help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1572
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/6/2300    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grundberg, E.
Right arrow Articles by Brändström, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grundberg, E.
Right arrow Articles by Brändström, H.
Related Collections
Right arrow Calcium and Bone Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 6 2300-2306
Copyright © 2007 by The Endocrine Society

The Impact of Estradiol on Bone Mineral Density Is Modulated by the Specific Estrogen Receptor-{alpha} Cofactor Retinoblastoma-Interacting Zinc Finger Protein-1 Insertion/Deletion Polymorphism

Elin Grundberg, Kristina Åkesson, Andreas Kindmark, Paul Gerdhem, Anna Holmberg, Dan Mellström, Östen Ljunggren, Eric Orwoll, Hans Mallmin, Claes Ohlsson and Helena Brändström

Departments of Medical Sciences (E.G., A.K., O.L., H.B.) and Surgical Sciences (H.M.), Uppsala University Hospital, 75185 Uppsala, Sweden; Department of Orthopaedics (K.A., P.G., A.H.), Malmö University Hospital, 20502 Malmö, Sweden; Center for Bone Research at the Sahlgrenska Academy (D.M., C.O.), Department of Internal Medicine, Göteborg University, 41345 Gothenburg, Sweden; and Bone and Mineral Research Unit (E.O.), Oregon Health and Science University, Portland, Oregon 97239

Address all correspondence and requests for reprints to: Elin Grundberg, Department of Medical Sciences, Uppsala University Hospital, Ing 70, 3 tr, 75185 Uppsala, Sweden. E-mail: elin.grundberg{at}medsci.uu.se.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Estrogens regulate bone mass by binding to the estrogen receptor (ER)-{alpha} as well as ER-ß. The specific ER{alpha} cofactor retinoblastoma-interacting zinc finger protein (RIZ)-1 enhances ER{alpha} function in the presence of estrogen.

Objective: The objective of the study was to determine whether a RIZ P704 insertion (+)/deletion (–) (indel) polymorphism modulates the impact of estradiol on bone mineral density (BMD) and study the association between the polymorphism and BMD in elderly subjects.

Design: This was a population-based, prospective, and cross-sectional study, the Swedish MrOS Study, and the Malmö OPRA Study, respectively.

Setting: The study was conducted at three academic medical centers: Sahlgrenska Academy in Gothenburg, Malmö University Hospital, and Uppsala University Hospital.

Participants: In total, 4058 men and women, aged 69–81 yr, were randomly selected from population registries.

Main Outcome Measures: BMD (grams per square centimeter) was measured at femoral neck, trochanter, lumbar spine, and total body.

Results: The RIZ P704+/+ genotype was associated with low BMD in both women (femoral neck, P < 0.001; trochanter, P < 0.01; lumbar spine, P < 0.05; total body, P < 0.01) and men (lumbar spine, P < 0.05). However, the association between the polymorphism and BMD was dependent on estradiol status. The positive correlation between serum estradiol and BMD was significantly modulated by the genotype with a stronger correlation in the P704+/+ group than the P704–/– group (r = 0.19 vs. r = 0.08, P < 0.05).

Conclusions: These large-scale studies of elderly men and women indicate that the ER{alpha} cofactor RIZ gene has a prominent effect on BMD, and the P704 genotype modulates the impact of estradiol on BMD. Further studies are required to determine whether this polymorphism modulates the estrogenic response to estradiol treatment.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE GENETIC CONTRIBUTION to bone mineral density (BMD) is well established in both twin and family studies (1, 2). However, identifying the genetic variants predisposing to BMD is more challenging. Many genetic association studies on candidate genes have been carried out over the past years, but the results have often been hard to replicate and results are conflicting (3). The primary explanation is probably lack of power because most association studies on BMD are based on small sample sizes (4). Moreover, several genetic linkage studies on BMD, mainly in mice, have also been performed. However, these are difficult to interpret because quantitative trait loci (QTL) exist on almost all chromosomal regions studied, and mice QTL linkage studies cannot simply be compared with human results. Furthermore, chromosomal areas identified are very large containing dozens if not hundreds of potential candidate genes.

Notably, one QTL region has been replicated in several studies, the 1p36 locus (5, 6), which contains among many other genes, the retinoblastoma-interacting zinc finger protein (RIZ) gene. The RIZ gene was initially characterized as a tumor suppressor gene (7, 8, 9), but its role as a specific coactivator of estrogen receptor (ER)-{alpha} is now well established. RIZ forms an active transcriptional complex with the ligand, the nuclear receptor, and its DNA response element and strongly enhances the function of the receptor complex (10, 11). The finding that RIZ protein is a coactivator of ER{alpha} has prompted studies of RIZ and its effect on bone metabolism because estrogen by binding to ER{alpha} is important for the skeletal growth and the maintenance of both the female and the male skeleton (12, 13, 14).

