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The Journal of Clinical Endocrinology & Metabolism 89(12):5898 –5907
Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2004-1717
MINIREVIEW
Aromatase Activity and Bone Homeostasis in Men
LUIGI GENNARI, RANUCCIO NUTI,
AND
JOHN P. BILEZIKIAN
Department of Internal Medicine, Endocrine-Metabolic Sciences, and Biochemistry, University of Siena (L.G., R.N.), Siena
53100, Italy; and Department of Medicine, Columbia University College of Physicians and Surgeons (J.P.B.), New York,
New York 10032
It is known that sex steroid hormones play an important role
in the maintenance of bone mass in males as well as in females.
Even though androgens are the major sex steroids in men,
their primacy in regulating male skeletal remodeling has been
increasingly questioned as direct and indirect evidence
emerged suggesting that estrogens may also play a major role
in male skeletal health. Recent data suggested that a threshold level of bioavailable estradiol is needed to prevent bone
loss, and that with aging an increasing percentage of elderly
men begin to fall below this level. The testes account for, at
most, 15% of circulating estrogens in the male; the remaining
85% comes from peripheral aromatization of androgen precursors in different tissues, including bone. Human models of
aromatase deficiency were the first to demonstrate the critical importance of the conversion of circulating androgens
into estrogen in regulating male skeletal homeostasis. All four
cases of aromatase-deficient men reported to date showed an
identical skeletal phenotype, characterized by tall stature due
to continued longitudinal growth, unfused epiphyses, high
bone turnover, and osteopenia. Studies using knockout mice
S
EX STEROIDS ARE important for the acquisition and
maintenance of bone mass in both sexes. Alterations in
their levels can become relevant in the pathogenesis of osteoporosis, either because their deficiency (or resistance)
leads to suboptimal acquisition of peak bone mass or because
deficits in adulthood can directly lead to bone loss. Although
estrogens have been shown to be critically important in these
respects for the female skeleton, the role of estrogen in male
skeletal health has only recently become appreciated. This is
due in part to attributions of steroid specificity as a function
of sex: estrogens for women and androgens for men. The
assumption is rational, especially because androgens are the
major circulating class of sex steroids in men. Moreover,
alterations in androgen levels in the growing male skeleton
or in the context of the aging male skeleton have been associated with osteoporosis in men. Although androgen undoubtedly plays a major role in male skeletal health, its
primacy has been increasingly questioned as direct and indirect evidence has emerged suggesting that estrogens may
also play a major role. Application of knowledge that anAbbreviations: AIS, Androgen insensitivity syndrome; ArKO, aromatase knockout mouse; BMD, bone mineral density; KO, knockout;
PPAR, peroxisomal proliferator-activated receptor; WT, wild type.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
along with experimental observations in rats treated with an
aromatase inhibitor provided useful information about the
importance of aromatase in the male skeleton. Confirmatory
evidence comes from recent interventional studies in adult
men using aromatase inhibition, which confirmed that estrogens are critically important to the male skeleton by helping
to control rates of bone remodeling. Intriguingly, common
polymorphisms at the human aromatase (CYP19) gene have
been associated with differences in aromatase activity, bone
turnover, and rates of bone loss in elderly men, suggesting
that variations in aromatase efficiency may also be relevant
for skeletal homeostasis. Several additional mechanisms have
been proposed in which aromatase activity could be modulated under certain circumstances in different tissues. Additional studies are needed to identify how these genetic, environmental, pathological, and pharmacological influences
might modulate aromatase activity in vivo, increasing or reducing estrogen production in males and thereby affecting
skeletal health. (J Clin Endocrinol Metab 89: 5898 –5907, 2004)
drogens are metabolized to estrogens in men as well as
women via the aromatase enzyme system was a first step in
appreciating a potential role for estrogens in men. The subsequent demonstration in humans and animals that alterations in estrogen production or responsiveness in men are
associated with adverse skeletal affects provided additional
evidence that estrogens are a critically important factor for
male skeletal development and homeostasis (1–5). This review summarizes the evidence that aromatase activity plays
an important role in the male skeleton health.
Aromatase, the Aromatase Gene, and Estrogen
Production in Males
Aromatase is a specific component of the cytochrome P450
enzyme system that catalyzes three consecutive hydroxylation reactions converting both adrenal and testicular C19
androgen precursors into C18 estrogenic steroids. This reaction converts the ⌬4 –3-one A ring of androgens into the corresponding phenolic A ring characteristic of estrogenic compounds. The testes account for, at most, 15% of the circulating
estrogens in the male; the remaining 85% come from peripheral aromatization of circulating androgen precursors in different tissues, including fat, brain, skin, endothelium, and
bone (6). Testicular androgen precursors contribute more to
the total amount of circulating estradiol than adrenal androgens (7), because dexamethasone-induced suppression of
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Gennari et al. • Minireview
J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
adrenal steroid synthesis decreases estradiol levels only
moderately (8), whereas orchidectomy leads to a more dramatic suppression of plasma estradiol concentrations (9, 10).
