Anitta Mahonen

Early postmenopausal bone loss is associated with PvuII estrogen receptor gene polymorphism in finnish women : Effect of hormone replacement therapy

Genetic factors regulate bone mineral density (BMD) and possibly the development of osteoporosis. An association between estrogen receptor (ER) polymorphism, BMD, and postmenopausal hormone replacement therapy (HRT) has not been established. Therefore, we studied the influence of the ER genotype on BMD before and after a 5‐year HRT in a placebo‐controlled, population‐based, randomized group of 322 early postmenopausal women. The participants were randomized into two treatment groups: the HRT group (n = 145) received a sequential combination of 2 mg estradiol valerate and 1 mg CPA with or without vitamin D3, 100–300 IU + 500 mg calcium lactate/day (equal to 93 mg Ca2+), and the non‐HRT group (n = 177) received calcium lactate, 500 mg alone or in combination with vitamin D3, 100–300 IU/day. PvuII restriction fragment length polymorphism (RFLP) of the ERα was determined using polymerase chain reaction (PCR). BMDs of the lumbar spine (L2–4) and proximal femur were measured by using dual‐energy X‐ray absorptiometry (DXA). At the baseline, there were no significant differences in the lumbar or femoral neck BMDs between the three ER PvuII genotype groups (PP,Pp,pp). After 5 years, the BMD of the femoral neck remained unaltered and that of the lumbar spine increased by 1.7% in the HRT group, whereas both BMDs were decreased by 4–5% in the non‐HRT group. The ER genotype did not modulate the femoral neck BMD change during the follow‐up. In contrast, in the non‐HRT‐group the lumbar spine BMD decreased more in subjects with the ER genotypes PP (6.4%) and Pp (5.2%) than in subjects with the pp genotype (2.9%) (p = 0.002). In the HRT group, the relative changes of the lumbar spine BMD were similar in all three ER genotype groups. Thus without HRT, the pp genotype was associated with a smaller decrease in the lumbar spine BMD than the Pp and PP genotypes. Long‐term HRT seemed to eliminate the ER genotype‐related differences in the BMD. We conclude that subjects with the ER PvuII genotypes PP and Pp may have a greater risk of relatively fast bone loss after menopause than those with the pp genotype and that they may preferentially derive benefit from HRT. (J Bone Miner Res 2000;15:315–321)

Effect of 1,25(OH)2D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells

Hormone-dependent accumulation of specific binding sites for 1,25(OH)2D3 and changes in human 1,25-dihydroxy-vitamin D receptor (hVDR) mRNA levels were examined in cell lines (MG-63, SaOs-2 and U2-Os) derived from human bone. Osteocalcin synthesis and secretion as well as alkaline phosphatase activity were also characterized as biochemical markers of the osteoblastic phenotype. Specific binding sites for 1,25(OH)2D3 were quantified by incubating cultured intact cells with [3H]1,25(OH)2D3 at 37 degrees C. Based on the uptake of 1,25(OH)2D3, there were about 3000 to 4000 receptor molecules per cell with apparent dissociation constants varying between 0.02 to 0.03 nM. The binding was saturated with 1,25(OH)2D3 in 3 to 6 h after the hormone addition and further exposure to the hormone resulted in an upregulation of the bindings sites. The levels were elevated by as little as 10 to 200 pM 1,25(OH)2D3, and maximal binding was achieved with 0.2-0.7 nM 1,25(OH)2D3. Treatment with 1,25(OH)2D3 also resulted in a clear increase (about 3-fold) in hVDR mRNA by 24 h in all three cell lines. The increase in hVDR mRNA level was time- and dose-dependent. MG-63 cells responded with 2- and 15-fold increases, respectively, in intracellular and secreted levels of osteocalcin after the 1,25(OH)2D3-treatment. In dot-blot hybridization assay, MG-63 cells expressed osteocalcin mRNA which was inducible with 1,25(OH)2D3 while, in SaOs-2 and U2-Os cells, osteocalcin mRNA was not detected under the same circumstances. Also, no secretion of osteocalcin was detected in SaOs-2 and U2-Os cells with or without addition of 1,25(OH)2D3.