The RIZ gene harbors a naturally occurring CCT insertion/deletion (indel) in exon 8 of a proline at codon 704 in the RIZ protein. To investigate the functional effect of the indel, we previously performed in vitro studies comparing the abilities of the RIZ P704 polymorphic variants to coactivate the ER{alpha} (15). The functional differences between the variant forms of RIZ were analyzed in a reporter gene system by using expression vectors containing either the P704– or the P704+ form of the RIZ gene. The results showed that the polymorphic variants of the P704 indel were functionally different in coactivating the receptor complex and that the P704– variant had reduced coactivating effect in the presence of estrogen in vitro. We also previously performed an association study between peak bone mass and the RIZ P704 indel in a female premenopausal population based cohort of 343 Swedish women (with relatively high serum estradiol levels) in which the P704–/– genotype was associated with low BMD (15).

To determine whether also the in vivo impact of RIZ P704 genotype is modulated by estradiol status, we here investigated the relationship among the RIZ P704 indel, and BMD in two population based cohorts of 3014 elderly men (known to have intermediate serum levels of E2), and 1044 postmenopausal women (known to have low endogenous serum levels of E2).


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The Swedish MrOS Study. The osteoporotoc fractures in men (MrOS) study is a multicenter prospective fracture epidemiology investigation involving elderly men from different sites around the world, including Sweden. The Swedish part consists of 3014 men aged 69 yr or older. Approximately 1000 participants were recruited at each of three academic medical centers: Sahlgrenska Academy in Gothenburg, Malmö University Hospital, and Uppsala University Hospital. The participants were randomly selected from the population registries and invited by mail. To be eligible for the study, the subject had to be able to walk without aids. At the clinic visit, participants completed standardized questionnaires about medical history, current medication use, lifestyle characteristics, and previous fractures. Six percent (n = 193) of the subjects reported at least one previous fracture after 50 yr of age at any of the major osteoporosis-related sites (hip, vertebrae, radius, and humerus). Height and weight were measured and blood samples for DNA analyses were collected. Informed consent was obtained for all subjects and the study was approved by the local ethics committees (Dnr Gbg M 014-01, LU 693-00, Ups 01-057) and conducted in accordance with the guidelines in the Declaration of Helsinki.

The Malmö OPRA Study. The Malmö Osteoporosis Prospective Risk Assessment study (OPRA) study consists of 1044 women, all 75 yr of age, randomly selected from the population registry of the city of Malmö, Sweden. Inclusions were made during 1995–1999 by sending out letters to 1604 women; 1044 women attended the study (65%). All women were 75 yr old (range, 75.0–75.9) at the initial visit. All women attending were ambulatory and living independently. At baseline, all women answered an extensive questionnaire on their health and lifestyle. Height and weight were measured and blood samples for DNA analyses were collected. Written informed consent was obtained for all subjects and the study was approved by the local ethics committee (Dnr LU 200-95) and conducted in accordance with the guidelines in the Declaration of Helsinki.

Assessments of BMD

The Swedish MrOS Study. BMD was measured using dual-energy x-ray absorptiometry (DXA) in all MrOS participants. A QDR 4500/A-Delphi (QDR 4500 W; Hologic, Inc., Waltham, MA) were used in Gothenburg and a Lunar Prodigy DXA (GE Lunar Corp., Madison, WI) in Malmö and Uppsala.

The coefficients of variation (CVs) for the BMD measurements ranged from 0.5% to 3%, depending on application. To be able to use DXA measurements performed with equipment from two different manufacturers (Lunar Prodigy and Hologic QDR 4500/A-Delphi), standardized BMD (sBMD) was calculated at the lumbar spine, femoral neck, trochanter, and total hip using previously reported algorithms (16, 17).

The Malmö OPRA study. In the total cohort, BMD was assessed using DXA in at least one site in 995 women with a DPX-L (Lunar, Madison, WI). The technical quality was continuously checked according to standard procedure and with phantom measurements. The precision of the equipment has been determined to 0.5% (lumbar spine), 1.6% (femoral neck), and 2.2% (trochanter).

Assessment of sex hormones

The Swedish MrOS Study. Total estradiol was measured using an ultra sensitive RIA (Orion Diagnostics, Esboo, Finland; limit of detection, 5 pmol/liter; intraassay CV, 3%; interassay CV, 6%). Total testosterone was measured using RIA (Orion Diagnostics; limit of detection, 0.1 nmol/liter; intraassay CV, 6%; interassay CV, 6%). SHBG was measured using immunoradiometric assay (Orion Diagnostics; limit of detection, 1.3 nmol/liter; intraassay CV, 3%, interassay CV, 7%).