Importantly, extragonadal sites of estrogen biosynthesis, unlike ovaries and testes, lack the ability to synthesize C19
precursors from cholesterol; hence, their estrogen-producing
activity depends solely on the availability of these circulating
C19 steroids (11–13). Apart from contributing to the circulating estrogen pool, the estrogen synthesized within extragonadal tissue compartments may be locally active in a
paracrine or intracrine fashion (6, 13, 14). Thus, although the
total amount of estrogen synthesized at any site could be
small, local tissue concentrations, in contrast, could be substantial. In human bone, aromatase has been reported to be
expressed in osteoblast or osteoblast-like cells from fetal and
normal tissues (15–18), in articular cartilage chondrocytes, in
adipocytes adjacent to bone trabeculae, and in osteocytes, but
not in osteoclasts (17). Both aromatase activity and amounts
of aromatase-specific mRNA in bone have been shown to
vary widely among subjects in some studies (17, 18).
Aromatase is encoded by the CYP19 gene located at chromosome 15q21.2 (19). Cloning of this gene has demonstrated
several unusual features compared with other genes of the
P450 enzyme system. In a tissue-specific fashion, a number
of untranslated initial exons are found in aromatase transcripts due to differential splicing by multiple tissue-specific
promoters (13, 20 –29). Only the 30-kb 3⬘ region of the gene
encodes aromatase, whereas a larger 93-kb 5⬘-flanking region
serves as the regulatory unit of the gene (13, 24, 25). Exon I
is not translated, but different splicing patterns lead to transcripts that are all translated as the same protein. To date, at
least nine tissue-specific promoters defining their respective
first exons have been reported in the human aromatase gene.
Their use in different tissues is summarized in Table 1. In
ovary and testes, aromatase expression is mediated by a
proximal promoter, PII; in the placenta, it is mediated by a
powerful distal promoter, I.1; and in mesenchymal cells of
adipose tissue and skin, aromatase expression is regulated by
another distal promoter, I.4, which is located between promoters I.1 and PII (6, 13, 24). In bone cells, the major promoter
is promoter I.4. Promoters I.3 and I.6 are expressed to a lesser
extent in bone (26). Some minor transcripts of promoter PII
and I.1 have been also described (26). Importantly, the various tissue-specific aromatase promoters are influenced by
different hormonal classes that, in turn, use different signal-
5899
ing pathways (Fig. 1). Thus, the overall control of aromatase
activity between and within different tissues is a complex
interplay of factors acting via the expression of different
promoters. This complex control system can be important in
pathological conditions, such as breast cancer. Although in
normal breast tissue, aromatase expression is stimulated primarily by class I cytokines through promoter I.4, local estrogen production in breast cancer adipose tissue can be
increased due to overexpression of promoter II-specific transcripts by prostaglandin E2 produced by the breast cancer
itself (13, 27, 28). The endothelial-type promoter I.7 also appears to be up-regulated in breast cancer (29).
Aromatase Deficiency and the Male Skeleton
Apart from indirect evidence showing that estrogen plays
a dominant role in the regulation of bone growth and mineralization in men (1–3, 30 –35) (Table 2), several clinical and
experimental studies recently underscored the importance of
aromatization of androgens into estrogen for male skeletal
homeostasis.
FIG. 1. Aromatase gene (CYP19) promoters and untranslated first
exons. The various tissue-specific promoters employ different signaling pathways (11–29). Exon 2a sequence, originally found as an extra
exon, has not been characterized as an independent promoter.
TABLE 1. Tissue-specific promoters of the aromatase CYP19 gene and their utilization in different tissues
Adipose tissue
Bone
Brain
Fetal intestine
Fetal liver
Fetal lung
Muscle
Ovary
Placenta
Prostate
Skin fibroblasts
Testis
Endothelium
I.1
I.4
I.5
I.7
1f
I.2
I.6
I.3
PII
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(⫹)
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J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
Gennari et al. • Minireview
TABLE 2. Indirect evidence illustrating the importance of
aromatase in bone metabolism in men
Model
Summary results
Mutation in the ER␣
gene in a man
Defect in skeletal growth and
mineralization
Aromatase-deficient
men
Continuous growth, unfused epiphyses,
no growth spurt, osteopenia.
