Full text of DOI:10.1007/BF03343837

J. Endocrinol. Invest. 24: 166-172, 2001
Age and gender specific stimulation of creatine kinase
specific activity by gonadal steroids in human bone-derived
cells in culture
S. Katzburg*1, A. Ornoy*, D. Hendel**, M. Lieberherr***, A.M. Kaye****,
and D. Somjen*****
*Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem,
**Department of Orthopaedics, Shaarei Zedek Hospital, Jerusalem, Israel; ***CNRS, UPR 1524, Paris,
France; ****Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot and
*****Institute of Endocrinology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel
ABSTRACT. We previously reported a non-enzy-
matic method for isolation of human bone cells in
culture that display osteoblastic features and re-
spond to 1,25 dihydroxy vitamin D (1,25) and to
parathyroid hormone (PTH). The present study
was undertaken to analyze the response of cul-
tured human bone cells to 17β-estradiol (E2) and
to dihydrotestosterone (DHT) as a function of
gender and age. Cultured human bone cells, ob-
tained from biopsies during orthopedic surgery,
were divided into four groups defined by gen-
der and age: pre- and post-menopausal healthy
non-osteoporotic women that were not under
hormone replacement therapy (HRT) and mature
(<55-year-old) and older (>60-year-old) men. We
found gender specific responses to gonadal
steroids using the specific activity of the brain
type (BB) isozyme of creatine kinase (CK) as a re-
sponse marker. Constitutive levels of CK activity
did not change with age or gender and the en-
zyme extracted from cells from the different sex-
es and ages did not respond to either proges-
terone (P) or to 1,25. CK from the different cells
responded to gonadal steroids in a gender spe-
cific manner, i.e. CK from female derived cells
responded to E2 only and the enzyme from male
derived cells responded to DHT only. In female
derived cells the response to E2 declined signif-
icantly with age, while the response to DHT in
CK from male derived cells did not vary with age.
This may be due to either decreased proportion
of mature osteoblasts and/or their differentia-
tion state and/or changes in the levels of estro-
gen receptor(s), coactivators or corepressors in
these cells. These results extend our knowledge
of human osteoblast biology (beyond murine
cells) and are therefore more relevant for devel-
oping models for treatment of human metabol-
ic bone diseases such as post-menopausal os-
teoporosis.
(J. Endocrinol. Invest. 24: 166-172, 2001)
©2001, Editrice Kurtis
INTRODUCTION
osteoporosis and its resulting fractures. Estrogen
replacement therapy can delay or prevent bone
loss (1-4). Most of these deleterious changes are
thought to be the result of low estrogen levels, but
lack of progesterone (P) may also contribute (5). In
human males, although there is no cessation of tes-
ticular function comparable with the menopause in
women, both total and free testosterone levels de-
cline with age (6) and a common secondary cause
of osteoporosis in men is hypogonadism. It has re-
cently been demonstrated that estrogens as well
as androgens are also necessary for the mainte-
nance of the male skeleton (6, 7).
Gonadal steroids play a crucial role in skeletal de-
velopment and maturation (1-4). Estrogen deple-
tion after menopause is associated with a reduction
in bone mass and is recognized as a risk factor for
1In partial fulfillment of the requirements for a Ph.D. Thesis of the
Hadassah Medical School, Hebrew University, Jerusalem.
This project was supported in part by private donation to D. Somjen in
memory of the late Haim Ben-Yitzhaq
Key-words: Bone, human, gonadal steroids, creatine kinase.
Correspondence: Dr. Dalia Somjen, Institute of Endocrinology, Tel-Aviv
Sourasky Medical Center 6, Weizmann St. Tel-Aviv 64239, Israel.
The failure for many years to demonstrate either es-
trogen receptors in bone cells (8) or direct estrogen
E-mail: dalias@tasmc.health.gov.il
Accepted August 25, 2000.