Free testosterone and free estradiol were calculated according to the method described by Vermeulen et al. (18) and Van den Beld et al. (19) taking the concentrations of total testosterone, total estradiol, and SHBG into account and assuming a fixed albumin concentration of 43 g/liter. All samples were analyzed in duplicate in one laboratory.

Assessment of vertebral fractures

The Swedish MrOS Study. Spine x-ray analyses were performed in the Swedish MrOS Study in 1349 randomly selected individuals. X-rays were taken of both the thoracic and lumbar spine, and the lateral view was examined by an experienced radiologist. The shape of each vertebrae was evaluated: if the vertebral body had a reduced height of 3 mm or more, compared with the vertebrae above, it was classified as a vertebral fracture (vertebrae increase in height in caudal direction). The spine x-ray analyses revealed that 16.2% of the subjects had at least one prevalent vertebral fracture (1349 subjects investigated and 218 fractures diagnosed).

Genotyping

Whole blood was obtained from 1008 women and 2959 men and genomic DNA was isolated from EDTA stabilized blood. PCR of the RIZ gene was performed with the forward primer 5'-CT GGT CCG CCT CTT AAA CCT ACT-3' and the reverse primer 5'-AA CAA CAG AGG CAG TGT CTT TCC-3' (Sigma Genosys, Sigma-Aldrich House, Suffolk, UK) and carried out according to the following protocol: denaturation for 10 min at 95 C, 40 cycles of denaturation for 30 sec at 95 C, annealing at 60 C for 30 sec, and elongation at 72 C for 1 min and an extension step at 72 C for 7 min. Genotyping was performed using the AcycloPrime-II single-nucleotide polymorphism detection kit (PerkinElmer Inc., Boston, MA) (20, 21) with an single-nucleotide polymorphism detection primer (5'-CAA ACC CAA GAT AAA CTA ACT CCT-3'; Sigma Genosys, Sigma-Aldrich House, Suffolk, UK). The primer extension reaction was performed with an initial denaturation at 95 C for 2 min followed by 30 cycles of denaturation for 10 sec at 95 C and annealing for 30 sec at 54 C. Approximately 10% of random DNA samples were reanalyzed and no discrepancies, compared with the initial analyses, were found.

In total, 2933 men (99.1%) and 994 women (98.6%) were successfully genotyped for the RIZ P704 polymorphism.

Statistical analysis

All statistical analyses were performed using STATISTIKA software (StatSoft Inc., Tulsa, OK) and SAS 9.1 software (SAS institute Inc. Cary, NC). ANOVA compared the means of the genotype groups for the differences of the continuous variables and Fisher’s least square difference (LSD) post hoc test for pair wise tests. General linear model (GLM) was used to analyze designs with any combination of categorical independent factors and continuous predictors. Levene’s test for homogeneity of variance was used in the GLM procedure. Pearson correlation coefficient, R, was used for measurements of the degree of linear relationship between two continuous variables and R2 for the proportion of variability observed in the data explained by the regression line. The Fisher’s z transformation was used for testing for equality of population correlations. The Pearson {chi}2 statistic was used for measurements of the association between two nominal variables, e.g. fractures and genotype group. Binary logistic regression was performed to characterize the relationship between a categorical variable and one or more predictor variables. The odds ratios indicated how much more likely a certain event occurs in one group relative to its occurrence in another group.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
RIZ P704+/+ genotype as a predictor of low BMD in elderly men and postmenopausal women

RIZ P704 genotype groups were denoted P704–/– (absence of the P704 on both alleles), P704+/+ (presence of P704 on both alleles), and P704+/– (heterozygous), and allele frequencies were consistent with Hardy-Weinberg equilibrium in both study cohorts.

Because vertebral deformities in elderly subjects can overestimate BMD interpretation (22, 23), individuals with lumbar spine BMD values of +3.5 SD from the two different study population means, respectively, were excluded (MrOS Sweden, n = 12; Malmö OPRA, n = 7). Of the 12 individuals that were excluded from the Swedish MrOS Study, three were homozygous for the P704– allele and nine were heterozygous. Of the seven individuals that were excluded from the Malmö OPRA Study, two were homozygous for the P704– allele, four were heterozygous, and one was homozygous for the P704+ allele. In total of 2918 men and 987 women were included in the association analyses, respectively.

The clinical characteristics based on RIZ genotype groups of both study populations are presented in Table 1Go. In total of 2918 men and 987 women were included in the association analyses, respectively. BMD of the lumbar spine (L1–L4) was higher in men with the RIZ P704–/– genotype, compared with individuals in the RIZ P704+/+ genotype group (P = 0.02, ANOVA, and P = 0.02, LSD). No effect was seen of the RIZ genotype on hip BMD in men (P > 0.05, ANOVA).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Subject characteristics and BMD measurements according to RIZ P704 genotype1

 
In women, the effect of the RIZ P704 indel on BMD was more pronounced than in men and seen at all sites. Women with the RIZ P704+/+ genotype had 5.2% lower BMD in the femoral neck (P = 0.005, ANOVA, and P = 0.0007, LSD) and 5.7% lower BMD in the trochanter (P = 0.009, ANOVA, and P = 0.0012, LSD), compared with the RIZ P704–/– genotype group. Moreover, the RIZ P704 genotype was significantly associated with total body BMD (P = 0.006, ANOVA, and P = 0.002, LSD) and lumbar spine BMD (P = 0.02, ANOVA, and P = 0.007, LSD).

