Effects of dexamethasone on proliferation, activity, and cytokine secretion of normal human bone marrow stromal cells: possible mechanisms of ...
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371
Effects of dexamethasone on proliferation, activity, and cytokine
secretion of normal human bone marrow stromal cells: possible
mechanisms of glucocorticoid-induced bone loss
C-H Kim1, S-L Cheng2 and G S Kim3
1
Division of Endocrinology, Department of Internal Medicine, Soonchunhyang University College of Medicine, Seoul, Korea
2
Division of Bone and Mineral Diseases, Department of Medicine, Washington University, School of Medicine, St Louis, Missouri, USA
3
Division of Endocrinology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
(Requests for offprints should be addressed to G S Kim, Divison of Endocrinology, Asan Medical Center, Song-Pa P O Box 145, Seoul 138–600, Korea)
Abstract
It is well documented that glucocorticoid excess causes non-collagen protein (65·28·4% of control), with a
bone loss, but the mechanisms of these effects remain greater effect on CDP. Northern blot analysis indicated
poorly defined. To understand further the mechanisms of that á1(I)-collagen mRNA level was decreased by dexa-
glucocorticoid-induced osteoporosis, we investigated the methasone to 27·69·0% of the control value after 1 day
effects of glucocorticoids on bone formation and bone of exposure, and to 55·26·2% after 7 days. Dexametha-
resorption by examining the proliferation, functional sone markedly suppressed basal production of interleukin
activities, and cytokine secretion of cultured human bone (IL)-6 and IL-11 and that stimulated by parathyroid
marrow stromal cells (hBMSC). Treatment with dexa- hormone (PTH), IL-1á, or tumour necrosis factor-á in a
methasone for 24 h at the concentration of 10 8 M dose-dependent manner. These results suggest that the
significantly suppressed [3H]thymidine incorporation and glucocorticoid-induced bone loss is derived at least in part
further inhibition was observed with longer treatment via inhibition of bone formation, which includes the
(8 days) or higher concentration (10 7 M). Alkaline suppression of osteoblast proliferation and collagen syn-
phosphatase activity of hBMSC was markedly stimulated thesis. As both basal and PTH-stimulated production of
with addition of dexamethasone (10 8 M), to 19122% IL-6 and IL-11 are decreased by dexamethasone, the
(after 4 days) and 31746% (after 7 days) of control. increased bone resorption observed in glucocorticoid-
Dexamethasone (10 8 M) treatment for 48 h decreased induced osteopenia does not appear to be mediated by
the incorporation of [3H]proline into collagenase- IL-6 or IL-11.
digestible protein (CDP; 43·77·9% of control) and Journal of Endocrinology (1999) 162, 371–379
Introduction culture, but decreases these parameters after 96 h (Dietrich
et al. 1979, Canalis 1983, Kream et al. 1997). In addition,
Osteoporosis is one of the most serious side effects of both stimulation and inhibition of cell proliferation by
long-term high-dose glucocorticoid therapy, but the glucocorticoids have been reported in these organ
mechanisms of bone loss remain poorly defined. Previous cultures (Tenenbaum & Heersche 1985, McCulloch &
studies have suggested that glucocorticoid excess causes Tenenbaum 1986, Kream et al. 1997). In isolated
bone loss by two mechanisms: suppression of bone forma- osteoblast-like cell populations, glucocorticoids decrease
tion and stimulation of bone resorption (Canalis 1996, cell proliferation but increase alkaline phosphatase activity
Reid 1997). The data regarding the effects of glucocorti- (Chen et al. 1977, Wong et al. 1990, Cheng et al. 1994).
coids on osteoblast activities in vitro, however, are conflict- The effects of glucocorticoids on collagen expression in
ing. They appear to depend upon whether physiological or osteoblast-like cells, however, remain controversial. Both
pharmacological concentrations of the hormone are used, stimulation and inhibition have been reported depending
on the time and duration of exposure to the drug, and on on the osteoblast systems and culture conditions (Wong
the particular system investigated (Bellows et al. 1987). 1979, Kasugai et al. 1991, Shalhoub et al. 1992, Fernandez
Experiments using organ cultures of fetal or neonatal bone & Minguell 1997).