Aerobic exercise and bone mineral density in middle-aged finnish men: a controlled randomized trial with reference to androgen receptor, aromatase, and estrogen receptor α gene polymorphisms☆

Abstract Our aim was to investigate associations of the polymorphic loci of androgen receptor (AR), aromatase CYP19, and estrogen receptor α (ERα) genes with bone mineral density (BMD) in a four-year controlled randomized exercise intervention trial in Finnish middle-aged men. Additionally, we studied whether the gene polymorphisms affect circulating testosterone (T), estradiol (E 2 ), and sex hormone-binding globulin concentrations. The polymorphic CAG repeat of the AR gene, the TTTA repeat of the human aromatase gene, and the Pvu II site of the ERα gene were analyzed. BMDs of the lumbar spine (L2–L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). In the exercise group, the subjects with the ERα gene PP or Pp genotypes showed an increase (+6.5 and +5.1%, respectively) in lumbar spine BMDs ( P = 0.007; repeated measures ANOVA) during intervention, while there was no change in the subjects with the pp genotype. The long TTTA repeat (TTTA 9–12 ) in aromatase gene was associated with greater height ( P = 0.026) and lower BMI ( P = 0.029) values than the short TTTA repeat (TTTA 6–8 ). With regard to the AR gene, no statistically significant differences in bone properties were found between the genotypes. There were no significant associations of any analyzed polymorphic sites with the serum sex steroid hormone concentrations in the exercise or reference group. In conclusion, the Finnish middle-aged men with ERα PP or Pp genotypes appear to have increased BMD values in the lumbar spine. This increase may reflect a predisposition to age-related degenerative changes in the spine. In addition, the AR CAG repeat and aromatase TTTA repeat do not modify the effect of regular aerobic exercise on BMD.

The Protective Effect of Hormone‐Replacement Therapy on Fracture Risk Is Modulated by Estrogen Receptor α Genotype in Early Postmenopausal Women

Genetic factors regulate bone mineral density (BMD) and possibly development of osteoporosis. It has been suggested that estrogen receptor α (ERα) genotype is associated with BMD, but the association between ERα genotype, fracture risk, and postmenopausal hormone replacement therapy (HRT) has not been studied. Therefore, we evaluated whether ERα polymorphism is associated with fracture risk in a 5‐year trial with HRT in a population‐based, randomized group of 331 early postmenopausal women. The participants consisted of two treatment groups: the HRT group (n = 151) received a sequential combination of 2 mg of estradiol valerate (E2Val) and 1 mg of cyproterone acetate with or without vitamin D3, 100‐300 IU + 93 mg calcium as lactate per day; and the non‐HRT group (n = 180) received 93 mg of calcium alone or in combination with vitamin D3, 100‐300 IU/day. All new symptomatic, radiographically defined fractures were recorded. Pvu II restriction fragment length polymorphism of the ERα was determined using polymerase chain reaction (PCR). In all, 28 women sustained 33 fractures during the approximately 5.1‐year follow‐up. In the HRT group, the ERα genotype (PP, Pp, and pp) was not significantly associated with fracture risk (p = 0.138; Cox proportional hazards model). When the genotype was dichotomized (PP + Pp vs. pp), the incidence of new fractures in the HRT group was significantly reduced in women with the P allele (p = 0.046) with the relative risk (HR) of 0.25 (95% CI, 0.07‐0.98), in comparison with the non‐P allele group. After adjustment for time since menopause and previous fracture, the association between the dichotomous genotype and fracture risk persisted with HR of 0.24 (95% CI, 0.06‐0.95; p = 0.042). In the non‐HRT group, the ERα genotype was not significantly associated with fracture risk. During HRT, women with the pp genotype have a greater fracture risk than those with the P allele. The results suggest that the pp genotype is a relatively hormone‐insensitive genotype, and it appears that women with the P allele may benefit more from the protective effect of HRT on fracture risk than women with the pp genotype.

Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo.

Osteoporosis is a major problem in elderly population. We tested the hypothesis whether vascular endothelial growth factor (VEGF‐A) gene transfer is an appropriate way to enhance bone formation and recruitment of osteoblasts in vivo. Adenovirus vectors containing VEGF‐A or lacZ cDNAs (1.4×1010 pfu) were injected locally into right distal femurs of New Zealand White rabbits. Saline was injected into all contralateral distal femurs. One and three weeks after the gene transfers femurs were collected for analyses. X‐Gal staining showed that up to 20% of the bone marrow cells were transfected although gene transfer also resulted in biodistribution of the vector and expression of the transgene in liver and spleen. Trabecular bone hard tissue histomorphometry of the distal femurs was performed to analyze the effect of gene transfer on bone turnover. When compared with unilateral lacZ transfected trabecular bone at one‐week and three‐week time points, VEGF‐A gene transfer significantly increased bone formation parameters, such as osteoblast number, osteoid volume, and bone volume. Also, bone resorption surface was greatly reduced. It is concluded that injection of adenovirus vector can transfect bone marrow cells in vivo with a relatively high efficiency. Our results suggest that adenovirus‐mediated VEGF‐A gene transfer induces bone formation via increasing osteoblast activity and may be useful for the treatment of osteoporosis and other diseases that require efficient osteogenic therapy.

Association Between Exercise and Pubertal BMD Is Modulated by Estrogen Receptor α Genotype

Genetic and environmental factors contribute to bone mass, but the ways they interact remain poorly understood. This study of 245 pre‐ and early pubertal girls found that the PvuII polymorphism in the ER‐α gene modulates the effect of exercise on BMD at loaded bone sites.