In males, study site, body mass index (BMI), age, and genotype were included in a multivariate model as independent predictors of lumbar spine BMD. The association between RIZ genotype and lumbar spine BMD was independent of these covariates (GLM, P < 0.05).

In females, BMI and users of hormone replacement therapy were considered independent predictors of BMD and included together with genotype in the models. Because the women were all at the same age, age was not included in the model. The effect of RIZ genotype was independent of the covariates and the associations remained significant for all sites (GLM, P < 0.05).

Regarding percentage of self-reported osteoporotic fractures in men after 50 yr of age, no significant difference was seen between the RIZ genotype groups (Table 2Go).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Self-reported osteoporotic fractures above 50 yr of age in the Swedish MrOS Study

 
RIZ P704 genotype modulates the positive correlation between serum estradiol and BMD in elderly men

Total serum estradiol levels were measured in 2914 men in the Swedish MrOS Study and grouped into quartiles (Table 3Go). In the lower quartile, the P704+/+ genotype group was highly associated with not only low BMD at lumbar spine (P704+/+ 1077.8 ± 199.1 mg/cm2 vs. P704–/– 1140.3 ± 207.0 mg/cm2, P = 0.002 GLM, P < 0.01 LSD, but also low BMD at total body (P704+/+ 1.091 g/cm2 ± 0.171 vs. P704–/– 1.136 mg/cm2 ± 0.151, P = 0.007 GLM, P < 0.001 LSD) and with the same trend for total hip (P704+/+ 928.9 ± 141.0 mg/cm2 vs. P704–/– 947.3 ± 148.3 mg/cm2, P = 0.07 GLM, P < 0.05 LSD) (Fig. 1Go). In contrast to the lower quartile, an opposite moderate association was shown in the upper quartile, where the P704–/– genotype was associated with low BMD in total hip (P704–/– 971.6 ± 146.7 mg/cm2 vs. P704+/+ 995.6 ± 160.3 mg/cm2, P = 0.10 GLM) and femoral neck (P704–/– 845.0 ± 128.3 mg/cm2 vs. P704+/+ 871.4 ± 148.4 mg/cm2, P = 0.06 GLM, P < 0.05 LSD) (Fig. 1Go) and similar with the findings in premenopausal women (18). The effect of the RIZ genotype on BMD was independent of study site, BMI, and age.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Total serum estradiol levels (pmol/liter) in the Swedish MrOS Study in quartiles

 

Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 1. RIZ P704 genotype in relation to BMD in the lower (Q1) and upper (Q4) quartiles of total serum estradiol levels in the MrOS Study. A, The RIZ genotypes P704+/+, P704+/–, and P704–/– in relation to total hip sBMD (milligrams per square centimeter) in Q1 (left) and Q4 (right). B, The RIZ genotypes P704+/+, P704+/–, and P704–/– in relation to femoral neck sBMD (milligrams per square centimeter) in Q1 (left) and Q4 (right). C, The RIZ1 genotypes P704+/+, P704+/–, and P704–/– in relation to total body BMD (grams per square centimeter) Q1 (left) and Q4 (right). D, The RIZ1 genotypes P704+/+, P704+/–, and P704–/– in relation to lumbar spine sBMD (milligrams per square centimeter) Q1 (left) and Q4 (right). Data are presented as mean ± 95% confidence interval, and P < 0.05 is considered as statistically significant.

 
Moreover, we recently demonstrated that serum estradiol is positively related to BMD at several bone sites in the Swedish MrOS Study in which the strongest correlation was seen at lumbar spine (24). To determine whether this relationship might be modulated by RIZ P704 genotype, the correlation between total serum estradiol and lumbar spine BMD was analyzed separately in the different genotype groups. Interestingly, total serum estradiol was weakly associated with BMD at lumbar spine in the P704–/– genotype group (r = 0.08, P = 0.02), whereas a significantly stronger association was seen in the P704+/– (r = 0.15, P < 0.0001) and P704+/+ (r = 0.19, P < 0.0001) genotype groups (r = 0.19 vs. r = 0.08, P < 0.05, Fisher’s z test) (Fig. 2Go). The same trend of stronger association between estradiol and BMD in the P704+/– and P704+/+ genotype groups, compared with the P704–/– group, was also seen at the hip (data not shown). Because there were some outliers of total serum estradiol levels, the variable was log transformed but without affecting the association.