166

Gonadal steroid action in cultured human bone cells
responsiveness of bone in organ or cell cultures (9,
10) led to the notion that estrogens exert their effects
indirectly by mechanisms concerned mainly with de-
creased bone resorption. However, estrogen recep-
tors and their mRNA transcripts have been found in
cultured murine and human bone cells (11-13) as well
as in human embryonic bone (13). Estrogen has also
been shown to influence the proliferation and differ-
entiation of rodent osteoblast-like cells and human
osteosarcoma cell lines (14-16). Androgen receptors
have been identified in normal human osteoblast-like
cells (17) and it has been shown that androgens stim-
ulate bone cells proliferation in vitro (18, 19). The
presence of P receptors, as well as the effects of P on
cell proliferation, have also been demonstrated in hu-
man osteoblast-like cell lines (5, 19-21).
The brain type (BB) isozyme of creatine kinase (CK) is
an enzyme involved in the “energy buffer” system,
which regulates cellular concentration of ATP and
ADP. It is the major component of the “E2-induced
protein” found in rat uterus and other tissues con-
taining 17β-E2 (estradiol) receptors (22). CK stimula-
tion is an efficient response marker to detect bio-
logical activity of E2 (22-24) particularly in the case
of osteoblasts which contain low concentrations of
E2 receptors (11, 12). The stimulation of CK in cul-
tured bone cells requires the higher end of the phys-
iological range of estrogen concentrations (19). CK is
also a general marker for the action of hormones,
which, like estrogens, cause increased utilization of
energy for processes such as cell growth (22-25).
Although it is not a specific marker for bone, it is a
very useful marker for the activity of gonadal steroids
and was shown to be correlated with markers of
bone formation in vivo (26).
The stimulatory effects of gonadal steroids on bone
are gender specific, since estrogens are active on-
ly in females and androgens only in males. This
specificity was found in rat diaphyseal bone (18, 27)
and in mice epiphyseal cartilage and diaphyseal
bone (28), as well as in bone-derived cells such as
rat embryo calvaria cells in culture (19). This was al-
so true for human osteoblasts in culture such as
SaOS-2 cell lines (29) and primary human bone cells
in culture (30). Rat cartilage cells in culture (19) or rat
cartilage in vivo (18) did not show any gender spe-
cific response to gonadal steroids although there
are sex dependent effects of E2 on chondrocyte
differentiation in culture (28, 29, 31).
Recently, we established a primary human bone-
derived osteoblast-like cell culture system, exhibit-
ing major osteoblastic characteristics, i.e. high lev-
el of basal alkaline phosphatase activity and re-
sponsiveness to 1,25 (OH)2D3 (1,25) and stimulat-
ed cAMP formation in response to PTH, and high
levels of osteocalcin and its stimulated production
by 1,25 (32).
In our model, only cells derived from female bone
showed minor age-related changes. Higher basal ac-
tivity of alkaline phosphatase and higher responsive-
ness to 1,25 as well as higher level of osteocalcin were
found in pre-menopausal cells compared to post-
menopausal cells. Osteoblastic cultures derived from
male bones at similar age groups did not show any
age dependent differences.
Using this culture system we analyzed whether these
cells respond to gonadal steroids, using the stimula-
tion of CK as a response marker, and whether these
effects are gender and/or age dependent.
We present evidence that the effects of both es-
trogen and androgen on normal human cultured
osteoblasts are gender specific and that the re-
sponses are age-dependent in females, but not in
males in similar age groups.
MATERIALS AND METHODS
Cell isolation and culture
We used a modified procedure of Beresford et al.
