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Journal of Molecular Endocrinology (2008) 40 273-279    DOI: 10.1677/JME-08-0003
© 2008 Society for Endocrinology
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Tumour necrosis factor superfamily member 11 gene promoter polymorphisms modulate promoter activity and influence bone mineral density in postmenopausal women with osteoporosis

 Simona Mencej, Omar M E Albagha1, Janez Prezelj2, Tomaz Kocjan2 and Janja Marc

Faculty of Pharmacy, Chair of Clinical Biochemistry, University of Ljubljana, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia 1 Rheumatology Section, Molecular Medicine Centre, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XU, UK 2 Department of Endocrinology, Diabetes and Metabolic Diseases, University Medical Centre, Zaloska cesta 2, SI-1000 Ljubljana, Slovenia

(Correspondence should be addressed to J Marc Email: janja.marc{at}ffa.uni-lj.si)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumour necrosis factor superfamily member 11 (TNFSF11) gene, that codes for receptor activator of nuclear factor-{kappa}B ligand, is one of the candidate genes for the genetic susceptibility to osteoporosis. As variations in the TNFSF11 gene promoter could alter its expression, the aim of the study was to evaluate the functional influence of three polymorphisms in the promoter and to investigate their association with bone mineral density (BMD) and biochemical markers in postmenopausal women. A total of 404 postmenopausal women were genotyped for the presence of TNFSF11 gene promoter polymorphisms –290C>T, –643C>T and –693G>C. Two common haplotypes, CCG and TTC, which occur in 44.3 and 49.3% of subjects respectively, were subjected to functional analysis. Amplified fragments were cloned into pGL3-basic reporter plasmid, which was co-transfected with pRL-TK plasmid into HEK293 cells. Dual luciferase reporter assay was performed. BMD and biochemical markers were measured. Reporter gene analysis showed significantly higher luciferase activity in CCG than in TTC haplotype (P=0.018). Both showed association with lumbar spine BMD (BMD-ls; P=0.005 and 0.007 for TTC and CCG respectively), whereas in femoral neck there was no association with BMD. In postmenopausal osteoporosis, association with BMD-ls was established in –290C>T, –643C>T and –693G>C (P values: 0.001, 0.041 and 0.013 respectively). Association with femoral neck BMD was shown in –693G>C (P=0.049). No association was found with biochemical markers in any of the groups. Our results suggest that in postmenopausal osteoporosis, TNFSF11 gene promoter polymorphisms –290C>T, –643C>T and –693G>C play a functional role in the genetic regulation of BMD.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The number of identified single nucleotide polymorphisms (SNPs) is rapidly increasing through SNP discovery in the HapMap Consortium (International HapMap Consortium 2007). However, the functional relevance of SNPs is still unknown. The data arising from the research on the functional role of SNPs are important and could further be used to allow a rational choice of SNPs for inclusion in genetic epidemiological studies or to help the interpretation of apparent disease-associated SNPs (Knight 2003). Osteoporosis is a disease with a strong genetic component (Ralston & Crombrugghe 2006) and its development is due to the persistent excess of osteoclastic bone resorption over osteoblastic bone formation (Troen 2003).

Genetic ablation experiments have shown that members of the tumour necrosis factor (TNF) ligand and receptor (TNFR) superfamilies are essential factors of osteoclast biology and bone metabolism, namely receptor activator of nuclear factor-{kappa}B ligand (RANKL; Lacey et al. 1998), RANK (Anderson et al. 1997) and osteoprotegerin (OPG; Simonet et al. 1997). The TNF superfamily member 11 (TNFSF11) gene encodes for RANKL protein and is located on human chromosome 13q14 (Walsh & Choi 2003).

RANKL expression is regulated by various hormones (glucocorticoids, vitamin D, oestrogen and parathyroid hormone), cytokines (transforming growth factor-β, TNF-{alpha}, interleukins 1, 4, 6, 11 and 17, and prostaglandin E2) and various mesenchymal transcription factors (TFs) such as cbfa-1 and peroxisome proliferators-activated receptor {gamma} (Hofbauer & Heufelder 2001). The osteoblastic protein of the Wnt signalling pathway, β-catenin, also regulates RANKL and OPG expression in mice and is therefore involved in the process of osteoclastogenesis (Glass et al. 2005, Holmen et al. 2005).

