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.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture, and the trait is under genetic control by a large number of genes. It is recognized that estrogen plays an important role in the maintenance of bone mass by binding to estrogen receptor alpha (ERalpha). RIZ1 has previously been shown to be a specific ERalpha coactivator and strongly enhances its function both in vivo and in vitro. We performed in vitro studies comparing the abilities of RIZ1 P704 polymorphic variants (homozygous presence, P704+; absence, P704-; heterozygosity P704(+/-) of a proline at position 704) to coactivate the ERalpha and also examined the polymorphism associated to BMD of 343 Swedish women, aged 20-39 yr. The expression vector containing P704- RIZ1 showed an impaired response in coactivating ERalpha in a ligand- and dose-dependent manner compared with P704+ RIZ (P < 0.0001). The genotype frequencies were 19% (P704+), 32% (P704-), and 49% (P704(+/-)) and were in Hardy-Weinberg equilibrium. BMD at the heel was higher in the P704+ genotype group than in the P704(+/-) group (P = 0.02), which was evident also after corrections for fat and lean mass (P = 0.03). We conclude that RIZ1 may be a new candidate gene for involvement in the variation seen in BMD.

A single nucleotide polymorphism in the promoter region of the osteoprotegerin gene is related to intima-media thickness of the carotid artery in hypertensive patients. The Swedish Irbesartan Left Ventricular Hypertrophy Investigation vs Atenolol (SILVHIA).

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor receptor family, and in previous studies has been shown to regulate osteoclast activity and differentiation. Ablation of the OPG gene in mice results in calcification of the aorta and renal arteries. We have previously reported an association between a single nucleotide polymorphism in the promoter region of OPG and vascular morphology and function in healthy humans. The objective with this study was to confirm our previous results in a larger population, and in addition, to study subjects with hypertension. The OPG genotype was determined by restriction fragment length and the intima-media thickness (IMT) of the common carotid artery was measured by ultrasound in 100 patients with hypertension and left ventricular hypertrophy, and 75 healthy normotensive control subjects. In the hypertensive group subjects with the CC genotype (n=24) showed a significantly increased IMT compared to those with the TC (n=52, p=0.007) and TT (n=24, p=0.009) genotype, in the hypertensive group only (mean +/- SD for TT=0.88 +/- 0.21 mm, TC=0.90 +/- 0.16 mm, CC=1.05 +/- 0.31 mm). The allele distribution did not differ between hypertensive and control individuals. The present study confirms our previous finding and shows that polymorphism in the promoter region of OPG is associated with vascular morphology in hypertensive subjects.

Single nucleotide polymorphisms in the human gene for osteoprotegerin are not related to bone mineral density or fracture in elderly women.

Osteoprotegerin (OPG), a secreted member of the tumor necrosis factor receptor family, is a potent inhibitor of osteoclast activation and differentiation. In animal models OPG prevents bone loss, and in humans bone resorption can be reduced by injections of OPG. OPG may also play a role in cardiovascular disease since mice lacking the OPG gene display arterial calcification. In a screening effort of the OPG gene, we recently discovered a single nucleotide polymorphism in the promoter region of OPG (T950C), and reported an association with vascular morphology and function in 59 healthy individuals. Due to the pronounced effect of OPG on bone turnover, the present study was conducted to investigate whether OPG polymorphisms are also associated with bone mineral density or with fracture. The relationship between single nucleotide polymorphisms in the promoter region of OPG (T950C) and the first intron (C1217T), and bone mineral density, measured by DXA in the hip or spine or ultrasound of the heel, was investigated in the Malmö OPRA-study of 1044 women, all 75 years old. The possible relation to fracture incidence was also analyzed. Among the 858 and 864 individuals respectively, genotyped, no significant associations between the investigated single nucleotide polymorphisms and bone mineral density measurements (T950C P = 0.50-0.64, C1217T P = 0.51-1.00), quantitative ultrasound measurements of the calcaneus, or fractures (T950C P = 0.61-0.66, C1217T P = 0.14-0.33) were found. Thus, our results show that polymorphisms in the OPG gene, one of which has previously been found to be associated with cardiovascular morphology and function, are not associated with bone mineral density in elderly Swedish women.