(33). Normal human bones were obtained from biop-
sies of patients undergoing corrective surgery fol-
lowing accidental injury or hip replacement. The ex-
perimental groups were divided into male and fe-
male. The male groups consisted of 17 healthy ma-
les: mature (under 55-year-old, no.=12) and older
(over 60-year-old, no.=5). The female groups con-
sisted of 23 healthy, non-osteoporotic females that
were not receiving hormone replacement therapy
(HRT): pre-menopausal (under 50-year-old, normally
menstruating, no.=7) and post-menopausal (over 55-
year-old, not menstruating, no.=16). The trabecular
surface of the iliac crest or long bones were crushed
into small (1 mm3) pieces, then extensively and re-
peatedly washed with phosphate buffered saline
(PBS) to remove blood components. The explants
were seeded in 100 mm tissue culture dishes and in-
cubated in DMEM medium without Ca++ containing
10% fetal calf serum (FCS) and antibiotics. Cell out-
growth from the trabecular bone surface was appar-
ent after 6 days and the cells were then cultured un-
til sub-confluence in phenol red free DMEM with
10% charcoal stripped FCS. Cells were seeded at a
density of 3x105 cells/35 mm tissue culture dishes.
Hormonal treatment
Sub-confluent cell cultures were incubated in tripli-
cate for 24 h with increasing concentrations of E2,
P, dihydrotestosterone (DHT) or vehicle (0.01%
ethanol). At the end of incubation CK was extract-
ed and assayed as described previously (18, 19).
167

S. Katzburg, A. Ornoy, D. Hendel, et al.
Enzyme extraction and assay
Dose-dependent stimulation of CK by gonadal
steroids in human cultured bone cells
At the end of incubation cells were washed with
cold PBS, homogenized by freezing and thawing
three times in cold extraction buffer (50 mM Tris
NaCl, 5 mM magnesium acetate, 2.5 mM dithio-
threitol, 0.4 mM EDTA, and 250 mM sucrose, pH
6.8). Homogenates were centrifuged at 14,000 g
for 5 min at 4 C. The supernatant extracts were
stored at -20 C. CK activity was measured at 340
nm in a Uvicon, Kontron automatic computerized
recording spectrophotometer, using the Sigma
coupled assay kit (18). A unit of enzyme activity was
defined as the amount yielding 1 μmole ATP/min
at 30 C and the specific activity as nmol/min/mg
protein. Protein was assayed by the Coomassie
blue method using BSA as standard (34).
Bone-derived cells obtained from females showed a
dose dependent stimulation by E2 of CK specific acti-
vity with a maximum at 10 nM in both female groups
(Fig. 1). Cells from pre-menopausal females respon-
ded even to a lower dose of E2 (1 nM). On the other
hand, treatment with DHT caused no significant chan-
ge in bone cells derived from females in any age
group. Bone-derived cells obtained from males, sho-
wed a dose dependent stimulation by DHT of CK spe-
cific activity at 100 nM and 1000 nM in both age
groups, but no stimulation at any age or concentra-
tion by E2 (Fig. 2; Table 2).
Maximal responsiveness of cultured bone cells:
gender and age specific stimulation
The stimulations of CK activity by E2 in both cell
types compared to untreated cells was significant
Statistical analyses
Wilcoxon’s signed-ranks test was performed to test
the effect of the treatment within groups. Dose-de-
pendent differences within the group were analyzed
using Bonferroni test. Non-parametric Mann-Whitney
U-test for two independent samples analyzed differ-
ences between age groups of each gender.
Post-menopausal females
E2
DHT
3
2
RESULTS
Constitutive CK activity in cultured bone cells
**
The basal activity of CK in cells derived from pre-
and post-menopausal women showed no gender
or age changes (22.6 3.25 and 20.8 3.7 nmol/min/
mg protein, respectively). Similarly, male derived
cells from similar age groups showed no significant
difference in basal activity of CK between age
groups of mature males compared to older males
(28 6.5 and 24.6 4.0 nmol/min/mg protein, re-
spectively) (Table 1).
*
1
3
Pre-menopausal females
**
2
1
*
*
Table 1 - Age and gender effect on basal creatine kinase (CK)
specific activity.
