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Variability in the Level of Aromatase Activity: Effects on Bone Metabolism
AROMATASE ACTIVITY AND BONE LOSS
Genotype L/L and L/S
LS-BMD loss (Δ %/4yrs)
P < 0.05 ANOVA
FIG. 6. Rates of bone loss at 4 years in elderly men with long or short (TTTA)n repeat
genotype of the CYP19A1 aromatase gene. Subjects were grouped according to short (S; TTTA
9) and long (L; TTTA > 9) repeats number.
underpowered, more recent meta-analyses and large-scale studies generally
evidenced an association between polymorphisms in the CYP19A1 gene and
circulating estradiol levels or osteoporotic risk in both genders as well as with
breast cancer risk in females [144–148]. Thus, it is possible that the presence
of particular CYP19A1 variants could be responsible of higher aromatase
activity and increased estrogen production. If so, these polymorphisms
should be protective for bone loss in elderly men or postmenopausal
women while potentially also increasing the risk of estrogen-related cancer.
Interestingly, these associations appear to be modulated by fat mass,
particularly in men. Differences between CYP19A1 genotypes were greater
in male subjects with a normal BMI, while the association progressively
decreased in magnitude when overweight and obese men were analyzed
 (Fig. 7A and B). This point suggests that fat mass may be a mitigating
factor in the expression of CYP19A1 genotypes on bone. It is possible that
with more adipose tissue, the associated overall increase in adipose aromatase activity dominates any effect of the polymorphisms on intrinsic aromatase activity. This effect could be less important in postmenopausal females,
where the amount of circulating androgen precursors for aromatization into
estrogen is low. Moreover, the observed interaction between CYP19A1
polymorphism and BMI is also consistent with the hypothesis of a threshold
estradiol level for skeletal sufficiency [1–4]. In fact, either the presence
of more efficient allelic variants or the abundance of adipose tissue
(with increased source for aromatization) could each operate in a similar
manner to maintain sufficient amounts of circulating estradiol to prevent
GENNARI ET AL.
Lumbar BMD (g/cm2)
Normal weight (BMI ≤ 25)
Overweight (BMI > 25)
P < 0.05 ANOVA
FIG. 7. Lumbar BMD values according to CYP19A1 (TTTA)n repeat genotype in subjects
divided by BMI in (A) normal (BMI 25) and (B) overweight or obese groups (BMI > 25).
Subjects were grouped according to short (S; TTTA 9) and long (L; TTTA > 9) repeats
number (adapted from Ref. ).
Given the importance of estrogen in bone accrual, it is likely that deleterious CYP19A1 polymorphisms exert even a greater role in young individuals.
Despite an early study in 140 middle-aged Finnish men that revealed an
association between the number of TTTA repeat sequences and height and
BMI but not with BMD , a larger analysis confirmed that CYP19A1
polymorphisms significantly affect the attainment of peak bone mass .
In a well-characterized cohort of 1068 men at the age of peak bone mass
(18.9 Ỉ 0.6 years), both the TTTA repeat variation and a silent G/A polymorphism at Val80 of the CYP19A1 gene were predictors of areal BMD of
the radius, lumbar spine, total body, and cortical bone size (cortical crosssectional area and thickness) of both the radius and tibia.
To date, the molecular mechanisms through which the different CYP19A1
variants affect aromatase activity and bone metabolism remain in great part
unknown. In a preliminary study in elderly men, higher in vitro aromatase
efficiency and greater estrogen production were observed in fibroblasts from
subjects with a high TTTA repeat sequence genotype in comparison to
fibroblasts from a low TTTA repeat sequence genotype . However,
due to its location in intron 4 of the CYP19A1 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 gene or with a nearby gene. Indeed, a different 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 . In that study, the T allele was associated
AROMATASE ACTIVITY AND BONE LOSS
with a higher number of TTTA repeat sequences and showed a high activity
phenotype, with increased aromatase activity, increased aromatase mRNA
levels, and with a switch in promoter usage from adipose tissue promoter to
the more active ovary promoter. Different studies also evidenced a functional
role of other polymorphisms located within the complex promoter region of
the CYP19A1 gene. In particular, a C/G polymorphisms in promoter I.2
(rs1062033) was shown to influence gene transcription by interacting with
CEBPb , a transcription factor acting either as a stimulator or inhibitor
of aromatase expression in different tissues [152,153]. In fact, in experiments
of transient transfections of osteoblastic cell lines with luciferase reporters,
inserts with the rs1062033 region stimulated the expression of the reporter
gene, in an allele-specific way. The expression of the reporter gene was
significantly higher in constructs bearing the G allele (which was also associated with higher BMD in population studies) than in those with the C allele.
In the same model, cotransfecting with CEBPb increased luciferase expression by the aromatase constructs, especially in those with the G allele.
Furthermore, evidence for differential allelic expression was found in bone
tissue samples, again indicating the G allele as the more overexpressed.
Although these studies, in the aggregate, provide data to argue for the
importance of polymorphisms in CYP19A1 as determinants of estrogen
production and bone strength, larger and more definitive studies are needed
before any firm conclusions can be drawn.
Finally, recent evidence also suggested that CpG methylation represents
an important epigenetic mechanism for regulating CYP19A1 expression and
that different methylation patterns may be responsible for the observed
interindividual variation in promoter-driven expression of aromatase, at
least in skin fibroblasts . In fact, unmethylated constructs showed consistently higher promoter activity than methylated constructs.