have shown that exposure to glucocorticoids increases Glucocorticoids have been shown to stimulate
collagen synthesis and alkaline phosphatase activity – osteoclast-like cell formation directly (Kaji et al. 1997) and
parameters of osteoblast differentiation – after 24 h of to increase bone resorption in neonatal mouse calvaria
Journal of Endocrinology (1999) 162, 371–379 Online version via http://www.endocrinology.org
0022–0795/99/0162–371 1999 Society for Endocrinology Printed in Great Britain
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via free access372 C-H KIM and others · Effects of dexamethasone on human bone marrow stromal cells
(Conaway et al. 1996). In addition, glucocorticoid admin- hBMSC possess many of the phenotypic characteristics of
istration causes secondary hyperparathyroidism as a result differentiated osteoblasts, such as production of osteocalcin
of impaired gut calcium absorption and hypercalciuria and deposition of mineralized matrix (Cheng et al. 1994,
(Canalis 1996, Reid 1997). As parathyroid hormone 1996, Kim et al. 1997).
(PTH) is an important stimulator of osteoclast activities
mediated via osteoblasts (Suda et al. 1995), it is possible
[3H]Thymidine incorporation
that glucocorticoids may also induce bone resorption
indirectly via PTH. Consistent with this possibility, PTH Cell proliferation was assessed by the measurement of
responses in osteoblast-like cells are increased by gluco- [3H]thymidine incorporation. hBMSC were seeded in
corticoids (Chen & Feldman 1978) and dexamethasone 24-well plates at a density of 2104 cells/well. After 48 h
enhances PTH-stimulated osteoclast-like cell formation of culture, dexamethasone (10 9-10 7 M) was added to
and bone resorption (Kaji et al. 1997). Recently, the the media, and the cells were cultured for an additional
osteoclast-activating effects of PTH are shown to be 24 h. [3H]Thymidine (New England Nuclear, Boston,
mediated by interleukin (IL)-6 and IL-11 secreted by MA, USA) was added to the media at 1·0 µCi/ml for the
osteoblasts (Manolagas et al. 1994). However, the effects of final 4 h and the incorporation of [3H]thymidine into
glucocorticoid on IL-6 and IL-11 production in human trichloroacetic acid (TCA)-precipitable material was
osteoblastic cells have not been thoroughly explored. measured by standard methods (Benz et al. 1977).
Although these accumulated data indicate that gluco-
corticoids have profound effects on osteoblast activities
Alkaline phosphatase activity
that, in turn, alter osteoclast activities, it is still unclear
which of these glucocorticoid-induced changes contribute hBMSC were seeded into 12-well plates at a density of
to osteoporosis in human bone. It has been well docu- 4104 cells/well and cultured for 2 days in á-MEM
mented that bone marrow stromal cells include actively containing 5% FBS. Then the media were exchanged for
proliferating osteoprogenitor cells, and these cells can be fresh medium containing 0·1% bovine serum albumin
induced to differentiate into cells exhibiting osteoblast (BSA) with or without addition of dexamethasone
phenotype by dexamethasone (Haynesworth et al. 1992, (10 8 M). After 72 h of culture, the medium was re-
Vilamitjana-Amedee et al. 1993,Cheng et al. 1994). To moved and the alkaline phosphatase activity of the cell
investigate the mechanisms of bone loss incurred by layer was measured by the p-nitrophenyl phosphate
glucocorticoid administration in humans, we studied the hydrolysis method (Puzas & Brand 1985). The data were
effects of dexamethasone on the proliferation, differenti- normalized with cellular protein contents to represent the
ated activities, and production of bone-resorbing cytokines alkaline phosphatase activity as nmol/µg protein per min.
in normal human bone marrow stromal cells.