As RANKL is the final effector of the osteoclastogenesis pathway, it is definitely one of the candidate genes for the regulation of susceptibility to osteoporosis (Ralston & Crombrugghe 2006). In a 2 kb 5'-flanking region of human TNFSF11 gene, several motifs with significant sequence similarity to TFs (>85%) have already been identified (Roccisana et al. 2004). SNPs in the promoter region could influence the binding of TFs and gene expression, and finally impact on protein synthesis. A preliminary study of TNFSF11 gene promoter SNPs in 115 postmenopausal women showed the association of –290C>T SNP with femoral neck bone mineral density (BMD-fn), whereas SNPs –643C>T and –693G>C showed no association with BMD (Mencej et al. 2006). Their genetic influence on BMD has been evaluated, but no functional analysis of the studied SNPs has been performed. Therefore, we investigated whether the studied SNPs are the functional cause of the associated phenotype or, possibly, just in linkage disequilibrium (LD) with another site. Thus, the aims of the present study were: 1) to assess the functional role of certain TNFSF11 gene promoter haplotypes in transcriptional regulation of TNFSF11 gene expression and 2) to explore the potential associations of TNFSF11 gene promoter haplotypes and SNPs with BMD and biochemical markers of bone turnover in osteoporotic and non-osteoporotic postmenopausal women.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects

We evaluated 404 postmenopausal women, aged 42–91 years, who were referred to the outpatient departments of the University Medical Centre, Ljubljana, or the General Hospital, Celje, for BMD measurement. Each patient was examined clinically and routine biochemical tests were performed to exclude systemic and metabolic bone diseases other than primary osteoporosis. None had previously taken any drug known to influence bone metabolism. Osteoporosis was defined according to the World Health Organization criteria (WHO 1994). The study was approved by the Ethical Committee of the Republic of Slovenia and written informed consent was obtained from each patient participating in the study.

Genotyping

Genomic DNA was isolated from peripheral blood leukocytes using standard methodology. To detect –643C>T (rs9533156) and –693G>C (rs9533155) SNPs, TNFSF11 gene promoter fragment (–888 to –337 bp) was amplified by PCR, followed by restriction fragment length polymorphism analysis as described previously (Mencej et al. 2006). Genotyping of –290C>T (rs9525641) SNP was performed by the TaqMan allelic discrimination method, using the TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA) under the following conditions: 10 min at 95 °C, followed by 43 cycles of 15 s at 95 °C and 1 min at 60 °C. Reactions were performed on ABI Prism 7000 sequence detection system.

BMD measurement

BMD measurements at the lumbar spine (L1–L4), total hip, and femoral neck were performed using dual-energy X-ray absorptiometry (DXA; QDR-4500, Hologic, Inc., Waltham, MA, USA) in Ljubljana and Celje. Cross-calibration study of the precision of measurements between the centres had previously been performed. A correction factor was not considered necessary.

Biochemical markers of bone turnover

Serum-free soluble RANKL (sRANKL) was measured by an enzyme immunoassay (sRANKL ELISA, Biomedica, Vienna, Austria). Serum C-terminal cross-linking telopeptides of type I collagen (CTX) were measured by enzyme immunoassay (Serum CrossLaps ELISA, Nordic Bioscience Diagnostics A/S, Herlev, Denmark). Osteocalcin (OC) in heparinised plasma was measured by a solid-phase, two-site chemiluminescent enzyme-labelled immunometric assay (Immulite Osteocalcin, Diagnostic Product Corporation, Los Angeles, CA, USA).

Luciferase reporter assay

The selected TNFSF11 gene promoter sequence spans nucleotides –1715 to +94 relative to the transcription start site. Human genomic DNA from two individuals with the two most common haplotypes, CCG and TTC, was amplified by PCR, using the primers: 5'-ATTATGGTACCGTCAAGGAGCAGGGAGAGAATG-3' and 5'-TCGGAGATCTCGCTTCGGAGCTCTCCTC-3'. Primers were designed according to the GenBank sequence AF544022 with the addition of the KpnI and BglII recognition sequence at the 5'-end. PCR was carried out using Taq polymerase (Qiagen) under the following conditions: 94 °C for 4 min, followed by 33 cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C for 2 min. The resulting PCR products were digested with KpnI and BglII (New England Biolabs, Beverly, MA, USA) and inserted into pGL3-basic luciferase reporter vector (Promega) digested at the same sites.