Polymorphisms in the CYP19 and AR genes--relation to bone mass and longitudinal bone changes in postmenopausal women with or without hormone replacement therapy: The Danish Osteoporosis Prevention Study.

Polymorphisms in the androgen receptor ( AR) gene and genes encoding enzymes involved in synthesis of sex steroids (e.g., the CYP19 gene encoding aromatase) have recently received attention in osteoporosis research. In the Danish Osteoporosis Prevention Study, recent postmenopausal women were allocated to either hormone replacement therapy (HRT) or no treatment. We genotyped 1792 women for the CYP19 (TTTA)(n) repeat [short (TTTA)(n 7)] the CYP19 C(1558)-T, and the AR (CAG)(n) repeat polymorphism [short (CAG)(n < 22), long (CAG)(n >or= 22)], and investigated associations with bone mineral density (BMD) and 5-year change in BMD. The CYP19 polymorphisms were in strong linkage disequilibrium. Perimenopausal bone mass or bone loss in untreated women was not associated with the CYP19 polymorphisms. In hormone-treated women, BMD increase in the femoral neck was highest (+0.3%/year) for long CYP19 alleles, lowest (-0.09%/year) for short alleles, and intermediate (-0.002%/year) in heterozygous women, P = 0.015. Differences were also significant in the lumbar spine, total hip, and ultradistal forearm. The C(1558)-T T-allele was associated with a more pronounced response to HRT ( P = 0.04, total hip). AR genotype was not related to BMD, but a modifying effect of sex hormone-binding globulin (SHBG) was present. In the highest SHBG quartile (SHBG > 95 nmol/1, n = 222), AR genotype was associated with baseline BMD (femoral neck: P < 0.001, total hip: P = 0.008), but without a clear gene dosage effect. We have demonstrated that polymorphisms in the CYP19 gene are associated with the magnitude of bone gain in response to HRT and that the (CAG)(n) repeat polymorphism in the AR gene is associated with bone mass in women with high levels of SHBG. These findings emphasize the complexity of the genetics of bone mass and bone loss.

A poly adenosine repeat in the human vitamin D receptor gene is associated with bone mineral density in young Swedish women.

Peak bone mass (PBM) and subsequent bone loss are important risk factors for development of osteoporosis later in life, and twin studies have reported strong genetic influence on PBM. The genetic factor influencing PBM is polygenetic, and many genes most likely exert relatively small effects on bone mass. The poly adenosine (A) microsatellite in the 3' untranslated region (UTR) of the VDR gene has been associated with both prostate and breast cancer risk but little is known about the effect of bone mineral density (BMD). In this report the poly A microsatellite and the linked BsmI SNP have been investigated in a population-based cohort of 343 Swedish women, aged 20-39. BMD was measured by dual x-ray absorptiometry at the spine, proximal femur, total body and heel and by quantitative ultrasound at the heel. Correlations were found between VDR genotypes and BMD at lumbar spine L2-L4, (ss versus LL, P = 0.03 and BB versus bb, P = 0.02, respectively), with a similar pattern concerning total hip (ss versus LL, P = 0.12 and BB versus bb, P = 0.16 respectively). After corrections for age, height, fat and lean mass, the VDR BsmI genotype was still associated to BMD at the lumbar spine (BB versus bb, P = 0.03). The polymorphisms were in linkage disequilibrium (Chi-square = 566, P < 0.0001). In conclusion, genetic variation in the VDR is associated with BMD in premenopausal women, and further studies are needed to evaluate a possible functional role of the VDR 3'UTR poly A repeat, a region that has shown to be of important for mRNA stability.

Interleukin (IL)-13 and IL-4 inhibit proliferation and stimulate IL-6 formation in human osteoblasts: evidence for involvement of receptor subunits IL-13R, IL-13Ralpha, and IL-4Ralpha.