8.2. ACQUIRED VARIATION IN AROMATASE ACTIVITY
Besides genetic or epigenetic considerations, several additional mechanisms have been proposed in 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 PPARg receptor (i.e., troglitazone) could
also lead to downregulation of aromatase gene and a reduction in aromatase
activity. Of course, if there are more adipocytes, there could be more aromatase activity even with reduced production of estrogen per fat cell. In vitro
studies support this hypothesis [155–157]. Similarly, phthalates, ubiquitous
GENNARI ET AL.
environmental toxins found in plasticizers, have been reported to activate the
PPARg and PPARa pathways and to decrease aromatase activity, mRNA
and protein levels in ovarian granulosa cells . The clinical relevance of
these environmental modulators on global aromatase activity and estrogen
production in man remains unknown. Of interest, the activation of PPARa
pathway by fenofibrate in female mice significantly reduced aromatase
mRNA and activity, resulting in decreased femoral BMD and uterine size
. Several other contaminants may affect aromatase activity and estrogen
production. In particular, glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines that are widely used across the world.
Their residues are frequent pollutants in the environment and are spread on
most eaten transgenic plants, modified to tolerate high levels of these compounds in their cells. While up to 400 ppm of their residues are accepted in
some feed, recent experimental studies demonstrated that aromatase transcription and activity were disrupted with subagricultural doses and with
residues from 10 ppm .
In addition, cycloxygenase (COX) inhibitors, by reducing PGE2 production, may inhibit aromatase activity, at least in breast cancer cells, and in
some studies showed strong chemopreventive activity against mammary
carcinogenesis [161,162]. However, PGE2 appears also involved in the regulation of bone turnover , and its inhibition by the combination of
relative COX-2 selective nonsteroidal anti-inflammatory drugs and aspirin
was associated with high and not low BMD at multiple skeletal sites both in
men and women .
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 . It has
been estimated that daily consumption of 125 ml of red wine would provide
adequate amounts of procyanidin B dimers to suppress in situ aromatase in
an average postmenopausal woman. Similarly, myosmine, a minor tobacco
alkaloid widely occurring in food products of plant and animal origin,
inhibits the conversion of testosterone to estradiol by human aromatase
with potential implications for sex hormone homoeostasis .
Another important and well-recognized modulator of aromatase efficiency
in bone cells is vitamin D that has been shown to stimulate glucocorticoidinduced aromatase activity in cultured osteoblasts . The magnitude of
this effect varies largely among individuals, depending on the level of vitamin
D receptor . Of interest, vitamin D receptor knock-out mice showed
reduced aromatase activity with respect to WT animals .
Finally, aromatase efficiency may be influenced by pathological conditions. It is known that increased androgen aromatization can be caused by
hepatocellular carcinoma , adrenocortical tumors , and testicular
AROMATASE ACTIVITY AND BONE LOSS
tumors [172,173]. In these neoplastic conditions, inappropriate amounts of
aromatase enzyme are expressed and estrogen levels are increased. Elevated
plasma estradiol concentrations have also been described in men with liver
cirrhosis together with decreased plasma testosterone [173,174]. 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 a
preliminary study on elderly men, significant differences in estradiol levels
in relation to Helicobacter pylori infection were observed, independently from
circulating testosterone levels . Levels of estradiol in infected CagApositive patients were significantly lower than in infected CagA-negative
patients and this variation was associated with differences in bone turnover.
The mechanism underlying this association is unknown and deserves further
investigations. Indeed, aromatase activity and production of estradiol were
recently demonstrated in gastric parietal cells . Recent observations also
suggested that diabetes negatively affects expression levels of aromatase both
in ovary and testis [177,178]. However, the effects of this condition on major
extragonadal sites of aromatase activity including bone remains to be determined. Moreover, experimental studies also evidenced that metformin, an
oral antidiabetic agent, inhibits aromatase expression in both granulosa luteal
cells and breast adipose cells while insulin stimulates aromatase mRNA
expression in different cell lines [179,180]. Since a recent study evidenced
that metformin-induced inhibition of aromatase expression occurs via downregulation of promoter II, I.3, and 1.4 , its potential negative effects on
estrogen production and skeletal health should be investigated.
9. Summary and Conclusions
Aromatase, the enzyme responsible for the transformation of androgens
into estrogens, has a complex, tissue-specific regulation. The regulation of the
level and activity of this enzyme determines the concentrations of estrogens
that have endocrine, paracrine, and autocrine effects on several tissues including bone. Importantly, extraglandular aromatization of circulating androgen
precursors is the major source of estrogen not only in men but also in women
after the menopause. Several lines of clinical and experimental evidence now
clearly indicate that aromatase activity and estrogen production are necessary
for longitudinal bone growth, attainment of peak bone mass, the pubertal
growth spurt, epiphyseal closure, and normal bone remodeling in young
individuals. Moreover, with aging, individual differences in aromatase activity may significantly affect bone loss and fracture risk in both genders.
GENNARI ET AL.
Further studies are needed to better understand the role of glandular
versus peripheral aromatization, to clarify the androgen contribution on
bone homeostasis, and to identify how genetic, environmental, pathologic,
and pharmacological influences might modulate aromatase activity, increasing or reducing estrogen production in ageing individuals, and thereby
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