Estrogen, but not testosterone,
restores bone mass and arrests
longitudinal growth
ERKO, DERKO; male
mice
Osteopenia and impaired skeletal
growth
Cross-sectional studies
in males
Better correlation of BMD with
estradiol than testosterone
Longitudinal studies
in males
Correlation of bone loss with estradiol
levels, but not testosterone
Androgen
supplementation in
eugonadal men with
osteoporosis
Change in BMD positively correlates
with change in estradiol, but not
with testosterone levels
Raloxifene in men
Acts as an agonist when estrogen
levels are low; as an antagonist
otherwise
ER␣, Estrogen receptor ␣; ERKO, estrogen receptor ␣ KO; DERKO,
double estrogen receptor ␣/␤ KO.
Aromatase deficiency in men
Human models of aromatase deficiency preceded the construction of aromatase knockout (ArKO) animals and, in fact,
provided the first seminal insights into the role of estrogens
and aromatase in male skeletal physiology. To date, four
cases of complete aromatase deficiency in young males have
been reported. The skeletal phenotypes of men described in
these four reports are virtually identical. Common characteristics include tall stature due to continued longitudinal
growth, unfused epiphyses, delayed bone age, lack of pubertal growth spurt, eunuchoid skeletal proportions, genu
valgum, elevated markers of bone remodeling, and severe
osteopenia (36 – 40). Testosterone administration to some of
these subjects was of no benefit, an understandable observation because aromatase deficiency is not associated with
low testosterone levels. In fact, in two of the four reports (36,
39) testosterone levels were markedly elevated. In contrast,
estrogen treatment in all cases was associated with marked
improvements (Fig. 2). Epiphyses closed quickly, and longitudinal growth ceased. Bone mass increased dramatically
(37– 40); in two cases, it was related to dose (40, 41).
Aromatase deficiency in young men also suggests a key
role of estrogen in pubertal skeletal acquisition and the pubertal growth spurt, although uncertainties still remain because detailed data are not available, and growth curves are
incomplete. In this regard, the recent discovery of a novel
aromatase mutation in a male infant with no apparent skeletal abnormalities at birth will improve our knowledge about
this issue (42). The use of estrogen in adult men with aromatase deficiency and open epiphyses indicate that estrogen
in the growing skeleton may serve as an anabolic agent, in
FIG. 2. Change in lumbar spine (LS), femoral neck (FN), and radial
(RD) BMD with estrogen therapy in a man with aromatase deficiency
(adapted with permission from Ref. 107).
contrast to the way in which estrogen is regarded in the adult
female skeleton, i.e. as an antiresorptive agent.
There is a counterpart to the knockout of the human aromatase gene, namely the syndrome in which the aromatase
gene is overexpressed (43). The observations made with aromatase overexpression are opposite those made in the setting
of aromatase deficiency. The characteristic findings in aromatase overexpression are accelerated growth, advanced
bone age, and short final stature, all consistent with the
precocious presence of estrogen in an immature male skeleton. Other common characteristics of this syndrome include
prepubertal gynecomastia and mild hypogonadotropic hypogonadism. Despite increased estrogen levels and testosterone levels that are low or in the lower end of the normal
range, males with aromatase excess appear to be fertile. In
some cases, short-term treatment with an aromatase inhibitor
(testolactone or anastrozole) reduced serum estrogen levels,
restored gonadotropins and testosterone to normal levels,
and arrested additional skeletal aging (43– 45). Although at
least five families, with 13 affected male subjects, and two
sporadic cases of aromatase excess have been described, the
effects of estrogen excess on bone turnover and bone density
in men are still not known. The only available data in this
respect relate to bone density, which has been described to
be in the upper limits of the normal range in a male with
familial hyperestrogenism. Considering the additional point
that the man had hypogonadism, this is a remarkable observation (45).
Another counterpart of the estrogen-deficient male is the
androgen insensitivity syndrome (AIS). These subjects
present with different degrees of androgen resistance due to
mutations in the androgen receptor gene but their estrogen
levels and sensitivity are normal. Indeed, most patients with
AIS show high serum concentrations of both testosterone and
estradiol. Impaired to complete lack of response to endogenous or exogenous androgens does not delay epiphyseal
closure, but some anthropometric features, such as height
and bone size, appeared intermediate between typical male
and female patterns (46). Moreover, in the largest AIS sample
collected to date (22 complete and six high grade partial AIS),
moderately reduced bone mineral density (BMD) values
were described, with the patients who have complete AIS
showing lower BMD than those with partial defects (47).