The HEK293 cells were maintained in {alpha}-minimal essential medium ({alpha}-MEM) supplemented with L-glutamine (1% v/v), fetal calf serum (10% v/v) and penicillin/streptomycin (1% v/v) (Gibco). For transfection, cells were plated at a density of 100 000 cells/well in 24-well tissue culture plates. After 24 h, the cells were transfected in six replicates with a mixture of Fugene HD transfection reagent (Roche Applied Science) at a ratio (µl reagent:µl DNA) of 3:2, {alpha}-MEM and 500 ng DNA/well, including 100 ng/well of pRP-TK control reporter vector. Cells were harvested 48 h post-transfection, and the luciferase assay performed using Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured using a BIO-TEK Synergy HT multidetection microplate reader (Fisher Scientific, Pittsburgh, PA, USA).

Statistical analysis

Hardy–Weinberg equilibrium was tested for each SNP and group of participants using the {chi}2 test. In each studied SNP, frequency distributions of genotypes in osteoporotic and non-osteoporotic postmenopausal women were compared using the {chi}2 test. The standardized measures of LD, denoted as D' and r2, were assessed using the EMLD software (University of Texas, Houston, TX, USA), which calculates pairwise LD based on SNP genotype data from unrelated individuals (epi.mdanderson.org/~qhuang/Software/pub.htm). For two independent loci, the difference (D) is that between the actual and expected gametic frequencies and is usually expressed as a standardized difference (D'). D measures the statistical association of alleles in forming gametes and is related to the Pearson correlation coefficient (r). D' is used to assess the probability of historical recombination, whereas r2 is the most relevant measure in the association studies (Mueller 2004). PHASE program was used to estimate haplotypes from genotype data for individual participants (Stephens et al. 2001). For the purpose of statistical analysis, we used only haplotype data in which the probability of correct haplotype assignment by PHASE in an individual participant was estimated to be 95% or greater. Kolmogorov–Smirnov normality test was conducted before association analysis and data transformation was performed where appropriate. In haplotype analysis, subjects were coded according to whether they had two copies, one copy or no copies of the haplotype under study. Differences in BMD and biochemical markers between the genotype and haplotype subgroups were tested using ANOVA. For comparison of luciferase activities, the two-sample t-test was used. Statistical analyses were carried out using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA) and P values <0.05 were considered statistically significant.

Possible TF binding sites at the sites of our nucleotide changes in TNFSF11 gene promoter were searched using TFSerach ver 1.3 (Heinemeyer et al. 1998).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The frequencies of –290C>T, –643C>T and –693G>C SNPs in the TNFSF11 gene promoter were determined by screening DNA samples from 404 postmenopausal women. Subject characteristics are presented in Table 1.


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  		Table 1 Characteristics of the non-osteoporotic and osteoporotic postmenopausal women

 
The observed genotype frequency distributions were not significantly different from the Hardy–Weinberg distribution at the 5% level. In Hardy–Weinberg distribution testing of –290C>T, –643C>T and –693G>C, P values were as follows: 0.711, 0.090 and 0.195 for all postmenopausal women; 0.345, 0.586 and 0.791 for non-osteoporotic women; and 0.659, 0.053 and 0.130 for osteoporotic women. No significant differences were found between the frequencies of non-osteoporotic and osteoporotic postmenopausal women in any of these SNPs.

Evidence of strong LD was obtained within each of the three possible pairs: –290C>T and –643C>T, D'=0.96, r2=0.87; –290C>T and –693G>C, D'=0.93, r2=0.84; –643C>T and –693G>C, D'=0.92, r2=0.78.

In the group of postmenopausal women, the haplotypes, defined by –290C>T, –643C>T and –693G>C TNFSF11 gene promoter SNPs, were distributed as follows: TTC (49.3%), CTC (0.6%), TCC (2.6%), CCC (1.5%), TTG (1.2%), CTG (0.1%), TCG (0.4%) and CCG (44.3%). All eight possible haplotypes were found, but TTC and CCG accounted for 93.6% of the alleles observed.