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 2. The correlation between total serum estradiol and lumbar spine sBMD according to P704 genotype groups. Univariate analyses of total serum estradiol and lumbar spine sBMD according to RIZ P704 genotype groups in the MrOS Study. The P704+/+ group consists of 574 individuals (20%); the P704+/– group consists of 1397 individuals (48%), and the P704–/– group consists of 922 individuals (32%). The R2 value explains the model’s relative contribution to the variation of BMD.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
BMD is considered a complex genetic trait, which means that several genetic and environmental factors are involved. Often the contribution of a genetic factor is small, and therefore, genes are difficult to detect. However, interactions between a gene and an environmental factor may exist. In osteoporosis the risk associated with a polymorphism may depend for a large part on interaction with other genetic or environmental risk factors.

In this report we present data on associations between the indel polymorphism in the RIZ gene and BMD in two large-scale, population-based cohorts including 4058 elderly Swedish men and women. The polymorphism causes an alteration in the amino acid sequence of the final protein, resulting in presence (+) or absence (–) of a proline at position 704. Here the RIZ P704+/+ genotype was significantly associated with low BMD in both study populations. RIZ is known to be a specific coactivator of the ER{alpha} (10), and we considered the gene as a putative candidate gene for determining BMD in both men and women. Estrogens regulate bone turnover in women (25) and treatment with estrogen reverse bone loss, as seen in the Women’s Health Initiative randomized, controlled trial. In that trial, estrogen combined with progesterin increased BMD at both lumbar spine and total hip and reduced the risk of fracture (26). Mechanisms by which the transcriptional activity of the ER is altered might therefore be of central importance in the understanding of the pathophysiology of osteoporosis. This concept might also prove to be a future drug target. As reported here, women in the RIZ P704+/+ genotype group had 5.2% lower BMD at the femoral neck and 5.7% lower BMD at the trochanter, compared with the RIZ P704–/– genotype group. Furthermore, women with the RIZ P704+/+ genotype had significantly lower total body and lumbar spine BMD, compared with the P704–/– genotype group.

The ER{alpha} is important for not only the maintenance of the female skeleton but also the male skeleton (27, 28). Evidence of this includes data that androgens are metabolized to estrogens via the aromatase enzyme (CYP 19) system (29) and that aromatase deficiency in men results in osteopenia. A case report of a man, with a disruptive mutation in the ER{alpha} gene, had a failure of bone mass development and is considered a proof that the ER{alpha} is implemented also in male osteoporosis (30). Our data show main influence of the RIZ genotype in 3014 elderly men on BMD at lumbar spine. In line with the findings in women, the RIZ P704+/+ group had significantly lower BMD, compared with the P704–/– group. The power calculations of the study confirmed that the MrOS Study had more than 80% power to detect genotypic differences in BMD with SD ranges of 0.10–0.25. This indicates that the lack of association between RIZ genotype and hip BMD in men rather is due to a biological effect than a power issue. Interesting, there might exist differences between men and women concerning the effect of estrogens on bone. Changes in bone density in men in response to estrogen have reported to be most dramatic at sites rich in trabecular bone, such as the spinal region (31). Other gender differences regarding genetic effects on BMD at various sites have previously been reported. There is evidence to suggest a gender-specific genetic component to overall genetic variance at some skeletal regions, although the proportion of bone strength variance explained by genetic factors is similar for men and women (32). These findings have been confirmed in both humans and mice (33, 34, 35, 36). Duncan et al. found that lumbar spine BMD correlated more strongly in male-male comparisons and hip BMD in female-female comparisons (33). These data are in agreement with our findings of RIZ genotype on BMD sites in men and women.

The RIZ gene variant was, however, not associated with an increased risk of fractures. Because the importance of BMD in osteoporotic fractures is well established and the heritability of BMD is substantial, one could suggest that the genetic liability to osteoporotic fractures might reflect that for BMD. However, twin studies have demonstrated that the genetic influence on the risk of fractures was not significantly reduced when BMD was included as a covariate, indicating that fracture and low BMD have their own specific genetic risk factors that are unlikely to be shared between the two traits (37, 38). However, the specific ER{alpha} cofactor RIZ gene variants may predispose not only low BMD but also to other biological mechanisms underlying increased fracture risk as bone size. This, because an association of ER{alpha} variants with increase in fracture risk found in a large-scale population-based study, was not explained by the relatively modest difference in BMD between the genotypes. In that study it was observed that the ER{alpha} genotype association with vertebral fractures was independent of BMD and of age at menopause (39).

