The expression of IL-6 by osteoblasts is increased in healthy elderly individuals: stimulated proliferation and differentiation are unaffected by age.

Increasing age is associated with reduced bone mineral content and increased risk of fractures. This is caused by a relative insufficiency of osteoblasts compared with osteoclasts. We therefore wanted to examine the potential differences in proliferation, differentiation, and expression of cytokines between human osteoblasts (hOBs) obtained from young and elderly individuals. Cultures of hOBs were obtained from 11 elderly (73-85 years) and 15 young (21-27 years) healthy individuals. The cells were stimulated with hGH, IGF-I, hGH + IGF-I, and TGF-ß1. Proliferation was evaluated by thymidine incorporation, and differentiation was evaluated by alkaline phosphatase, OPG, and PINP production. Expression of IL-6, TGF-ß1, OPG, and RANKL was investigated using real-time PCR and three carefully selected housekeeping genes. Combined stimulation with hGH and IGF-I increased proliferation without differences between hOBs obtained from young and elderly individuals. hOBs from young individuals responded to stimulation with vitamin D with a more pronounced increase in alkaline phosphatase: 107 ± 17% vs. 43 ± 5%, P < 0.01. Stimulation with TGF-ß1 decreased OPG production by hOBs from elderly individuals but not from young individuals, P < 0.05. hOBs from elderly individuals expressed significantly higher amounts of IL-6 mRNA (P < 0.05) and less OPG and TGF-ß1 mRNA (P = 0.08 and P = 0.08, respectively) compared with hOBs from young individuals. In conclusion, hOBs from elderly individuals express more IL-6 mRNA and less OPG and TGF-ß1 mRNA than hOBs from young individuals. This could partly explain the reduced bone mass and increased fracture risk seen in the elderly. hOBs from young and elderly individuals responded similarly to short-term stimulation of proliferation and differentiation.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with osteoporotic vertebral fractures, but is a weak predictor of BMD.

Osteoporosis is a common disease with a strong genetic component. Linkage studies have suggested linkage between BMD and loci on chromosome 1. The MTHFR gene is located on chromosome 1. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, which is used for homocysteine methylation to methionine. The rare genotype (TT) of the C677T polymorphism has previously been demonstrated to be associated with increased plasma homocysteine levels in individuals with inadequate plasma folate levels. Recently, the TT genotype has been found to be associated with reduced bone mass. We therefore examined if the C677T polymorphism in the MTHFR gene is associated with changes in bone mass and risk of osteoporotic fractures in 388 osteoporotic patients and 336 normal individuals. The distributions of the genotypes CC, CT and TT in women with osteoporotic vertebral fractures and normal controls were 43.5%, 42.2% and 14.3% and 52.0%, 42.0% and 8.0%, respectively, chi2 = 5.62, P = 0.06. Since studies of the functionality of this polymorphism have revealed that only the TT genotype is associated with biochemical changes, we also compared the prevalence of the TT genotype versus the CT- and CC genotypes in patients and controls and found that the TT genotype is significantly more common in women with vertebral fractures (14.3%) compared with normal controls (8.0%), chi2 = 4.31, P < 0.05. Logistic regression analysis demonstrated that vertebral fractures were significantly associated with BMD (lumbar spine) and height but only marginally with the MTHFR genotype (P = 0.06). Multiple linear regression analysis revealed that weight, age and the MTHFR polymorphism were predictors of lumbar spine BMD in women. However, age- and gender-corrected BMD of the lumbar spine and the hip was not significantly different between MTHFR genotypes. Furthermore, individuals with the TT genotype did not have BMD significantly lower than the combined group of individuals with the CT- or CC genotypes. In conclusion, we have demonstrated that the rare TT genotype of the C677T polymorphism in the MTHFR gene is associated with increased risk of osteoporotic fractures in women and a weak predictor of lumbar spine BMD.

Polymorphisms in the transforming growth factor beta 1 gene and osteoporosis.