Interleukin-13 (IL-13) inhibits cell proliferation and stimulates interleukin-6 (IL-6) formation in isolated human osteoblasts (hOBs). Because the related cytokine, interleukin-4 (IL-4), is known to exert effects similar to IL-13 in other tissues, and because IL-4 has been implicated as a regulator of bone metabolism, we compared the effects of IL-13 and IL-4 on cell proliferation, IL-6 synthesis, the expression of osteoblastic phenotypic markers in hOB cultures. Also, the receptor proteins mediating these effects in hOBs have been partly characterized. IL-4 and IL-13 dose-dependently inhibited [(3)H]-thymidine incorporation into the DNA of human osteoblasts and stimulated secretion of IL-6 into culture supernatants. IL-13 and IL-4 also increased the mRNA levels of IL-6, as measured by RNAse protection assay. Furthermore, IL-13 and IL-4 dose-dependently enhanced alkaline phosphatase (ALP) activity, but did not affect osteocalcin or collagen type I synthesis. IL-4 was tenfold more potent than IL-13 in inducing both ALP activity and IL-6 secretion, whereas the cytokines were equipotent as inhibitors of cell proliferation. The expression of mRNA for receptor subunits previously implicated in IL-4 and IL-13 signaling was investigated by reverse transcriptase-polymerase chain reaction. IL-13R, IL-13Ralpha, and IL-4Ralpha mRNA were repeatedly detected in hOBs, whereas mRNA for IL-2Rgamma(C) was not detected. Receptor-blocking antibodies to IL-4Ralpha inhibited the induction of IL-6 formation by both IL-4 and IL-13, indicating that both cytokines utilize this receptor subunit in signaling. However, the antibodies did not affect the IL-4/-13-induced inhibition of [(3)H]-thymidine incorporation or the stimulation of alkaline phosphatase (ALP), suggesting that IL-4Ralpha does not mediate these effects of IL-4/-13 in hOBs. We conclude that the cytokines IL-13 and IL-4, through sharing of receptor components, induce similar effects on hOBs, causing inhibition of cell proliferation, stimulation of IL-6, and enhanced ALP activity.

Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells.

Osteoprotegerin (OPG) is a soluble receptor for receptor activator of NF kappa B-ligand, a factor required for osteoclastogenesis. OPG secreted from bone marrow stromal cells is believed to inhibit osteoclast differentiation and several agents known to influence bone resorption have been demonstrated to regulate mRNA levels of OPG. In this report we have investigated the secretion of OPG protein from primary cultures of human bone marrow stromal cells. An ELISA was developed for measuring the concentration of OPG in culture medium. OPG secretion was decreased by 50% when the human bone marrow stromal cells were treated with 1 microM of prostaglandin E(2), possibly through activation of the protein kinase A-pathway since stimulation of protein kinase A by forskolin also inhibited OPG secretion. Treatment with phorbol 12,13 di butyrate, an activator of the protein kinase C-pathway, potently stimulated the secretion of OPG from human bone marrow stromal cells. The cells were also stimulated with inflammatory mediators and glucocorticoids. Treatment with interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) stimulated OPG secretion to 500% and 400% of control whereas dexamethasone decreased OPG production by 40%. In conclusion, an ELISA measuring OPG in cell culture media was developed. Using this ELISA, the amount of OPG secreted from human bone marrow stromal cells was clearly detectable, and the secretion of OPG-protein was potently regulated by prostaglandin E(2), forskolin, phorbol 12,13 di butyrate, IL-1 alpha, TNF-alpha, and dexamethasone.

Osteoprotegerin mRNA is expressed in primary human osteoblast-like cells: down-regulation by glucocorticoids.