Previously we reported that the RIZ P704 polymorphic variants are functionally different in coactivating the ER{alpha} in a reporter system in vitro (15). Both RIZ protein variants, P704+ and P704–, were able to enhance the ER{alpha} in a ligand- and dose-dependent manner in vitro. These findings are in line with several reports showing that RIZ has the capacity, in the presence of estradiol, to enhance the ER{alpha} activation of transcription in vitro (10, 11, 40). However, the RIZ P704– polymorphic variant was associated with a reduced enhancing effect in ER{alpha} coactivation, compared with the RIZ P704+ variant. In the same report, the RIZ P704+/+ genotype was associated with high BMD in a cohort of 343 premenopausal women (15).

These results are in conflict with the data in the present report that clearly show that the RIZ P704+/+ genotype is a risk factor for low BMD in elderly subjects. However, it has been shown that RIZ both have corepressing and coactivating activities, depending on estrogen status. Carling et al. (11) demonstrated in an extensive study that, in the presence of estrogen, RIZ interacts with ER{alpha} cofactors and coactivated ER target gene transcription. However, in the absence of estrogen, RIZ1 was bound to the promoter of the ER-target gene and inhibited gene transcription (11). Taken together, this indicates that estrogen-bound ER may turn the RIZ corepressor into a coactivator. This was studied in the present study in which the male subjects were grouped into quartiles regarding total serum estradiol and the RIZ genotype in the lower and upper quartiles were subsequent associated with BMD. Interestingly, in the lower quartile, the P704+/+ genotype group was highly associated with not only low BMD at lumbar spine but also low BMD at total body and total hip. However, in the upper quartile, an opposite moderate association was shown in which the P704+/+ genotype was associated with high BMD in total hip and femoral neck and similar to the effect in premenopausal women (15). Moreover, to determine whether the known positive correlation of serum estradiol with BMD (24) might be modulated by RIZ genotype, the correlation between total serum estradiol and lumbar spine BMD was analyzed separately in the different genotype groups. Notably, the correlation of total serum estradiol was significantly stronger in the P704+/+ genotype group (r = 0.19), compared with the correlation in the P704–/– genotype group (r = 0.08), indicating that RIZ genotype modulates the estrogenic response in bone. Taken together, one could speculate that RIZ may act as a corepressor in postmenopausal women and men with low estradiol levels. In contrast, in premenopausal women and men with high levels of estradiol, RIZ may act as a coactivator and influences the effect of estradiol on bone. This may explain the contradictory effect of the RIZ indel in the different study populations.

In conclusion, a common polymorphism in the ER{alpha} cofactor RIZ gene is an independent and strong predictor of BMD in two large-scale, population-based cohorts of elderly Swedish men and women. The association between this polymorphism and BMD is dependent on estradiol status. Furthermore, our results demonstrate that this polymorphism modulates the positive correlation between serum estradiol levels and BMD, indicating that it modulates the estrogenic response in bone. Further studies are required to determine whether this polymorphism modulates the estrogenic response to E2 treatment.


   Footnotes
 
This work was supported by grants from the Swedish Society for Medical Research, the Swedish Research Council, Seda and Signe Hermanssons Foundation, and Börjessons Foundation.

Disclosure Statements: E.G., A.K., A.H., D.M., E.O., H.M., C.O., and H.B. have nothing to declare. K.Å. consults for Roche and Nycomed and received lecture fees from Merck Sharp & Dohme. P.G. received lecture fees from Merck. Ö.L. consults for Nycomed and Lilly and received lecture fees from Merck Sharp & Dohme and Amgen.

First Published Online March 13, 2007

Abbreviations: BMD, Bone mineral density; BMI, body mass index; CV, coefficient of variation; DXA, dual-energy x-ray absorptiometry; ER, estrogen receptor; GLM, general linear model; indel, insertion/deletion; LSD, least square difference; QTL, quantitative trait loci; RIZ, retinoblastoma-interacting zinc finger protein; sBMD, standardized BMD.

Received July 19, 2006.