To study whether different haplotypes have functional relevance, transient transfections with two different plasmids, containing these two most common haplotypes were performed for transcriptional activity evaluation. Forty-eight-hours post-transfection Renilla and firefly luciferase activities were measured and expressed as a ratio of firefly to Renilla activity. The ratio was 2.966 and 2.543 in the CCG and TTC haplotypes respectively, the difference between the ratios being statistically significant (P=0.018).

Analysis of TF binding motifs revealed numerous possible TF binding sites in the TNFSF11 gene promoter, two of which include two of the studied SNPs (Fig. 1). Polymorphism –290C>T lies in a potential TF binding motif for heat shock factors (95.3% homology) and –693G>C in ADR1 (87.7% homology).


Figure 1
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  		Figure 1 Nucleotide sequence analysis of the –744 to –205 human TNFSF11 gene promoter with TFSearch software. SNPs are marked in bold font and grey colour, and both nucleotides were given. Possible transcription factors binding motifs with significant homology (>85%), containing one of the studied SNPs, are underlined in bold.

 
In osteoporotic postmenopausal women, statistical analysis revealed an association with lumbar spine BMD (BMD-ls) for all studied genotypes and haplotypes. Data for BMD-fn and total hip BMD (BMD-th) showed no association with studied genotypes and haplotypes (Table 2). In non-osteoporotic women, statistical analysis showed no associations with BMD (Table 3).


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  		Table 2 Bone mineral density in osteoporotic women in different tumour necrosis factor superfamily member 11 (TNFSF11) gene promoter genotype and haplotype subgroups

 

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  		Table 3 Bone mineral density in non-osteoporotic women in different tumour necrosis factor superfamily member 11 (TNFSF11) gene promoter genotype and haplotype subgroups

 
The concentrations of OC, CTX and RANKL were measured in 130 postmenopausal women – 85 non-osteoporotic and 45 osteoporotic. No association was found between biochemical markers and studied genotypes or haplotypes. RANKL concentrations, observed in women with two copies of CCG haplotype and no copies of TTC haplotype, were higher than those in women with one copy of each allele and those with two copies of TTC allele (0.828 vs 0.372 and 0.444 pmol/l respectively) (P=0.222).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Functional analysis showed a difference in promoter activities of the TNFSF11 gene with the two most common haplotypes CCG and TTC. In postmenopausal osteoporosis, the less active TTC haplotype was associated with higher BMD-ls values, and the more active CCG haplotype with lower BMD-ls values. In osteoporotic women, BMD-ls was found to be associated with all three studied SNPs (–290C>T, –643C>T and –693G>C) and BMD-fn with –693G>C SNP. No other statistically significant associations with BMD were found for the studied SNPs and haplotypes in the studied groups. No association with biochemical markers of bone turnover was found in any of the studied groups.

As the RANKL/RANK/OPG system plays a crucial role in osteoclast development, the TNFSF11 gene is certainly a candidate for the genetic regulation of bone mass and remodelling. In men, SNP rs9594782 has shown an association with BMD (Hsu et al. 2006). In Korean postmenopausal women, SNPs rs12721445 and rs2277438 in TNFSF11 gene have been studied and the latter was related with BMD, but only in combination with G1181C SNP in the OPG gene (Kim et al. 2007). In a study on Slovenian postmenopausal women, three polymorphic nucleotide variations (–290C>T, –643C>T and –693G>C) were studied in the TNFSF11 gene promoter. In –290C>T, an association was shown with BMD-fn in postmenopausal women (Mencej et al. 2006). Therefore, we focused our research on the SNPs –290C>T, –643C>T and –693G>C, which have already been studied in Caucasian postmenopausal women. All three studied SNPs in the TNFSF11 gene promoter region were found to be in strong LD. However, it is possible that the three studied SNPs are in LD with some other nearby sequence variation that is the actual functional cause of the association with BMD. To evaluate the functional role of the studied SNPs, luciferase reporter gene experiments were performed. Comparison of the two most common inferred haplotypes, CCG and TTC, showed a small but significant difference in their transcriptional activities, CCG being more active. Thus, higher serum RANKL concentrations are expected in the CCG haplotype.

