Department of Biochemistry and Molecular Biology (T.C.S., M.S.) Division of Endocrinology, Department of Internal Medicine (B.L.R., S.K.) Mayo Foundation Rochester, Minnesota 55905
INTRODUCTION
The discoveries that sex steroid receptors for estrogen [estrogen receptor (ER)], androgen [androgen receptor (AR)], and progesterone [progesterone receptor (PR)] are present in the bone-forming osteoblasts (OB) and bone-resorbing osteoclasts (OCL), and that these cells are targets for sex steroid action, have generated much excitement in the field of bone biology. The subsequent discoveries that the sex steroids regulate the production of growth factors and cytokines in these OB and OCL cells and that these factors mediate much of the steroid action on the skeleton have added to the excitement and complexity of the system. Because of the required brevity of this minireview, we will discuss only selected aspects of this field using a limited number of references and review articles. For more information in this field, readers are referred to several recent books and chapters that review bone biology, bone diseases, and the role of sex steroids in bone biology and disease (1, 2, 3, 4, 5, 6, 7, 8).
As a brief background to this unique system, the skeletal maintenance
involves a continuous remodeling at discrete sites termed
bone-remodeling units (BRU). As outlined in Fig. 1, the multinucleated bone-resorbing OCLs
first dissolve bone at these BRU sites, resulting in resorption
cavities. While the mechanism(s)/signal(s) that initiates resorption at
a given site is unknown, there appears to be a marked regulation of
overall bone resorption via the regulation of the OCL differentiation
and activity (5, 6, 7, 8). These processes are discussed in more detail
below. The bone-forming OBs are then recruited by unknown signals, to
replace the previously resorbed bone. The identities of the primary
factors that couple these processes of resorption to formation to
maintain bone mass are also addressed later in this review and are
still the subject of much debate. During periods of skeletal growth in
children, the increase in bone formation largely involves bone modeling
(accretion of bone at surfaces in the absence of bone resorption). The
response to mechanical stresses also utilizes mostly modeling. During
early adulthood (2040 yr), bone resorption and formation are in
balance and bone mass is maintained. The increased serum estrogen (E)
level, which occurs at the onset of puberty in girls, is accompanied by
an increase in growth velocity and, ultimately, in the closure of the
epiphyseal growth plate and the cessation of linear growth (9). E is
the primary hormone responsible for maintaining bone mass in adult
women and may serve a similar role along with androgen, in adult men.
Finally, during periods of bone loss, such as in postmenopause in women
and aging in both genders, bone resorption outpaces bone formation and,
in some cases, the bone loss is sufficient to cause osteoporosis.
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E ACTION ON OB DIFFERENTIATION and ACTIVITIES
Action of E on Mature OB Activities
That bone cells, especially OB cells, contain ER is fairly well
established. Using sensitive techniques, such as immunofluorescence,
semiquantitative RT-PCR, in situ hybridization, and in
situ RT-PCR, both the ER mRNA and ER protein have been identified
in periosteal cells, OBs, osteocytes, and OCLs, at levels less than the
reproductive organs, but equivalent to those in liver (9, 10, 11, 12, 13).
Moreover, Bodine et al. (14), using RT-PCR and primary
cultures of rat calvarial OBs, found that ER concentrations increased
markedly during OB differentiation.
It is fairly well established that E has major effects on OB cells. As
summarized in Table 1, E acts directly on
OB cell proliferation and expression of genes coding for enzymes, bone
matrix proteins, hormone receptors, transcription factors, as well as
growth factors/cytokines (4, 5, 6, 7, 8, 15, 16). However, results on the
specific genetic and metabolic alterations induced by E in these
in vitro systems have been conflicting. As outlined in Table 2
, these discrepancies could be caused by
differences in the receptor concentrations, animal species, skeletal
locale of the OB cells, the stages of OB differentiation, and the
concentrations of the two ER species (ER-
and ER-ß) in the OB
cells. Although E inhibits the proliferation of mature human OB cells
in culture (15, 16), the possibility that E may stimulate OB
proliferation under some circumstances has not been excluded. In mature
OB cells in culture, E has been shown to induce the synthesis of
transforming growth factor-ß (TGF-ß), insulin-like growth factor-I
(IGF-I), and IGF-binding proteins, and to inhibit the synthesis of
interleukin (IL)-1, IL-6, and IL-11 (5, 6, 7, 8). E deficiency increases the
production of IL-1 and IL-6, as well as macrophage-colony stimulating
factor (M-CSF) in bone marrow stromal cells. Further, E has also been
reported to inhibit the IL-1 or tumor necrosis factor-
(TNF-
)-induced IL-6 production in human OB and rat bone marrow
stromal cells (5, 6, 7, 8, 17, 18). More recently, E has been shown to
regulate the synthesis of bone matrix proteins, bone morphogenetic
protein-6 (BMP-6), osteoprotegerin (OPG), as well as transcription
factors, such as TIEG (TGF-ß inducible early gene), nuclear
factor-
B and c/EBP-ß, and c-fos in human
OBs (5, 8, 19, 20, 21, 22). E has also been shown to regulate the activities of
the estrogen response element (ERE)-reporter genes and the IGF-I gene
promoter-reporter genes transfected into primary rat or human OB cells
in culture, as well as the inositol phosphate receptor gene
promoter-reporter in normal and transformed OB cells (5, 6, 7, 8).