Accepted March 6, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Arden NK, Spector TD 1997 Genetic influences on muscle strength, lean body mass, and bone mineral density: a twin study. J Bone Miner Res 12:2076–2081[CrossRef][Medline]
  2. Christian JC, Yu PL, Slemenda CW, Johnston Jr CC 1989 Heritability of bone mass: a longitudinal study in aging male twins. Am J Hum Genet 44:429–433[Medline]
  3. Ioannidis JP, Ntzani EE, Trikalinos TA, Contopoulos-Ioannidis DG 2001 Replication validity of genetic association studies. Nat Genet 29:306–309[CrossRef][Medline]
  4. Ioannidis JP, Trikalinos TA, Ntzani EE, Contopoulos-Ioannidis DG 2003 Genetic associations in large versus small studies: an empirical assessment. Lancet 361:567–571[CrossRef][Medline]
  5. Devoto M, Specchia C, Li HH, Caminis J, Tenenhouse A, Rodriguez H, Spotila LD 2001 Variance component linkage analysis indicates a QTL for femoral neck bone mineral density on chromosome 1p36. Hum Mol Genet 10:2447–2452[Abstract/Free Full Text]
  6. Wilson SG, Reed PW, Bansal A, Chiano M, Lindersson M, Langdown M, Prince RL, Thompson D, Thompson E, Bailey M, Kleyn PW, Sambrook P, Shi MM, Spector TD 2003 Comparison of genome screens for two independent cohorts provides replication of suggestive linkage of bone mineral density to 3p21 and 1p36. Am J Hum Genet 72:144–155[CrossRef][Medline]
  7. Huang S 1999 The retinoblastoma protein-interacting zinc finger gene RIZ in 1p36-linked cancers. Front Biosci 4:D528–D532
  8. Jiang G, Liu L, Buyse IM, Simon D, Huang S 1999 Decreased RIZ1 expression but not RIZ2 in hepatoma and suppression of hepatoma tumorigenicity by RIZ1. Int J Cancer 83:541–546[CrossRef][Medline]
  9. Fang W, Piao Z, Simon D, Sheu JC, Huang S 2000 Mapping of a minimal deleted region in human hepatocellular carcinoma to 1p36.13-p36.23 and mutational analysis of the RIZ (PRDM2) gene localized to the region. Genes Chromosomes Cancer 28:269–275[CrossRef][Medline]
  10. Abbondanza C, Medici N, Nigro V, Rossi V, Gallo L, Piluso G, Belsito A, Roscigno A, Bontempo P, Puca AA, Molinari AM, Moncharmont B, Puca GA 2000 The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action. Proc Natl Acad Sci USA 97:3130–3135[Abstract/Free Full Text]
  11. Carling T, Kim KC, Yang XH, Gu J, Zhang XK, Huang S 2004 A histone methyltransferase is required for maximal response to female sex hormones. Mol Cell Biol 24:7032–7042[Abstract/Free Full Text]
  12. Slemenda CW, Longcope C, Zhou L, Hui SL, Peacock M, Johnston CC 1997 Sex steroids and bone mass in older men. Positive associations with serum estrogens and negative associations with androgens. J Clin Invest 100:1755–1759[Medline]
  13. Khosla S, Melton 3rd LJ, Riggs BL 2002 Clinical review 144: estrogen and the male skeleton. J Clin Endocrinol Metab 87:1443–1450[Abstract/Free Full Text]
  14. Khosla S, Melton 3rd LJ, Riggs BL 2001 Estrogens and bone health in men. Calcif Tissue Int 69:189–192[CrossRef][Medline]
  15. Grundberg E, Carling T, Brandstrom H, Huang S, Ribom EL, Ljunggren O, Mallmin H, Kindmark A 2004 A deletion polymorphism in the RIZ gene, a female sex steroid hormone receptor coactivator, exhibits decreased response to estrogen in vitro and associates with low bone mineral density in young Swedish women. J Clin Endocrinol Metab 89:6173–6178[Abstract/Free Full Text]
  16. Hui SL, Gao S, Zhou XH, Johnston Jr CC, Lu Y, Gluer CC, Grampp S, Genant H 1997 Universal standardization of bone density measurements: a method with optimal properties for calibration among several instruments. J Bone Miner Res 12:1463–1470[CrossRef][Medline]
  17. Lu Y, Fuerst T, Hui S, Genant HK 2001 Standardization of bone mineral density at femoral neck, trochanter and Ward’s triangle. Osteoporos Int 12:438–444[CrossRef][Medline]
  18. Vermeulen A, Verdonck L, Kaufman JM 1999 A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 84:3666–3672[Abstract/Free Full Text]
  19. van den Beld AW, de Jong FH, Grobbee DE, Pols HA, Lamberts SW 2000 Measures of bioavailable serum testosterone and estradiol and their relationships with muscle strength, bone density, and body composition in elderly men. J Clin Endocrinol Metab 85:3276–3282[Abstract/Free Full Text]
  20. Hsu TM, Law SM, Duan S, Neri BP, Kwok PY 2001 Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection. Clin Chem 47:1373–1377[Abstract/Free Full Text]
  21. Chen X, Levine L, Kwok PY 1999 Fluorescence polarization in homogeneous nucleic acid analysis. Genome Res 9:492–498[Abstract/Free Full Text]