Measurement of collagen synthesis
Materials and Methods Collagen synthesis was measured by the incorporation of
[3H]proline (Amersham, Arlington Heights, IL, USA) into
Human bone marrow stromal cell (hBMSC) culture collagenase (type VII, Sigma) digestible protein. Cells in
hBMSC were isolated from ribs obtained at the time of 12-well plates were grown to confluence (7 days),
open thoracotomy in patients without metabolic bone followed by treatment with medium containing 100 µg/ml
disease, as previously described (Cheng et al. 1994). This ascorbic acid, 100 µg/ml â-aminopropionitrile fumarate
study was approved by the Institutional Ethics Committee and 10 8 M dexamethasone or vehicle for 48 h. [3H]Pro-
of the Asan Medical Center, and all patients gave informed line (5 µCi/ml) was added for the last 24 h. The medium
consent. Briefly, the ribs were excised aseptically, cleaned was removed and the cell layers were harvested by scraping
of soft tissues, and opened longitudinally. The bone mar- into water containing l mg/ml BSA and the following
row cells were flushed out using several washes of serum- protease inhibitors: 2·5 mM N-ethylmaleimide, 0·2 mM
free á-minimum essential medium (á-MEM; Sigma, St phenylmethylsulfonyl fluoride, and 2·5 mM Na2EDTA.
Louis, MO, USA), and purified via Ficoll/Hypaque The cell layers were sonicated for 10 s and TCA was added
(specific gravity 1·077; Nycomed, Oslo, Norway) gradient to a final concentration of 15%. Samples were stored at
centrifugation. The cells were seeded into a 75 cm2 plastic 4 C overnight, followed by centrifugation at 3000g for
culture flask at a density of 4105 cells/cm2 and cultured 20 min at 4 C. The precipitates were washed three times
in á-MEM containing 10% fetal bovine serum (FBS; with 5% TCA, dissolved in 50 µl 1 M NaOH and adjusted
Gibco, Grand Island, NY, USA). When the bone marrow to 1 ml by adding 0·1 M HEPES, pH 7·3, containing
stromal cells had grown to 80% confluence, they were then 3·5 mM CaCl2 and protease inhibitors described above.
subcultured and the first- or second-passage cells were used Samples were divided into equal aliquots and incubated
for experiments. Previously, we have shown that, when with or without bacterial collagenase, the incorporation of
cultured to confluence in the presence of serum, the [3H]proline into collagenase digestible protein (CDP) and
Journal of Endocrinology (1999) 162, 371–379
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via free accessEffects of dexamethasone on human bone marrow stromal cells · C-H KIM and others 373 Figure 1 Effects of dexamethasone (Dexa) on [3H]thymidine incorporation of hBMSC. Left: Cells were treated with 10 8 M dexamethasone or vehicle (control) for 1 or 8 days. Right: hBMSCs were treated with vehicle (control, 0) or various concentrations of dexamethasone (10 910 7 M) for 5 days. Each bar represents means S.E.M. of 12 determinations. *P
374 C-H KIM and others · Effects of dexamethasone on human bone marrow stromal cells
representative data are shown in the figures. All data are
expressed as means... The significance of the differ-
ences were assessed by Mann–Whitney U-test between
two groups, and by analysis of variance (ANOVA) with
post hoc analysis by Duncan’s multiple range test among
three or more groups. Dose–response relationships were
examined by Spearman’s rank correlation analysis.