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Role of ER- and ER-ß in OBs
At least some of the variable actions of E on OB cells may be
attributed to different concentrations of ER in the OB lines or to
different ratios of ER- and ß. In humans, Smith et al.
(24) described a man with an inactive mutated ER-
who had osteopenia
and open epiphyses, thereby demonstrating the importance of E in
maintaining bone mass in men. In contrast, ER-
knock-out mice
displayed only a 10% reduction in mineralization, but otherwise had
normal skeletal development (25), suggesting that another ER species or
nonreceptor mechanisms may be playing major roles in the E action in
skeletal tissues. Significantly, a separate species, ER-ß, has been
identified in humans and in rat tissues, including OB cells, and found
to have partial homology to the extensively studied ER-
(26, 27).
The ligand-binding affinities and DNA-binding domains of ER-
and
ER-ß are similar, but their transactivation domains, tissue
distribution, and molecular sizes differ significantly. In some cell
systems, using transfection studies with reporter genes, the ER-ß was
shown to be a weaker regulator of gene transcription than is the well
known ER-
(26, 27, 28). Further, the ER-
was shown to activate,
whereas ER-ß inhibits, AP-1 regulatory elements (28). While both
ER-
and ER-ß are expressed in rat and human OB cells, the two
species are differentially expressed during OB differentiation (13, 29). Although the exact functions of ER-ß and ER-
in the human
skeleton must await further studies, their differential expression in
bone cells may help explain the different E responses among the
various OB preparations, as well as among the different actions
of the selective ER modulators (SERMs), described below.
ACTION OF E ON OCL DIFFERENTIATION AND ACTIVITY
Direct Action of E on OCL Activity
The major action of E on the skeleton in vivo is
believed to be an inhibition of bone resorption, mostly by indirect
actions of E. This indirect E action involves regulation of growth
factor and cytokine production in OBs and their precursor cells, which,
in turn, regulate OCL differentiation and activity (discussed further
below). However, some direct actions of E on mature OCL activities have
been reported. E must have some direct actions on OCLs since avian OCL
cells have been shown to contain E receptors, and E has been shown to
regulate bone-resorbing activity and specific gene expression by these
cells (10, 30). Table 3 summarizes the
results from several laboratories, which demonstrated that E directly
inhibits bone-resorbing activity in avian, rabbit, and human OCL via
the regulation of specific gene activities (5, 10, 30, 31, 32). E was shown
to antagonize the induction of a novel 150-kDa superoxide
dismutase-related membrane glycoprotein by OCL differentiating agents
(31). E was also shown by that laboratory to induce an IL-1 decoy
receptor and to decrease the expression of an IL-1-signaling gene (32).
Other studies using murine and rabbit OCL have substantiated the above
avian OCL studies (33, 34). Not only was ER found in rabbit OCL but E
inhibited (and anti-E blocked) effects on OCL-resorptive activity,
including the production of bone resorbing enzymes (33). E-induced
decreases in bone resorption may also be caused by the reduced OCL
numbers that are due to E-induced apoptosis (33, 34). This direct E
induction of apoptosis may also explain the E inhibition of bone
resorption by reducing the OCL life span and population. The E
induction of apoptosis may be further enhanced in vivo since
TGF-ß, whose production by OB is enhanced by E, was also shown to
induce OCL apoptosis in these cultures (34). It should be mentioned,
however, that some laboratories have not been able to detect ERs in,
nor E effects on, OCL cells (5, 35). As with the OB cells (Table 2
),
these contradictory results may potentially be explained by the age of
the animal (embryonic, neonate, or adult) or differences in the OCL
cell preparations used (5).
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The IL-1, IL-6, TNF- Story
As mentioned at the beginning of this minireview, the coupling
between the OB and OCL at the BRU sites infers a cross-talk by chemical
signals (factors) between the OB and OCL cells and their precursors
(see Fig. 1 and reviews in Refs. 4, 5, 6, 7, 8). As outlined in Fig. 2
, recent studies suggest that OB
production of bone-resorbing cytokines, such as interleukin (IL)-1,
TNF-
, M-CSF, IL-6, and PGE2 may mediate bone loss after
E deficiency via acting on OCL cell differentiation and activity
(6, 7, 8). Previous studies have shown that the production of IL-1 and a
specific competitive inhibitor of IL-1 action, the IL-1 receptor
antagonist, IL-1ra, by peripheral blood monocytes is increased in
oophorectomized and postmenopausal women and is suppressed by estrogen
replacement therapy (36). Moreover, the administration of the
IL-1ra to OVX rats decreased bone resorption and bone loss (37). Also,
the overexpression of the soluble TNF receptor (TNF-R) in transgenic
rats, or treatment of the animals with soluble TNF-R , prevented the
increase in bone resorption and bone loss after ovariectomy in rats
(37, 38). Thus, both IL-1 and TNF-
and their receptors play
important roles in mediating the bone loss after E deficiency.
Recently, IL-1
was reported to induce the expression in osteosarcoma
cells of OPG, an inhibitor of OCL differentiation (39). Another IL
family member, IL-11, as well as PGs and PTH, have been shown to
increase the production of the OPG-ligand (OPG-L), an inducer of OCL
differentiation, in murine OB cells (40). The important roles of OPG
and OPG-L are discussed in the next section.
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Osteoprotegerin and Its Ligand
Recently, a novel secreted glycoprotein, OPG, a soluble
non-membrane-bound member of the TNF-R superfamily, has been identified
by screening a fetal rat intestine cDNA library. After a series of
studies, OPG now is believed to play a major role in bone biology (42).
The OPG lacks a transmembrane domain, and thus is secreted as a soluble
factor. Both the rat protein and a human homolog have been identified,
and both are potent inhibitors of osteoclastogenesis (see Fig. 2) (43).
Transgenic mice that overexpress OPG display a phenotype of nonlethal
osteopetrosis associated with decreased osteoclastogenesis (43, 44).
Further, OPG knockout mice have markedly increased OCL activity and
severe osteoporosis characterized by decreased trabecular bone mass and
cortical thinning (45).
Recent studies have also identified the natural ligand for OPG termed
"OPG-L" (46, 47). OPG-L is a TNF-related cytokine that is
produced by a variety of cells, including bone marrow stromal cells and
OBs. Interestingly, OPG-L is identical to TRANCE/RANKL, which was
recently identified as a factor enhancing T cell growth and dendritic
cell function (48). As outlined in Fig. 2, many investigators speculate
that OPG-L is likely the long-sought ligand mediating an essential
signal from both marrow stromal cells and OBs to the OCL progenitor
cells to initiate their differentiation into mature OCLs. OPG-L, in the
presence of M-CSF, can replace the requirement for stromal cells in the
spleen coculture model for in vitro osteoclastogenesis (46, 47). The osteoclastogenesis effects of OPG-L are blocked in
vitro and in vivo by OPG. Interestingly, E treatment of
human OB cells increased OPG mRNA steady state levels and OPG protein
secretion, and these effects were inhibited by the E antagonist, ICI
182,780 (19).
Thus, while ablation or neutralization of certain bone-resorbing
cytokines (IL-1, IL-6, TNF-) in animal models prevents sex hormone
deficiency-induced bone loss (38, 49), OPG is unique because, in
contrast to other cytokines affecting osteoclastogenesis (42), its
production is strongly regulated by E, its overexpression by gene
transfection results in osteopetrosis, and its targeted deletion
results in osteoporosis (45). Taken together, the evidence is
compelling that the OPG/OPGL system acts downstream to IL-1, TNF, IL-6,
and PGE2 and thus may be the final effector mediating the
effects of cytokines on the regulation of osteoclastogenesis, and thus,
an important mediator of the effect of E deficiency on bone cell
functions. More studies to confirm this interesting possibility are
needed.
The TGF-ß and IGF Families
There are two other growth factor families that are believed to also
play major roles in bone cell coupling and E regulation. Both TGF-ß
and IGFs are found in large concentrations in the bone matrix. Space
limitations only allow brief review of their role, but more extensive
reviews are available (4, 5, 8, 50). In vivo studies have
demonstrated that TGF-ß is involved in osteogenesis and
chondrogenesis and that local injections of TGF-ß into rodents caused
increased bone mass (4, 8, 50). Interestingly, when injected
subperiosteally into the femur of young rats, TGF-ß induced the
differentiation of periosteal mesenchymal cells into OB cells (50).
Similarly, TGF-ß treatment of mice increased calvarial thickness and
mineralized bone formation. OBs and OCLs have been demonstrated to
synthesize TGF-ß in vitro, and this production has been
shown to be induced by E (5). The TGF-ßs, in turn, have been shown to
regulate gene expression and other cellular activities of the OBs and
OCLs (5, 50, 51). The BMPs compose an extensive portion of the TGF-ß
superfamily and are an integral component in growth and development.
Select members of this superfamily are thought to act sequentially in
regulating endochondrial bone development, and BMP-6 has been shown to
be regulated by E whereas E has no effect on the other BMPs (50). IGF-I
and II are also found in high concentrations in the skeleton, are
produced in OB cells, and are regulated by E (4, 5, 8). Interestingly,
E also regulates one of the binding proteins for the IGFs, IGFBP-4,
which in turn regulates the biological activity of the IGFs (4, 5). The
exact roles of TGF-ß and the IGFs in OB-to-OCL communication, cell
differentiation, and bone loss caused by E deprivation in the skeleton
remain to be defined.
ACTION OF OTHER ESTROGENS ON THE SKELETON
SERMs
Studies with synthetic estrogens, including SERMs, such as
raloxifene and tamoxifen, are not only providing new insights into the
actions of E in bone, but also are identifying new therapeutic
estrogens to prevent osteoporosis without the negative side effects of
the native estrogens (8, 52). These compounds have tissue-specific
actions and can serve as either E antagonists or agonists. Certain
SERMs mimic the action of E in bone cells and blood lipids, while
antagonizing E action in reproductive cells. Other SERMs, such as
raloxifene, are E agonists in bone but have a neutral effect on
endometrial tissue (8, 52). While SERMs bind the receptor at the same
site and often with the same affinity as E, they induce different
conformational changes in the receptor molecule which, in turn, alters
which transcriptional activation domains on the ER molecule will be
active (53). In addition, the SERM-ER complex can interact with
different coactivators/corepressors to generate different patterns of
gene expression than the native E-receptor complex. For instance, E
regulates the expression of the IL-6 gene not by direct interaction of
the ER complex with the ERE on DNA, but by binding and inhibiting the
activity of transcription factors, such as nuclear factor-kß and
C/EBPß, which are required for IL-6 gene transcription (8, 22). In addition, raloxifene has been shown to activate the gene for
transforming growth factor-ß3 in human osteosarcoma MG-63 cells
transfected with a mutated ER- that lacked the DNA-binding domain in
the ER-
receptor. Finally, both ER-
and ER-ß interact with AP-1
sites in DNA via interactions with the transcription factors,
fos and jun. However, when native estradiol is
present, ER-
activates transcription from the AP-1 site, while
ER-ß inhibits transcription (26, 28, 53, 54, 55, 56). In contrast, in the
presence of SERMs such as raloxifene, ER-ß stimulates transcription
from the AP-1 site, whereas ER-
inhibits transcription. Thus, the
presence of either ER-
or ER-ß, or different ratios of the two,
may have a significant impact on the response of the OBs or OCLs to the
particular estrogenic steroid. The exact mechanisms of action of these
SERMs appear to be complex, but may well provide new insights into E
action in the skeleton and other tissues.
E Metabolites
The possible role of E metabolites in bone biology should be
mentioned, since these can become the major serum estrogens in
postmenopausal women. Estradiol (E2) is metabolized mainly
to estrone, the primary estrogenic metabolite in the circulation of
postmenopausal women. The estrone is then hydroxylated, primarily at
the C-2 or C-16 sites. The C-16 hydroxylation leads to the
production of 16
-hydroxyestrone (16
-OHE), an E agonist that binds
the ER, enhances uterine weight in OVX rats, alters gonadotropin
secretion in OVX rats, stimulates breast cell proliferation, as does E
(57). Further, 16
-OHE, but not 2-OHE, decreased bone turnover and
tibial growth in OVX rats, similar to E, but had much less of an effect
than did E on increasing uterine weight and development. Interestingly,
there is some evidence that women who generate less 16
-OHE than
2-OHE are at greater risk for developing osteoporosis and show greater
bone loss than women who have high levels of 16
-OHE. Further, the
ratio of 16
-OHE to 2-OHE correlates positively with spinal bone
mineral density (58). These results indicate that 16
-OHE has E
agonist activities on the skeleton and may play a role in the incidence
(i.e. protection against) of postmenopausal
osteoporosis.
ACTIONS OF OTHER SEX STEROIDS
Only a brief mention of the actions of the other two classes of sex steroids, androgens and progesterone, can be made. The increase in bone mass at puberty in boys is associated with increases in markers of bone formation and is closely linked to pubertal stage, suggesting that testicular androgen production contributes to the pubertal increase in bone mass (8). As in the case of E, there is evidence that androgens inhibit bone resorption and have receptors in the bone-resorbing OCLs and inhibit their activity in vitro. Thus, orchiectomy, like ovariectomy, is associated with increased bone resorption and rapid bone loss (8). Since human OB and OCL cells have been demonstrated to have ARs and because dehydrotestosterone (DHT) has been shown to have mitogenic effects on normal and transformed OB-like cells in most OB cell lines (depending on the differentiation state of the OB), it is probable that androgens act on OB cells to regulate their activity and differentiation (8). Recent studies have shown that DHT can activate an androgen response element-chloramphenicol acetyltransferase reporter construct and regulate OB cell proliferation and bone matrix protein production (59). In addition, DHT and testosterone inhibited IL-6 production and the IL-6 receptor gene promoter activity in murine bone marrow-derived stromal cells (60), as well as IL-6 production by human OB cells (61). In addition to testosterone and DHT, there is some evidence that adrenal androgens may also have significant effects on bone metabolism (60, 62). The effects of progesterone on bone metabolism are much less clear than those of E or androgens. However, PRs have been identified in primary human osteoblastic cells, human osteosarcoma cells, and immortalized fetal OB cells (for a review, see Ref. 8). Clinical studies using progesterone to prevent bone loss, however, have found variable results, with some studies showing an effect of progesterone in prevention of spinal or cortical bone loss, whereas other studies have found no significant benefit of progesterone treatment on postmenopausal bone loss (8). Further studies are required to determine the exact role of progesterone and androgens in bone biology.
CONCLUSIONS
Figure 3 summarizes the potential
pathways of the E regulation of bone resorption, as described in this
minireview. Although the ultimate action of E in the skeleton appears
to occur at the level of bone resorption due to the regulation of OCL
activity as well as differentiation of the OCL precursors, one has to
consider the critical role of the OB cells. Most of the growth
factors/cytokines that mediate E action on bone resorption as well as
OCL differentiation and activity are produced by OB cells and are under
E regulation. Therefore, any defects in the production of these
OB-derived factors or in the regulation of this production by E, would
be reflected in the overall bone resorptive activity found in
oophorectomy, postmenopause, and bone disease. This review presents
only a sampling of the rapidly developing field of sex steroids and
cytokine action on bone. Readers are encouraged to read further in the
more extensive reviews and new papers listed in the reference section.
Since osteoporosis affects millions of women and men and costs the
United States approximately 14 billion dollars a year in medical care,
investigations into new drugs, both steroidal and other, to prevent and
even reverse bone loss, will be a challenging and exciting endeavor for
scientists in the years to come. To succeed in this endeavor, the
molecular actions and interactions of the sex steroids, SERMS, and
growth factors/cytokines on the skeleton and bone diseases need to be
fully elucidated. This field will present exciting challenges for basic
scientists and clinical investigators in the years to come.
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FOOTNOTES
Address requests for reprints to: Dr. Thomas Spelsberg, Department of Biochemistry and Molecular Biology, Mayo Clinic, 1601 A Guggenheim Building, Rochester, Minnesota 55905-0001.
This study was supported by NIH Grants AG-04875 and AR-43627.
Received for publication February 25, 1999. Revision received March 18, 1999. Accepted for publication March 22, 1999.
REFERENCES