  22. Krege JH, Miller PD, Lenchik L, Misurski DA, Chen P 2006 New or worsening lumbar spine vertebral fractures increase lumbar spine bone mineral density and falsely suggest improved skeletal status. J Clin Densitom 9:144–149[CrossRef][Medline]
  23. Paiva LC, Filardi S, Pinto-Neto AM, Samara A, Marques Neto JF 2002 Impact of degenerative radiographic abnormalities and vertebral fractures on spinal bone density of women with osteoporosis. Sao Paulo Med J 120:9–12[Medline]
  24. Mellstrom D, Johnell O, Ljunggren O, Eriksson AL, Lorentzon M, Mallmin H, Holmberg A, Redlund-Johnell I, Orwoll E, Ohlsson C 2006 Free testosterone is an independent predictor of BMD and prevalent fractures in elderly men: MrOS Sweden. J Bone Miner Res 21:529–535[CrossRef][Medline]
  25. Lanyon L, Armstrong V, Ong D, Zaman G, Price J 2004 Is estrogen receptor {alpha} key to controlling bones’ resistance to fracture? J Endocrinol 182:183–191[Abstract]
  26. Cauley JA, Robbins J, Chen Z, Cummings SR, Jackson RD, LaCroix AZ, LeBoff M, Lewis CE, McGowan J, Neuner J, Pettinger M, Stefanick ML, Wactawski-Wende J, Watts NB 2003 Effects of estrogen plus progestin on risk of fracture and bone mineral density: the Women’s Health Initiative randomized trial. JAMA 290:1729–1738[Abstract/Free Full Text]
  27. Vidal O, Lindberg MK, Hollberg K, Baylink DJ, Andersson G, Lubahn DB, Mohan S, Gustafsson JA, Ohlsson C 2000 Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice. Proc Natl Acad Sci USA 97:5474–5479[Abstract/Free Full Text]
  28. Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, Ohlsson C 2004 Androgens and bone. Endocr Rev 25:389–425[Abstract/Free Full Text]
  29. Simpson ER 2000 Role of aromatase in sex steroid action. J Mol Endocrinol 25:149–156[CrossRef][Medline]
  30. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331:1056–1061[Abstract/Free Full Text]
  31. Carani C, Qin K, Simoni M, Faustini-Fustini M, Serpente S, Boyd J, Korach KS, Simpson ER 1997 Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med 337:91–95[Free Full Text]
  32. Naganathan V, Macgregor A, Snieder H, Nguyen T, Spector T, Sambrook P 2002 Gender differences in the genetic factors responsible for variation in bone density and ultrasound. J Bone Miner Res 17:725–733[CrossRef][Medline]
  33. Duncan EL, Cardon LR, Sinsheimer JS, Wass JA, Brown MA 2003 Site and gender specificity of inheritance of bone mineral density. J Bone Miner Res 18:1531–1538[CrossRef][Medline]
  34. Beamer WG, Donahue LR, Rosen CJ 2002 Genetics and bone. Using the mouse to understand man. J Musculoskelet Neuronal Interact 2:225–231[Medline]
  35. Karasik D, Cupples LA, Hannan MT, Kiel DP 2003 Age, gender, and body mass effects on quantitative trait loci for bone mineral density: the Framingham Study. Bone 33:308–316[Medline]
  36. Ralston SH, Galwey N, MacKay I, Albagha OM, Cardon L, Compston JE, Cooper C, Duncan E, Keen R, Langdahl B, McLellan A, O’Riordan J, Pols HA, Reid DM, Uitterlinden AG, Wass J, Bennett ST 2005 Loci for regulation of bone mineral density in men and women identified by genome wide linkage scan: the FAMOS study. Hum Mol Genet 14:943–951[Abstract/Free Full Text]
  37. Michaelsson K, Melhus H, Ferm H, Ahlbom A, Pedersen NL 2005 Genetic liability to fractures in the elderly. Arch Intern Med 165:1825–1830[Abstract/Free Full Text]
  38. Andrew T, Antioniades L, Scurrah KJ, Macgregor AJ, Spector TD 2005 Risk of wrist fracture in women is heritable and is influenced by genes that are largely independent of those influencing BMD. J Bone Miner Res 20:67–74[CrossRef][Medline]
  39. van Meurs JB, Schuit SC, Weel AE, van der Klift M, Bergink AP, Arp PP, Colin EM, Fang Y, Hofman A, van Duijn CM, van Leeuwen JP, Pols HA, Uitterlinden AG 2003 Association of 5' estrogen receptor {alpha} gene polymorphisms with bone mineral density, vertebral bone area and fracture risk. Hum Mol Genet 12:1745–1754[Abstract/Free Full Text]
  40. Medici N, Abbondanza C, Nigro V, Rossi V, Piluso G, Belsito A, Gallo L, Roscigno A, Bontempo P, Puca AA, Molinari AM, Moncharmont B, Puca GA 1999 Identification of a DNA binding protein cooperating with estrogen receptor as RIZ (retinoblastoma interacting zinc finger protein). Biochem Biophys Res Commun 264:983–989[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
92/6/2300    most recent
Author Manuscript (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grundberg, E.
Right arrow Articles by Brändström, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grundberg, E.
Right arrow Articles by Brändström, H.
Related Collections
Right arrow Calcium and Bone Metabolism

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals