19.0 ± 0.7 pg/ml). Serum IL-6 values in
PTH-treated ovx animals pretreated with placebo pellets was
significantly higher (mean of 46 ± 2.1 pg/ml) than that seen in
animals implanted with estrogen pellets (P <
0.001).
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, the mean levels of urine collagen cross-links were
236 ± 13 µg/mmol creatinine in the PTH-treated ovx animals
compared with 88.5 ± 6 µg/mmol creatinine in the PTH-treated
sham/ovx animals (P < 0.0001). Estrogen replacement in
PTH-treated ovx animals returned values of urine collagen cross-links
to levels comparable to those in PTH-treated sham/ovx animals
(98.6 ± 3.6 µg/mmol creatinine; P < 0.0001 for
PTH-treated ovx animals vs. either PTH-treated sham/ovx
animals or PTH-treated ovx +17ß estradiol animals). Serum collagen
cross-links values were 17.4 ± 0.7 µg/liter for PTH-treated ovx
animals, 7.4 ± 0.2 µg/liter for PTH-treated sham/ovx animals
and 10.7 ± 0.5 µg/liter for PTH-treated ovx animals implanted
with estrogen pellets (P < 0.0001 for PTH-treated ovx
vs. either PTH-treated sham/ovx or PTH-treated ovx +17ß
estradiol animals). PTH-treated ovx animals implanted with placebo pellets had mean values of bone turnover markers similar to those in PTH-treated ovx animals precluding any nonspecific effect of pellet implantation (206 ± 7.9 vs. 236 ± 13 µg/mmol creatinine, for urine collagen cross-links and 19.6 ± 0.5 vs. 17.4 ± 0.7 µg/liter, for serum collagen cross-links).
Ovariectomy increased levels of bone resorption markers at baseline. Thus, mean levels for urine collagen cross-links were significantly higher at baseline in the ovx vs. sham/ovx animals 64.7 ± 4.9 vs. 29.9 ± 3.0 µg/mmol creatinine (P = 0.001) as were the serum collagen cross-links values 9.5 ± 0.5 vs. 4.3 ± 0.3 µg/liter (P < 0.001). Despite this, the mean increment in levels of urine collagen cross-links in PTH-treated ovx animals when compared with untreated ovx animals was greater than that observed in response to PTH treatment in sham/ovx animals vs. untreated sham/ovx animals (172 ± 18 vs. 59 ± 9 µg/mmol creatinine, P < 0.0001). Similarly, the mean increase in serum collagen cross-links in ovx animals in response to PTH treatment was greater than that observed in response to PTH treatment in sham/ovx animals (7.8 ± 0.9 vs. 3.1 ± 0.4 µg/liter, P = 0.0001). These data demonstrate that, in the absence of estrogen, the bone-resorbing activity of PTH in vivo is considerably augmented.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interleukin-6 has emerged as a pivotal factor in mediating increased bone turnover associated with states of PTH excess (11, 12, 13). Although several recent studies have focused on understanding the effects of estrogen on modulating cytokine-induced regulation of IL-6 (16, 17, 18, 19, 20, 21, 27), little is known about the mechanism(s) through which PTH induces IL-6 release from bone cells or the role of estrogen in this response. Further, the effect of estrogen on PTH-induced IL-6 production in vivo has not been examined. Passeri et al. have reported that estrogen withdrawal in vitro enhances PTH-stimulated IL-6 production by ex vivo cultures of bone marrow, although the cellular source of the IL-6 was uncertain (28).
In the current study, we demonstrate that PTH is a potent stimulator of IL-6 release by human osteoblast like Saos-2 cells. Further, estrogen-withdrawal dramatically up-regulates this response which was blocked by adding back estrogen. Estrogen also significantly attenuated PTH-induced IL-6 production in cultured primary human osteoblasts, although the effect was of a smaller magnitude. This may, in part, reflect the fact that primary human osteoblasts obtained by explant culture tend to have only a modest response to PTH, for example in cAMP production. The effect of estrogen on PTH-induced IL-6 production appears to be cytokine specific, as there was no significant effect of estrogen on PTH-induced production of two other proresorptive cytokines, CSF-1, and IL-11. In vivo, ovx mice demonstrated an exaggerated rise in circulating levels of IL-6 following PTH treatment. This augmented rise in circulating levels of IL-6 was significantly lower in PTH-treated sham/ovx animals and could be prevented by treatment of ovx animals with estrogen. The augmented rise in IL-6 was paralleled by a greater rise in markers of bone resorption. In view of these data and our earlier findings that IL-6 plays a key role in mediating the resorptive effects of PTH, it seems plausible that increased skeletal sensitivity to PTH seen with estrogen deficiency, is mediated in part, by greater IL-6 production.
The tissue source(s) of circulating IL-6 produced in response to PTH remains unknown. Our in vitro data suggest that bone may be one source. However, we have recently reported that PTH also increases IL-6 production in isolated rat livers (29). Studies are currently underway to determine if estrogen modulates IL-6 production in the liver.
In summary, the principal findings of this study are that PTH-induced IL-6 production is augmented following estrogen withdrawal in vitro and in vivo. The exaggerated rise in IL-6 following treatment of estrogen-deficient animals is accompanied by a greater increase in bone turnover markers, and both of these changes can be prevented by estrogen. Taken together with existing data, the current study suggests that IL-6 release induced by PTH may play a role in the accelerated bone loss that attends estrogen deficiency, particularly in states of parathyroid excess.
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Footnotes |
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Received October 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are elevated in primary hyperparathyroidism, and
correlate with markers of bone resorption. J Clin Endocrinol Metab 81:3450–3454[Abstract]
B binding sites in
interleukin-6 promoter inhibition by estrogens. Mol Endocrinol 10:713–722B and
C/EBPß. Mol Cell Biol 15:4971–4979[Abstract]
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