Dioxins interfere with differentiation of osteoblasts and osteoclasts

We have previously shown that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone growth, modelling and mechanical strength in vivo. In this study, we utilized differentiation of bone marrow stem cells to osteoblasts and osteoclasts as a model system to study the effects of TCDD on bones. Stem cells were isolated from bone marrow of femurs and tibias of rats and mice. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells using quantitative RT-PCR. TCDD significantly and dose-dependently decreased the mRNA levels of RUNX2, alkaline phosphatase and osteocalcin. Also the activity of alkaline phosphatase was significantly inhibited in both rat and mice cells. In the case of osteoclasts, TCDD decreased the number of TRACP+ multinucleated cells, with corresponding decreases in the number of F-actin rings and the area of resorption. Studies in AHR-knockout mice indicated that TCDD has no effect on the expression of osteoblastic differentiation markers suggesting that TCDD mediates its effects by AHR. Both osteoblastic and osteoclastic effects took place at very low doses of TCDD, as in most cases 100 fM TCDD was enough to significantly affect the differentiation markers. Therefore, differentiation of osteoblasts and osteoclasts from bone marrow stem cells seems to be a very sensitive target for TCDD. Disrupting effects in osteoblastic cells, in addition to disturbed osteoclastogenesis, may thus play a role in adverse effects on bone quality in TCDD exposed animals.

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

Relation of aromatase gene polymorphism and hormone replacement therapy to serum estradiol levels, bone mineral density, and fracture risk in early postmenopausal women*

BACKGROUND. After the menopause, estrogen synthesis from androgens and androgen precursors by aromatase is the main source of circulating estrogens. AIM. To evaluate whether aromatase gene (CYP19)polymorphism affects circulating estradiol (E[Formula: See Text])levels, bone mineral density (BMD), BMD change or fracture risk. METHODS. A 5-year randomized hormone replacement therapy (HRT) trial on 331 early postmenopausal women (mean baseline age 52.7 ± 2.3 years). The participants consisted of two treatment groups: the HRT group (n = 151) received a sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate with or without vitamin D[Formula: See Text], 100-300 IU + 93 mg calcium as lactate/day, and the non-HRT group (n = 180) received 93 mg calcium alone or in combination with vitamin D[Formula: See Text], 100-300 IU/day for 5 years. BMD was measured from lumbar spine and proximal femur (DXA) before and after the 5-year trial. All new symptomatic, radiographically defined fractures were recorded during the follow-up. The polymorphism (intron 4 TTTA repeat) of CYP19 was evaluated after PCR amplification of the polymorphic site. CYP19 polymorphism was divided into three repeat groups: short (length of 7 or 8 in both alleles; n = 135), long (length of 11 or higher in both alleles; n = 47), and medium (rest of the values; n = 149). RESULTS. Of the baseline characteristics, only physical activity was associated with CYP19 polymorphism (P = 0.04) and a borderline significance was observed with previous fractures (P = 0.05). In the HRT or non-HRT groups, the 5-year serum E[Formula: See Text] change was not associated with CYP19 polymorphism (P = 0.87 and 0.74, respectively). Further, the polymorphism did not influence the calculated annual changes of lumbar or femoral neck BMD during the 5-year follow-up in the HRT (P = 0.60 and 0.17, respectively) or non-HRT (P = 0.92 and 0.80, respectively) groups. In all, 28 women sustained 33 fractures during the follow-up. The CYP19 polymorphism was not significantly associated with fracture risk (P = 0.89 and 0.23 respectively; Cox proportional hazards model) in the HRT or non-HRT groups. CONCLUSIONS.CYP19 polymorphism was not associated with circulating E[Formula: See Text] levels, BMD values, or fracture risk in these early postmenopausal Finnish women. If such an association exists in women, it may become apparent in older age groups.

Type I procollagen synthesis is regulated by steroids and related hormones in human osteosarcoma cells

Changes in the synthesis of type I collagen, the major extracellular matrix component of skin and bone, are associated with normal growth, tissue repair processes, and several pathological conditions. Expression of the COL 1A1 gene is regulated by transcriptional and post‐transcriptional mechanisms. However, the hormonal regulation of type I collagen synthesis in human bone has not been well characterized. We have studied the influence of calcitriol, dexamethasone, retinoic acid, and estradiol on the COL 1A1 gene expression by determining the secretion of the C‐terminal propeptide (PICP) and the levels of α1(I) procollagen mRNA in cultured human MG‐63 and SaOs‐2 osteoblast‐like osteosarcoma cells. Similar experiments were also performed with respect to expression of the nuclear proto‐oncogenes, c‐fos and c‐jun, in MG‐63 cells.