Results
Effect of dexamethasone on [3H]thymidine incorporation
Treatment with dexamethasone for 24 h at the concen-
tration of 10 8 M suppressed [3H]thymidine incorpor-
ation to 824% of the control (PEffects of dexamethasone on human bone marrow stromal cells · C-H KIM and others 375
Figure 3 Effects of dexamethasone (Dex, Dexa) on the steady state mRNA levels of type I collagen in hBMSC. Cells
were treated with vehicle (control, C) or dexamethasone at 10 8 M for 1–28 days. The relative type I collagen mRNA
concentration on the autoradiograms was analyzed by densitometer and normalized with the â-actin mRNA level. The
ratio of type I collagen/â-actin for the control culture after 1 day of treatment was defined as 100% (n=8, *P376 C-H KIM and others · Effects of dexamethasone on human bone marrow stromal cells
Although long-term use of pharmacological doses of
glucocorticoids results in osteoporosis in vivo, it has been
reported that glucocorticoids induce differentiation of
osteoprogenitor cells into osteoblasts (Haynesworth et al.
1992, Vilamitjana-Amedee et al. 1993, Cheng et al. 1994).
These seemingly paradoxical effects of glucocorticoid can
be explained by the inhibition of proliferation of osteo-
progenitor cells. Although glucocorticoids stimulate the
differentiation of osteoprogenitor cells to osteoblasts, the
inhibition of their proliferation may diminish the supply of
these osteoprogenitor cells, and decrease the numbers of
osteoblasts and bone formation. It has also been suggested
that physiological levels of glucocorticoids are necessary for
osteoblast differentiation, and high levels of glucocorticoid
drastically reduce proliferation of the osteoblastic precur-
sors (Scutt et al. 1996). Our findings that dexamethasone
decreased [3H]thymidine incorporation and markedly
increased alkaline phosphatase activity are consistent with
such a concept.
Conflicting results have been reported on the effects of
glucocorticoids on the expression of type I collagen.
Glucocorticoid can stimulate, inhibit, or have a biphasic
effect on collagen mRNA expression, depending on the
osteoblast systems and culture conditions (Dietrich et al.
1979, Kasugai et al. 1991, Shalhoub et al. 1992, Yao et al.
1994). In rat BMSC, dexamethasone decreased the level of
type I collagen mRNA during the first 6 or 7 days of
Figure 4 Effects of dexamethasone on the steady-state mRNA treatment, followed by a rebound to near control on day 9
levels of type I collagen in hBMSC as a function of dose. hBMSC and, finally, an increase of four- to fivefold 14–28 days
were treated with vehicle (control, 0) or dexamethasone at
indicated concentrations for 7 days. The relative type I collagen
after treatment (Kasugai et al. 1991, Leboy et al. 1991, Yao
mRNA concentration on the autoradiogram was analyzed by et al. 1994). Our results showed that the expression of type
densitometer and normalized with the â-actin mRNA level. The I collagen mRNA in hBMSC was decreased 1 day after
ratio of type I collagen/â-actin for the control culture was defined treatment, and slightly rebounded in the next 7 days,
as 100% (n=8, *PEffects of dexamethasone on human bone marrow stromal cells · C-H KIM and others 377 Figure 5 Effects of dexamethasone on IL-6 production of hBMSC. Cells were stimulated with PTH (10 8 M), IL-1á (500 U/ml), or TNF-á (10 11 M) in the presence of various concentrations of dexamethasone for 72 h and the amounts of IL-6 secreted into the media were measured by ELISA (n=8; *P
378 C-H KIM and others · Effects of dexamethasone on human bone marrow stromal cells
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Acknowledgements Haynesworth SE, Goshima J, Goldberg VM & Caplan AI 1992
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This research was supported by grants from Asan Institute marrow. Bone 13 81–88.
for Medical Sciences, Seoul, Korea, and NIH 5P0l Haynesworth SE, Baber MA & Caplan AI 1996 Cytokine expression
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type I collagen cDNA, and Dr Bratin Saha (Emory Kaji H, Sugimoto T, Kanatani M, Nishiyama K & Chihara K 1997
University, Atlanta, GA, USA) for human â-actin cDNA. Dexamethasone stimulates osteoclast-like cell formation by directly
acting on hemopoietic blast cells and enhances osteoclast-like cell
formation stimulated by parathyroid hormone and prostaglandin E2.
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