Osteoprotegerin (OPG) is a recently cloned member of the tumour necrosis factor receptor family. It has been suggested that this secreted glycoprotein acts as an inhibitor of osteoclastic differentiation. Expression of OPG has previously been demonstrated in a number of tissues. However, it is still unclear whether or not OPG is expressed by human osteoblasts. We have used the RNase protection assay to demonstrate the OPG transcript in primary cultured human osteoblast-like cells, human marrow stroma cells and osteosarcoma cell lines. Furthermore, we have studied the effect of glucocorticoids on OPG mRNA levels in these cells. We demonstrate that glucocorticoids decrease the OPG transcript in a dose- and time-dependent manner. The time-course study reveals that hydrocortisone (10(-6) M) decreases OPG mRNA levels within 2 h. This decrease is transient, reaching control levels again after 24 h. Our findings demonstrate that human osteoblasts express the mRNA corresponding to OPG, an inhibitor of osteoclast differentiation. The finding that OPG mRNA levels are decreased by glucocorticoids indicates that a reduced production of OPG from osteoblasts and/or marrow stroma cells could, in part, explain glucocorticoid-induced bone resorption.

Interleukin-13 inhibits cell proliferation and stimulates interleukin-6 formation in isolated human osteoblasts.

Interleukin-13 (IL-13) is a recently identified cytokine that is secreted by activated T cells and regulates inflammatory responses. We have investigated the effects of IL-13 on isolated human osteoblast-like cells (hOB). IL-13 dose-dependently (1-100 pmol/L) reduced the incorporation rate of [3H]thymidine in hOB cells by more than 50%. Using a cell metabolic assay as well as direct cell counting, we found that treatment with IL-13 lead to a decrease in hOB cell number. The effect was both time and dose dependent, and after 12 days of culture, treatment with IL-13 (0.1 nmol/L) caused a 70% decrease in the number of cells. Also, IL-13 increased the levels of IL-6 messenger ribonucleic acid in hOBs, as measured by ribonuclease protection assay, and stimulated secretion of IL-6 into culture supernatants. In conclusion, IL-13 inhibits cell proliferation and increases IL-6 formation in human osteoblasts. Our findings suggest that IL-13 may cause bone loss due to impaired osteoblastic growth and IL-6-induced osteoclast recruitment.

Tumor necrosis factor-alpha and -beta upregulate the levels of osteoprotegerin mRNA in human osteosarcoma MG-63 cells.

Osteoprotegerin (OPG) is a recently cloned soluble member of the tumor necrosis factor receptor family. OPG has been shown to inhibit osteoclast recruitment by binding to OPG-ligand, an osteoclast differentiating factor on osteoblastic stromal cells, thereby blocking osteoclastogenesis. In this report we have examined the effect of tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on OPG mRNA levels in the human osteosarcoma cell line MG-63. We demonstrate that both TNF-alpha and TNF-beta dose- and time-dependently upregulate the mRNA levels of OPG. The effect is significant at and above 5 pM of TNF-alpha and 1 pM of TNF-beta. The stimulatory effect on OPG mRNA levels in MG-63 cells was detected after 2 hrs of incubation with TNF-alpha or TNF-beta. These data demonstrate that the expression of OPG in osteoblasts, with subsequent effects on osteoclastogenesis, is regulated by TNFs.

Regulation of osteoprotegerin mRNA levels by prostaglandin E2 in human bone marrow stroma cells.

The recently cloned osteoclastogenesis inhibitory factor, or osteoprotegerin (OPG), has been shown to be a potent inhibitor of osteoclast formation. The inhibition is believed to be mediated through specific binding of OPG to a cell surface ligand on osteoblastic stromal cells. In this report we have studied the effect of the bone resorbing agent prostaglandin E2 (PGE2) on OPG mRNA levels in primary cultures of human bone marrow stroma cells (hBMSC). PGE2 dose- and time-dependently down-regulated the mRNA levels of OPG, as measured by RNAse protection assay. After 4 hours of stimulation with 1 microM PGE2, OPG mRNA levels were significantly decreased. The inhibitory effect was seen at and above 1 nM of PGE2. To elucidate whether the OPG mRNA levels are regulated via the proteinkinase A and/or the proteinkinase C pathways we stimulated cells with either forskolin (FSK) or phorbolic ester (PDbu) respectively. FSK (10 microM) decreased OPG mRNA levels to 50 % of control, whereas PE (10 nM) upregulated the mRNA levels to 250 % of control. These data show that PGE2 down-regulates the expression of OPG mRNA in hBMSC, probably via an increase in cAMP. This mechanism might be involved in PGE2-induced bone resorption.