Gender
Females
Age
CK specific activity
(nmol/min/mg protein)
Pre-menopausal
no.=7
22.6 3.25
20.8 3.7
0.1
1
10
100
1000
Post-menopausal
no.=16
Hormone (nM)
Fig. 1 - Dose dependent stimulation of creatine kinase (CK) speci-
fic activity in cultured female human bone-derived cells. Bone cells
derived from pre- and post-menopausal women (no.=7 and 16 re-
spectively) were cultured and treated as explained in Materials and
Methods. Cells were treated with estradiol (E2), dihydrotestostero-
ne (DHT) or vehicle (C) for 24 h. CK was extracted and assayed as
described. The results are expressed as means SEM for experi-
mental/control (E/C). Statistical analysis by Bonferroni test. *p=0.05,
**p=0.01, **p=0.001.
Males
Mature
no.=12
Older
28.0 6.5
24.6 4.0
no.=5
Bone cells derived from pre- or post-menopausal women and mature or
older men were cultured as explained in Methods and Materials. CK was
extracted and assayed as described. The results are expressed as
mean SEM of the specific activity as nmol/min/mg protein.
168

Gonadal steroid action in cultured human bone cells
was significantly lower than in cells from pre-
menopausal female bone (p=0.05) (Table 2).
While the stimulation of CK activity by DHT, in each
male cell type, compared to untreated cells was sig-
nificant in both age groups (p=0.05 in mature and
p=0.001 in older cells at 100 nM) (Fig. 2; Table 2),
no difference was found between cells derived from
the two age groups (Table 2). Cells derived from
both males and females showed no significant stim-
ulation of their CK activity by either P (1 μM) or 1,25
(100 nM) (Table 2).
Older males
E2
DHT
2
1
**
*
DISCUSSION
**
Mature males
Our cell culture system of osteoblasts, derived from
human bone explants, expressed osteoblastic fea-
tures such as stimulation of cAMP formation by PTH,
high levels of alkaline phosphatase activity and in-
duction by 1,25(OH)2D3 and osteocalcin synthesis
and its induction by 1,25(OH)2D3 (32). Human os-
teoblasts (33), human embryonic bone and cartilage
(19) express receptors for gonadal steroids. Indeed,
estrogens and androgens are active in our cell cul-
tures in a gender specific manner (Figs. 1 and 2), i.e.
female-derived cells respond only to E2 and male-
derived cells only to DHT, using the specific activi-
ty of CK (BB CK) as a response marker to gonadal
steroids (24). Although this marker is not a specific
bone marker, it is one of the most sensitive markers
for gonadal steroid action (22) and it was also cor-
related in vivo with calciotropic hormone activity and
increased bone formation markers (26). This gender
specificity was shown previously for rat calvaria cells
in culture (18, 19), for primary human bone cells in
culture (32) and for the human osteoblasts-like cell
line SaOS2 (29). This specific response was abolished
upon gonadectomy and was retained upon treat-
ment with the hormones, probably via changes in
the concentrations of estrogen receptors (ERs) or
their sub-types (27).
2
1
*
0.1
1
10
100
1000
Hormone (nM)
Fig. 2 - Dose dependent stimulation of creatinine kinase (CK) spe-
cific activity in cultured male human bone-derived cells. Bone cells
derived from mature and older males (no.=12 and 5 respectively)
were cultured and treated as explained in Materials and Methods.
Cells were treated with estradiol (E2), dihydrotestosterone (DHT)
or vehicle (C) for 24 h. CK was extracted and assayed as described.
The results are means SEM for experimental/control (E/C).
Statistical analysis by Bonferroni test. *p=0.05, **p=0.005.
in both age groups (2.1 in pre- and 1.51 fold stim-
ulation in post-menopausal cells, p=0.05 for both)
(Fig. 1; Table 2). In cell cultures derived from post-
menopausal female bones, E2-induced CK activity
Table 2 - Maximal induction of creatine kinase by gonadal hormones. Results are experimental/control (E/C).