Transforming growth factor (TGF)-beta1 is the most abundant growth factor in human bone. It is produced by osteoblasts and inhibits osteoclast proliferation and activity and stimulates proliferation and differentiation of preosteoblasts. Several polymorphisms have been described in the TGF-beta1 gene. Previously, we and others have found associations between some of these polymorphisms and bone mass. We therefore wanted to examine if these polymorphisms are also predictors of osteoporotic fractures. The polymorphisms G(-1639)-A, C(-1348)-T, C(-765)insC, T(29)-C, G(74)-C, 713-8delC, C(788)-T, and T(816-20)-C were examined using RFLP and sequencing in 296 osteoporotic patients with vertebral fractures and 330 normal individuals. Bone mineral density (BMD) was examined at the lumbar spine and at the femoral neck by DXA. Genotype distributions were in H-W equilibrium. Linkage disequilibrium was found between the polymorphisms. The T(816-20)-C genotypes were distributed differently among osteoporotic patients and normal controls. The TT genotype was less common in individuals with osteoporotic fractures (chi(2) = 6.02, P < 0.05). BMD was higher in individuals with the TT-genotype (T(816-20)-C) at the lumbar spine, 0.960 +/- 0.173 g/cm(2) compared with individuals with the TC or CC genotypes: 0.849 +/- 0.181 g/cm(2) and 0.876 +/- 0.179 g/cm(2), respectively (P < 0.001, ANOVA). Similar differences between genotypes were found at the different hip regions as well as at the total hip. Individuals with the TT-genotype (C(-1348)-T) had higher bone mass at the femoral neck: 0.743 +/- 0.134 g/cm(2) compared with 0.703 +/- 0.119 g/cm(2) in individuals with TC or CC genotypes (P < 0.05). Individuals with the CC-genotype (T(29)-C) had higher bone mass at the femoral neck, 0.735 +/- 0.128 g/cm(2) compared with 0.703 +/- 0.120 g/cm(2) in individuals with TC or TT genotypes (P < 0.05) and at the total hip: 0.852 +/- 0.166 g/cm(2) vs. 0.818 +/- 0.149 g/cm(2), respectively (P < 0.05). None of the other polymorphisms were distributed differently in patients and controls and did not affect BMD. In conclusion, The TT genotype of the T(816-20)-C polymorphism is less common in patients with osteoporotic fractures and is associated with higher bone mass both at the lumbar spine and at the hip. The C(-1348)-T and T(29)-C polymorphisms were distributed similarly in osteoporotic patients and normal controls, however, the rare genotypes were associated with higher bone mass at the hip.

Polymorphisms in the osteoprotegerin gene are associated with osteoporotic fractures.

Osteoprotegerin (OPG) is a soluble receptor for RANKL and therefore a competitive inhibitor of osteoclast differentiation and activity. With this key role in the control of resorptive activity, we found that OPG is a candidate gene for genetic control of bone mass. We examined the promoter and the five exons with surrounding intron sequences of the OPG gene for polymorphisms in 50 normal patients and 50 patients with osteoporosis. We found 12 polymorphisms. Two sets of four and five polymorphisms, respectively, were in complete linkage. Subsequently, we examined the effect of the informative polymorphisms A163-G (promoter), T245-G (promoter), T950-C (promoter), G1181-C (exon 1), and A6890-C (intron 4) on the prevalence of osteoporotic fractures, bone mass, and bone turnover in 268 osteoporotic patients and 327 normal controls. In A163-G the variant allele G was more common among fracture patients: 34.0% versus 26.3% in normal controls (p < 0.05) and the odds ratio (OR) for a vertebral fracture, if an individual has the G allele, was 1.44 (1.00-2.08). In T245-G the variant allele G was more common in osteoporotic patients: 12.4% versus 6.5% (p < 0.02) and the OR for vertebral fracture, if an individual has the G-allele, was 2.00 (1.10-3.62). G1181-C is located in the first exon and causes a shift in the third amino acid from lysine to asparagine. The CC genotype was less common among fracture patients: 26.3% versus 36.7% in the normal controls (p < 0.01). T950-C and A6890-C were not distributed differently among patients with osteoporosis and normal controls. None of the polymorphisms affected bone mineral density (BMD) or biochemical markers of bone turnover in the normal controls. In conclusion, we have examined the human OPG gene for polymorphisms and found 12. The rare alleles of the A163-G and T245-G were significantly more common among patients with vertebral fractures.