However, the reduced bone mass in most of these subjects
may be related in part to inadequate treatment or compliance
Gennari et al. • Minireview
with estrogen replacement therapy after orchidectomy. Of
note, in the same study, AIS subjects who had undergone
gonadectomy in infancy and had not yet received estrogen
replacement showed a rather low lumbar BMD. In contrast,
normal BMD was reported for two subjects who had not yet
undergone gonadectomy and showed elevated estrogen levels. Even though an additive effect of androgen in maintaining bone mass is likely, as suggested from the AIS model and
other recent experimental observations (48), it is also clear
from observations in individuals lacking the capacity to synthesize or respond to estrogen that estrogen represents an
important hormonal component for skeletal homeostasis in
the male.
Animal models
Studies with knockout (KO) mice along with experimental
observations in rats treated with an aromatase inhibitor have
provided useful information on the importance of aromatase
in the male skeleton. In aged male rats, inhibition of estrogen
production by inhibition of androgen aromatization by the
nonsteroidal aromatase inhibitor, vorozole, increased bone
resorption and bone loss to an extent similar to that after
orchidectomy (49, 50). In these aged rats, bone turnover and
bone density were equally affected by orchidectomy, in
which both androgen and estrogen levels fell, or by aromatase inhibition, in which only estrogen levels were reduced (51). Importantly, in the same study, vorozole treatment did not significantly affect cortical thickness, in contrast
to orchidectomy, suggesting a direct role of androgens in this
geometric property of bone (51).
The skeletal consequences of aromatase deficiency are also
illustrated by studies of the ArKO model (52–54). Aromatase
gene inactivation in the ArKO mouse model is not lethal.
Furthermore, the newborn skeleton of ArKO animals demonstrated no consistent differences in bone and mineralized
cartilage from wild-type (WT) littermates (55). In contrast,
bone growth was significantly affected by aromatase inactivation to an extent similar to what was observed in animal
models of estrogen resistance (56). In a recent study, femur
length in ArKO mice was followed from the time of weaning
to 7 months of age (55). There was no significant difference
in femur length between WT and ArKO females, whereas
ArKO male mice showed reduced femur length growth with
an absence of the accelerated growth during puberty compared with WT (55). Moreover, radiological and densitometric analyses showed osteopenia in both male and female
ArKO animals (55, 56). In studies from a different group of
mice in which the aromatase gene was knocked out, 9-wk-old
male ArKO mice showed reduced BMD at the femoral neck
with respect to WT littermates. Estradiol replacement permitted these ArKO mice to achieve the same femoral neck
BMD as WT littermates (57). In the same study, older (32wk-old) ArKO animals showed reduced BMD and microcomputed tomographic parameters with respect to WT mice,
particularly at trabecular sites (57). Histologically, KO animals of both sexes showed an osteoporotic phenotype, characterized by significant reductions in trabecular bone volume, trabecular thickness, and cortical thickness (56).
Analysis of the effects of aromatase inactivation on bone
J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
5901
remodeling indexes in male ArKO mice show conflicting
results. In the first study on static and histomorphometric
parameters of the spine, adult ArKO males showed a low
turnover pattern, with significant reductions in osteoblastic,
osteoid, and eroded surfaces and with reduced mineralizing
surface, as observed by tetracycline uptake compared with
that in WT littermates (56). In contrast, adult ArKO females
showed a high turnover pattern, suggesting sexual dimorphism in bone remodeling (56). These observations were
confirmed in a subsequent study using fluorine-18 positron
emission tomography imaging of WT and ArKO males (55).
By this technique, ArKO males clearly demonstrated reduced vertebral remodeling compared with WT littermates,
and estradiol treatment in these mice increased fluorine-18
uptake to normal levels. Sexual dimorphism in bone remodeling in ArKO animals was not appreciated in another ArKO
mouse model, in which increased bone resorption was observed in both male and female animals (57). Treatment with
estrogen restored the increased parameters of bone remodeling to the WT level in both sexes (57). Although it is not
clear how to account for the differences in these observations,
what is apparent is that aromatase deficiency profoundly
influences processes associated with bone turnover.
Interruption of estrogen production in men by inhibition of
aromatase activity: clinical studies in men
Recently, three studies have reported short-term skeletal
effects of aromatase inhibition by pharmacological means in
adult men (58 – 60). Falahati-Nini et al. (58) examined the
differential effects of estrogen vs. testosterone replacement in
a group of men (mean age, 68 yr) rendered temporarily
hypogonadal by the use of a GnRH agonist (leuprolide acetate, Lupron, Takeda Chemical Industries, Osaka, Japan).