Treatment
Females
Males
Pre-menopausal Post-menopausal
Mature
0.7 0.23
Older
E2 (30 nM)
DHT (300 nM)
P (1 μM)
2.1 0.50**
0.5 0.10
1.1 0.20
1.12 0.16
1.5 0.10**
0.65 0.08
1.07 0.05
0.97 0.08
1.05 0.07
1.63 0.10*
1.2 0.25
1.2 0.06
1.67 0.10**
1.47 0.30
1.3 0.26
1,25 (10 nM)
Bone cells derived from pre- or post-menopausal women and mature or older men were cultured and hormonally treated as explained in Methods and
Materials. CK was extracted and assayed as described. Results are expressed as the ratio of hormonally treated compared to vehicle control treated;
E/C for no.=3-16. Basal CK activities: pre-menopausal women=24 6.7, post-menopausal women=24.5 4.0, mature males=32.6 6.1, older males=
26.1 5.2 nmol/min/mg protein. The results are expressed as mean of E/C SEM. Statistical analysis by Bonferroni test. *p=0.05, **p=0.01).
169

S. Katzburg, A. Ornoy, D. Hendel, et al.
Although the constitutive level of CK was similar in
bone cells derived from both pre- and post-meno-
pausal women, the response of pre-menopausal
cells to E2 was 50% higher than that of post-
menopausal cells (p<0.005) (Fig. 1) (Table 2). This
may indicate that under these culture conditions,
the growth of the cells from different origins is sim-
ilar, but the content of receptors to the gonadal
steroids and/or the co-activators and/or the co-re-
pressors are different, and are modulated in female
bones with age. This extent of CK stimulation par-
alleled the constitutive synthesis of osteocalcin, a
parameter of bone differentiation, which declined in
cells derived from bones of post-menopausal wom-
en (32). Our bone samples were obtained from pre-
menopausal and post-menopausal women without
osteoporosis in their medical history. The post-
menopausal cell populations, showing a decreased
response, could have less functional osteoblasts, or
they might have lost some of their ability to re-
spond to E2 by either a decrease in the number of
receptors or co-receptors and/or other factors.
However, Ankrom et al. (35) found in human post-
menopausal women osteoblasts strains a greater
concentration of ERα along with a reduction in their
induction of hydroxyproline by estrogen. They in-
terpreted this as a loss of receptor regulation and
diminution of ligand-receptor signal transduction
with increased age. Our results of age-associated
decrease of CK-response to E2 can support their
conclusions, provided that changes in ERβ are also
measured, since they are important in bone (36).
Whether this is due to decreased number of re-
ceptors or decreased functionality has to be further
investigated.
We have previously demonstrated that gonadec-
tomy of rats or of mice leads to the loss of gender
specific response in rat bone to gonadal steroids
and in both cartilage and bone in mice (27, 28). This
can be restored by replacement with the steroid
hormones (27). However, bone cells from post-
menopausal women in culture, which might be
equivalent to bone cells from ovariectomized rats or
mice, did not lose their sex specific response and
did not acquire responsiveness to DHT (Fig. 1),
even if their response to E2 was significantly re-
duced. One possibility for the difference between
species could be the relative loss of circulating es-
trogen in post-menopausal women, compared to
post-ovariectomy in rats or mice, and/or in the
threshold levels of the sex steroids needed for in-
duction of CK, although there is no evidence for
this since serum estrogen levels sharply decreased
after menopause in both types. Circulating E2 in
post-menopausal women (0-20 pg/ml) declines four
orders of magnitude compared to the levels in pre-
menopausal women (40-80 pg/ml) (37). The same
magnitude of decline was observed in ovariec-
tomized rats: 3.8 vs 13.7 pg/ml in sham rats (37,
38). Also, the same concentrations of E2 are need-
ed for the induction of CK activity in humans and
rats derived osteoblasts in culture (38).
The results presented in this study extend our knowl-
edge of human osteoblast biology (beyond murine
cells) and are therefore more relevant for developing
models for treatment of human metabolic bone dis-
eases such as post-menopausal osteoporosis.
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