To confirm the positive results from the functional analysis study, association analysis was performed. In 182 osteoporotic women, all studied SNPs in the TNFSF11 gene promoter were associated with BMD-ls, and one of them, –693G>C, also with BMD-fn. However, no association with BMD was found in 222 non-osteoporotic women. In the previous study on 115 postmenopausal women, only –290C>T was associated with BMD-fn. The present study was conducted on a larger number of participants and the homogeneity of groups was improved, as participants were involved according to their menopausal status and the presence of osteoporosis.

One of the focuses of the research was haplotype analysis. All eight possible haplotypes were inferred in our group. The two most common ones, TTC and CCG, showed an association with BMD-ls. Women carrying two alleles with TTC haplotype had higher BMD-ls than those without. The values of BMD-ls were similar for osteoporotic women with either one or two copies of the TTC allele. The CCG haplotype was also associated with BMD-ls, but BMD-ls values were lowest in individuals with two copies of CCG haplotype and highest in those with no CCG allele. The CCG and TTC haplotypes were not associated with BMD-fn, although women with two CCG alleles had lower BMD-fn.

Biochemical markers of bone turnover, which respond to changes more rapidly than BMD, did not show any association with the studied genotypes or haplotypes in any of the groups. Serum RANKL concentrations in osteoporotic women also showed no association with any of the studied genotypes or haplotypes. Higher transcriptional activity was observed in the CCG haplotype and, as expected, much higher RANKL concentrations were measured in 45 serum samples from osteoporotic women with two CCG alleles. As RANKL induces osteoclastogenesis, higher levels of RANKL could lead to increased bone loss and lower BMD. Indeed, osteoporotic women with two copies of CCG haplotype had lower BMD than those with two copies of TTC allele.

The transcriptional activity of the TNFSF11 gene could be influenced, if one or all SNPs were to lie in a TF binding site. So far, several putative TFs binding motifs with significant homology (>85%) have been identified in the human TNFSF11 gene promoter region including heat shock factor-responsive elements, CdxA, SRY, MZF-1, Nkx-2, NF-E2, AML-1a, C/EBP-β, ADR1, AP-4, GATA-1, GATA-2 and c-Myb (Roccisana et al. 2004). Our analysis showed that the two studied SNPs in the TNFSF11 gene promoter are located in possible binding sites for known TFs. Heat shock factors have already been functionally evaluated further upstream in human TNFSF11 gene promoter (Roccisana et al. 2004). However, no functional analysis of the ADR1 TF binding site has been performed on human TNFSF11 gene promoter.

In conclusion, the results of our study support the proposal that the TNFSF11 gene promoter region SNPs –290C>T, –643C>T and –693G>C have a functional role in osteoporosis development, due to the difference in transcription activity between two common haplotypes. Haplotype CCG had higher transcriptional activity and was associated with lower BMD-ls. Therefore, the studied haplotypes could be considered as risk factors for genetic susceptibility to postmenopausal osteoporosis, since a statistically significant association with BMD-ls has been demonstrated in all three, as well as in the two most common haplotype forms TTC and CCG. These findings were further supported by the association of –693G>C polymorphism with BMD-fn. These results provide important additional information on the functional and genetic role of TNFSF11 gene in bone density. However, the genetic effects of the TNFSF11 gene promoter SNPs need further confirmation on a larger group of subjects and the next step in the explanation of their functional role will require identification of the binding TF(s).


   Acknowledgements
 
We are grateful to M Kranjc, N Turk, J Zupan and I Kucak for their technical assistance. We thank Prof. Roger Pain for advice on the English language. We also thank the European Calcified Tissue Society for the support on functional analysis work. This work was supported by grant P3-0298 provided by Ministry of Education, Science and Sport of the Republic of Slovenia. The functional analysis work was supported by European Calcified Tissue Society Exchange Scholarship Grant. There is no conflict of interest that would prejudice its impartiality.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received in final form 13 March 2008
Accepted 28 March 2008
Made available online as an Accepted Preprint 28 March 2008




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