Selective replacement of testosterone, estrogen, or both in the
presence of the aromatase inhibitor, letrozole, permitted a
specific sex steroid assignment to the bone dynamics that
were observed (58). The increase in bone resorption markers
after induction of the hypogonadal state was almost completely prevented by estradiol, but not by testosterone therapy alone, indicating that the increase in bone resorption was
due primarily to estrogen loss, not testosterone loss. In the
case of bone formation indexes, there was evidence for independent effects of estrogen and testosterone. In a similar
study in younger individuals, Leder et al. (59) confirmed the
increase in bone resorption markers after induction of the
hypogonadal state by the GnRH agonist, goserelin acetate. In
this model, evidence was provided for independent effects of
testosterone and estrogen on bone resorption. Moreover, in
that study, bone formation markers appeared to be dependent upon both androgens and estrogens. These observations are in keeping with the increase in bone formation
markers described in aromatase-deficient or gonadectomized men (37– 40, 61). Finally, in a similar study by Taxel
et al. (60), treatment of elderly men with an aromatase inhibitor for 9 wk resulted in significant increases in bone
resorption and decreases in bone formation. Taken together,
all three studies using aromatase inhibition confirm that
estrogens are critically important to the male skeleton by
helping to control rates of bone remodeling. These results are
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J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
FIG. 3. Estradiol to testosterone ratio in osteoporotic and nonosteoporotic elderly men (adapted with permission from Ref. 62).
also compatible with concepts of accelerated bone remodeling in the female postmenopausal skeleton rendered deficient in estrogen.
In agreement with results from these short-term interventional studies in adult males, in a recent study of elderly men,
the ratio between estradiol and testosterone, plausibly an
indirect measure of aromatase activity, was significantly
lower in osteoporotic than in nonosteoporotic subjects (Fig.
3), suggesting an essential role of aromatase activity in maintaining bone mass in the aging male (62).
Not surprisingly, a similar approach to boys with constitutional delay of puberty has given different results. Suppression of estrogen production by aromatase inhibitor,
letrozole, for 1 yr during testosterone treatment did not negatively affect bone mineral content, BMD, or apparent BMD,
an estimate of true volumetric BMD, with respect to testosterone treatment alone (63). In testosterone alone and testosterone plus letrozole groups, bone mass parameters increased with respect to baseline values and those in the
untreated group. However, the increase in apparent BMD in
the testosterone plus letrozole group was statistically significant only at 18 months, 6 months after discontinuation of
letrozole treatment. Moreover, a decrease in BMD was observed in four letrozole-treated boys, but in only one of the
boys treated with testosterone alone. These findings suggest
that 1-yr treatment with an aromatase inhibitor in pubertal
boys is unlikely to have any major harmful effect on BMD
and the attainment of peak bone mass, but such short-term
studies do not diminish the clear effects of long-term estrogen deficiency (i.e. aromatase deficiency) on the developing
male skeleton.
Threshold estradiol hypothesis for skeletal sufficiency in
the male
Data from clinical and experimental studies of aromatase
deficiency support the hypothesis of a threshold estradiol
level for skeletal sufficiency in the male (1, 2). In particular,
a threshold estradiol treatment dosage was evident in men
with aromatase deficiency, with estradiol patch doses less
than 25 ␮g/d (corresponding to achieved serum estradiol
levels ⬍88 pmol/liter) being ineffective in preventing bone
loss (40, 41). Indeed, in a recent cross-sectional analysis, men
Gennari et al. • Minireview
in the lowest quartile for bioavailable estradiol level showed
significantly lower BMD at multiple sites compared with
men in the upper three quartiles (64). Moreover, in two recent
longitudinal studies in elderly men, rates of bone loss at
different skeletal sites were unrelated to serum estradiol
levels when the latter were above the median value, but they
were clearly associated with estradiol levels when these levels were below this value (62, 65). The threshold estradiol
hypothesis gains additional support from a recent study in
which raloxifene was given to men with varying estradiol
levels (66). Subjects with serum estradiol levels below 96
pmol/liter responded to raloxifene with a decrease in bone
resorption markers. In this group, raloxifene was serving as
an agonist. Above this estrogen value, raloxifene caused an
increase in bone resorption markers. In this group with
higher estrogen levels, raloxifene was acting as an estrogen
antagonist. The data argue that men need a sufficient concentration of estrogen, defined as a threshold value, for normal skeletal remodeling. In all of these studies, the required
concentration of bioavailable estradiol appears to be remarkably similar, ranging from 40 –55 pmol/liter. This apparent
threshold value is higher than typical estradiol concentrations for postmenopausal women who are not receiving exogenous estrogens. In contrast, premenopausal women and
young men are typically above this apparent threshold level.
Because about 50% of middle-aged men fall below this estradiol threshold, it could be a determinant in age-related
bone loss (1, 2, 62, 64, 65, 67– 69).
Because only a small fraction of circulating estradiol is
derived directly from the testes, it is likely that peripheral
aromatization of testicular and adrenal androgen precursors
into estrogen exert a key role in maintaining estradiol levels
above the threshold with ageing. A major unresolved issue
is which tissue site of aromatase activity is the most important in terms of bone metabolism in men. Moreover, is paracrine/intracrine estradiol or systemic estradiol the most important for bone physiology? The consistent association
among circulating estradiol levels, BMD, and fracture risk
reported in various cross-sectional and longitudinal studies
(62, 64, 65, 67– 69) suggests that although local aromatization
in bone may contribute significantly to skeletal homeostasis
(13, 70), a minimum circulating level of estradiol (derived
from the amount of peripheral produced estrogen that is
released into the circulation) is necessary to prevent bone loss
in elderly men. However, it is possible that circulating estrogen levels reflect estrogen status within bone, due to local
aromatase activity, and that locally produced estradiol exerts
an even greater impact on bone physiology than plasma
estradiol. This important issue needs more investigation.
Influence of Variability in Aromatase Activity Level
on Male Skeleton
The experiments of nature or of clinical research, in which
aromatase activity is absent or inhibited, have shown clearly
that estrogens are important factors in male skeletal health.
Although these models have established this important point
in the context of complete estrogen deficiency, they do raise
the possibility that significant differences in estrogen levels
might be present among males (17, 18), due to variability in
Gennari et al. • Minireview
aromatase activity, and that this variability may be important
for skeletal homeostasis. Such differences in aromatase activity, and hence estrogen levels, among men might become
particularly evident in elderly males, in whom age-related
declines in testicular and adrenal androgen precursors are
common. This aspect could become even more relevant in
postmenopausal women, in whom the availability of androgen precursors for aromatization to estrogen is much lower
than in men. In fact, testosterone levels are at least 1 order of
magnitude greater in men than in postmenopausal women,
whereas adrenal androgen levels are similar (13, 71).
Inherited variation in aromatase efficiency
Several polymorphic regions (including 74 single nucleotide polymorphisms) have been detected in the human
CYP19 gene that could be responsible for qualitative and/or
quantitative differences in gene expression of aromatase activity. The most widely studied include a silent polymorphism (G3 A at Val80) in exon 3, a tetranucleotide (TTTA)n
tandem repeat polymorphism in intron 4, an Arg264Cys
(C3 T) substitution in exon 7, and a single nucleotide change
(C3 T) in exon 10. In particular, the tetranucleotide repeat
polymorphism of the CYP19 gene has been shown to be
related to breast cancer and osteoporotic risk in postmenopausal women (72–75). Despite inconsistent associations
with breast cancer risk (76), it is possible that the presence of
longer TTTA repeats could be responsible for higher aromatase activity and increased estrogen production. If so,
these polymorphisms should be protective for bone loss in
postmenopausal women while potentially also increasing
the risk of breast cancer. Intriguingly, an association between
circulating estradiol levels and polymorphisms in the CYP19
gene has been recently proposed in postmenopausal women
(77). To date, three studies have examined the role of the
(TTTA)n repeat polymorphism on bone metabolism in elderly males (78 – 80). In the study by Remes et al. (78), the
number of TTTA repeat sequences in 140 middle-aged Finnish men was significantly associated with height and body
mass index, but not with femoral or lumbar BMD values. In
contrast, Van Pottelbergh et al. (79), in a study of communitydwelling elderly men from Belgium, showed that the same
CYP19 polymorphism was significantly associated with
BMD changes and with self-reported clinical fracture risk.
Subjects homozygous for the shortest observed length of
FIG. 4. Sex hormone levels according to CYP19 genotype in
Italian elderly men. Subjects were grouped according to
different TTTA repeat lengths: homozygous genotype 1-1
with two TTTA alleles with more than nine repeats, homozygous genotype 2-2 with two TTTA alleles with fewer
than nine repeats, and heterozygous genotype 1-2 with one
TTTA allele with fewer than nine repeats and one allele
with more than nine repeats (adapted with permission from
Ref. 80).
J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
5903
TTTA repeat sequences, the (TTTA)7 allele, showed increased rates of bone loss over a 4-yr period at the distal
forearm with respect to subjects with the other, longer TTTA
repeat sequences. Moreover, the (TTTA)7 polymorphism was
represented more frequently in elderly men with a history of
fractures as well as among men whose first degree relatives
reported fractures. These results were confirmed and extended by a recent longitudinal study of elderly Italian men
(80). A significant correlation between the number of TTTA
repeats and baseline BMD, circulating sex steroid hormones,
and bone turnover was demonstrated (80). Men with a high
number of repeat sequences (more than nine TTTA) had
higher 17␤-estradiol levels than those with a lower number
of repeats regardless of weight, sex hormone-binding globulin, or androgen levels (Fig. 4). Moreover, lower BMD values and increased bone loss were observed in subjects with
a low number of repeats compared with those with a high
number of repeats. Consistent with these clinical observations, higher in vitro aromatase efficiency and greater estrogen production were observed in fibroblasts from a high
TTTA repeat sequence genotype than in fibroblasts from a
low TTTA repeat sequence genotype (80).
The observations correlating the (TTTA)n polymorphism
of the CYP19 gene to estrogen levels and bone density in
males appear to be dependent on fat mass. When analyses are
restricted to subjects with a normal body mass index, the
differences in BMD between CYP19 genotypes were greater,
whereas such differences progressively decreased in magnitude when overweight and obese men were considered
among these polymorphic distribution profiles (80). This
suggests that fat mass may be a mitigating factor in the
expression of CYP19 genotypes on bone. It is possible that
with more adipose tissue, the associated overall increase in
adipose aromatase dominates any effect of the polymorphism on intrinsic aromatase activity. This latter consideration helps to explain the work of Van Pottelbergh et al. (79),
in which a significant association between the CYP19 genotype and BMD was observed only after excluding subjects
below the 25th and above the 75th percentiles for total fat
mass. Additionally, the observed interaction between the
(TTTA)n repeat polymorphism and body mass index is also
consistent with the hypothesis of a threshold estradiol level
for skeletal sufficiency in the elderly male (1). In fact, the
presence of a high number of TTTA repeat alleles (more
5904
J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
efficient) and the abundance of adipose tissue (as an enhanced source of aromatization) could operate in a similar
manner to maintain sufficient amounts of circulating estradiol to prevent bone loss.
The molecular mechanisms through which different
CYP19 (TTTA)n repeat alleles affect aromatase activity and
bone metabolism are still unknown. Due to its location in
intron 4 of the CYP19 gene, it is unlikely that this polymorphism directly affects aromatase activity. It is more likely that
the different TTTA alleles are in linkage disequilibrium with
other functional variants in the CYP19 gene or with a nearby
gene. Indeed, a recent study described a strong degree of
linkage disequilibrium between the (TTTA)n repeat polymorphism and the C-T substitution in exon 10, just 19 bp
downstream of the termination site of translation (72). In that
study the T allele was associated with a higher number of
TTTA repeat sequences and showed a high activity phenotype, with increased aromatase activity, increased aromatase
mRNA levels, and a switch in promoter usage from promoter
I.4 to promoter I.3 (72). More recently, other studies have
confirmed the high degree of linkage disequilibrium between the TTTA and C/T polymorphisms in the CYP19 gene
as well as the hypothesis that the T allele may have elevated
aromatase activity (81, 82).
Although data from these studies, in the aggregate, argue
for the importance of the (TTTA)n repeat polymorphism in
the CYP19 gene as a determinant of estrogen levels and
osteoporosis risk in men, larger and more definitive studies
are needed before any firm conclusions can be drawn.
Acquired variation in aromatase efficiency
Besides genetic considerations of the CYP19 aromatase
polymorphism, several additional mechanisms have been
proposed by which aromatase activity could be modulated
under certain circumstances in different tissues. It is known,
for example, that aromatase is a marker of the undifferentiated adipose mesenchymal cell phenotype and that, on a
per cell basis, it is more highly expressed in these cells than
in mature adipocytes. Thus, factors that stimulate adipocyte
differentiation, such as ligands of the peroxisomal proliferator-activated receptor-␥ (PPAR␥) receptor (i.e. troglitazone)
could also lead to down-regulation of aromatase gene and a
reduction in aromatase activity (Fig. 5). Of course, if there are
more adipocytes, there could be more aromatase activity
even with reduced production of estrogen per fat cell. In vitro
FIG. 5. Modulation of local estrogen production by PPAR␥ in adipose
tissue.
Gennari et al. • Minireview
studies in ovarian and adipose cell lines support this hypothesis (83– 87). Similarly, phthalates, ubiquitous environmental toxins found in plasticizers, have been reported to
activate the PPAR␥ and PPAR␣ pathways and to decrease
aromatase activity and mRNA and protein levels in ovarian
granulosa cells (88). The clinical importance of these environmental modulators on global aromatase activity and estrogen production in the male is not known. Of interest,
activation of the PPAR␣ pathway by fenofibrate in female
mice significantly reduced aromatase mRNA and activity,
resulting in decreased femoral BMD and uterine size (89).
Similarly, cyclooxygenase inhibitors, by reducing PGE2 production, may inhibit aromatase activity, at least in breast
cancer cells; in a recent study cyclooxygenase inhibitors
showed strong chemopreventative activity against mammary carcinogenesis (90 –92). However, prostaglandin E2
also appears to be involved in the regulation of bone turnover
(93), and its inhibition by the combination of relative cyclooxygenase-2-selective nonsteroidal antiinflammatory drugs
and aspirin was associated with high, not low, BMD at multiple skeletal sites in both men and women (94). A recent
study showed that phytochemicals such as procyanidin B
dimers, contained in red wine and grape seeds, inhibit aromatase activity in vitro and suppress aromatase-mediated
breast tumor formation in vivo (95). It has been estimated that
daily consumption of 125 ml red wine would provide adequate amounts of procyanidin B dimers to suppress in situ
aromatase in an average postmenopausal woman. Another
important modulator of aromatase efficiency in bone cells is
vitamin D, which has been shown to stimulate glucocorticoid-induced aromatase activity in cultured osteoblasts
through actions on promoter I.4 (96, 97). The magnitude of
this effect varies largely among individuals depending on the
level of the vitamin D receptor (98). Of interest, male vitamin
D receptor KO mice showed reduced aromatase activity
compared with wild-type animals (99).
Finally, aromatase efficiency may be influenced by pathological conditions. It is known that increased androgen aromatization can be caused by hepatocellular carcinoma (100),
adrenocortical tumors (101), and testicular tumors (100, 102).
In these neoplastic conditions, inappropriate amounts of aromatase enzyme are expressed, and estrogen levels are increased. Elevated plasma estradiol levels also have been described in men with liver cirrhosis together with decreased
plasma testosterone levels (103, 104). In these patients, the
metabolic clearance rate of estrogens seems to be unaltered,
suggesting that the observed hyperestrogenism could be
caused solely by an increase in androgen aromatization.
Much less is known about a possible negative influence of
pathological conditions on aromatase activity in men. In a
preliminary study of elderly men, significant differences in
estradiol levels in relation to Helicobacter pylori infection were
observed independent from circulating testosterone levels
(105). Levels of estradiol in infected cytotoxin-associated protein CagA-positive patients were significantly lower than
those in infected CagA-negative patients, and this variation
was associated with differences in bone turnover. The mechanism underlying this association is unknown and deserves
additional investigation. Indeed, aromatase activity and pro-
Gennari et al. • Minireview
J Clin Endocrinol Metab, December 2004, 89(12):5898 –5907
duction of estradiol were recently demonstrated in gastric
parietal cells (106).
15.
Summary and Conclusions
Extraglandular aromatization of circulating androgen precursors is the major source of estrogen in men. Several lines
of clinical and experimental evidence now clearly indicate
that estrogens in men are necessary for longitudinal bone
growth, attainment of peak bone mass, the pubertal growth
spurt, epiphyseal closure, and normal bone remodeling. In
adults, estrogens appear to be more important in maintaining male skeletal mass than androgens. With aging, individual differences in aromatase activity may help to distinguish
among men and their rates of bone loss. The concept that a
minimum circulating level of estrogen is needed to prevent
bone loss in men is supported by promising new data (1, 62).
Additional studies are needed to better understand the role
of glandular vs. peripheral aromatization, to clarify the contribution of androgens to bone homeostasis, and to identify
how genetic, environmental, pathological, and pharmacological influences might modulate aromatase activity, increasing or reducing estrogen production in males and
thereby affecting skeletal health.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Acknowledgments
Received August 27, 2004. Accepted September 28, 2004.
Address all correspondence and requests for reprints to: Dr. Luigi
Gennari, Department of Internal Medicine, Endocrine-Metabolic Sciences, and Biochemistry, University of Siena, Viale Bracci 1, 53100 Siena,
Italy. E-mail: [email protected].
This work was supported by the International Osteoporosis Foundation-Servier Young Investigator Award (to L.G.).
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