Meta-analysis identifies a MECOM gene as a novel predisposing factor of osteoporotic fracture.

BACKGROUND: Osteoporotic fracture (OF) as a clinical endpoint is a major complication of osteoporosis. To screen for OF susceptibility genes, we performed a genome-wide association study and carried out de novo replication analysis of an East Asian population. METHODS: Association was tested using a logistic regression analysis. A meta-analysis was performed on the combined results using effect size and standard errors estimated for each study. RESULTS: In a combined meta-analysis of a discovery cohort (288 cases and 1139 controls), three hospital based sets in replication stage I (462 cases and 1745 controls), and an independent ethnic group in replication stage II (369 cases and 560 for controls), we identified a new locus associated with OF (rs784288 in the MECOM gene) that showed genome-wide significance (p=3.59×10(-8); OR 1.39). RNA interference revealed that a MECOM knockdown suppresses osteoclastogenesis. CONCLUSIONS: Our findings provide new insights into the genetic architecture underlying OF in East Asians.

A higher serum gamma-glutamyl transferase level could be associated with an increased risk of incident osteoporotic fractures in Korean men aged 50 years or older.

Oxidative stress has detrimental effects on bone metabolism, and gamma-glutamyl transferase (GGT) is known to play an important role in the generation of free radical species through the extra-cellular hydrolysis of glutathione, the main cellular antioxidant. We performed a large longitudinal study with an average follow-up period of 3 years to investigate the association between baseline serum GGT levels and the development of future osteoporotic fractures (OFs) in men. A total of 16,036 Korean men aged 50 years or older who had undergone comprehensive routine health examinations were enrolled. Incident fractures at osteoporosis-related sites (e.g., hip, spine, distal radius, and proximal humerus) that occurred after baseline examinations were identified from the nationwide claims database of the Health Insurance Review and Assessment Service of Korea using selected ICD-10 codes. Among the study subjects, 156 cases (1.0%) developed incident OFs during the study period. The event rate was 32.7 (95% CI = 28.0-38.3) per 10,000 person-years. Multivariable adjusted Cox proportional hazard analyses adjusted for age, body mass index, lifestyle factors, and medical and drug histories revealed that the hazard ratio per standard deviation increase of the baseline GGT levels for the development of incident fractures was 1.115 (95% CI = 1.011-1.230). These data provide the first epidemiological evidence, in support of previous in vitro and animal studies, of the harmful effects of GGT on bone metabolism, and indicate that the serum GGT level may be a useful biomarker of poor bone health outcomes in men.

High serum total bilirubin as a protective factor against hip bone loss in healthy middle-aged men.

Bilirubin is known to have a physiologic role as an antioxidant that efficiently scavenges peroxyl radicals and suppresses oxidation, and oxidative stress has detrimental effects on bone metabolism. In the present study, we performed a 3-year longitudinal study of healthy middle-aged men, investigating the association between serum total bilirubin concentrations and annualized changes in bone mineral density (BMD). The study enrolled a total of 917 Korean men aged 40 years or older who had undergone comprehensive routine health examinations with an average follow-up interval of 3 years. BMD at proximal femur sites was measured with dual-energy X-ray absorptiometry using the same equipment at baseline and follow-up. The overall mean annualized rates of bone loss at the total femur, femoral neck, and trochanter were -0.25 %/year, -0.34 %/year, and -0.44 %/year, respectively. After adjustment for potential confounders, the rates of bone loss at all proximal femur sites were significantly attenuated in a dose-response fashion across increasing bilirubin concentrations (P = 0.006-0.046). Moreover, compared to subjects in the lowest bilirubin quartile category, those in the highest bilirubin quartile category showed significantly less bone loss at all proximal femur sites after adjustment for confounding factors (P = 0.010-0.048). This study provides the first clinical evidence that serum total bilirubin could be a protective marker against future bone loss, especially in subjects without liver diseases.

TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition.

Although recent clinical studies have suggested a possible role for sclerostin, a secreted Wnt antagonist, in the pathogenesis of postmenopausal osteoporosis, the detailed mechanisms how estrogen deficiency regulates sclerostin expression have not been well-elucidated. Bilateral ovariectomy or a sham operation in female C57BL/6 mice and BALB/c nude mice was performed when they were seven weeks of age. The C57BL/6 mice were intraperitoneally injected with phosphate-buffered serum (PBS), 5 µg/kg ß-estradiol five times per week for three weeks, or 10 mg/kg TNF-α blocker three times per week for three weeks. Bony sclerostin expression was assessed by immunohistochemistry staining in their femurs. The activity and expression of myocyte enhancer factors 2 (MEF2), which is essential for the transcriptional activation of sclerostin, in rat UMR-106 osteosarcoma cells were determined by luciferase reporter assay and western blot analysis, respectively. Bony sclerostin expression was stimulated by estrogen deficiency and it was reversed by estradiol supplementation. When the UMR-106 cells were treated with well-known, estrogen-regulated cytokines, only TNF-α, but not IL-1 and IL-6, increased the MEF2 activity. Consistently, TNF-α also increased the nuclear MEF2 expression. Furthermore, the TNF-α blocker prevented the stimulation of bony sclerostin expression by ovariectomy. We also found that there was no difference in sclerostin expression between ovariectomized nude mice and sham-operated nude mice. In conclusion, these results suggest that TNF-α originating from T cells may be at least in part responsible for stimulating the sclerostin expression observed in an estrogen-deficient condition.

(-)-Epigallocathechin-3-gallate, an AMPK activator, decreases ovariectomy-induced bone loss by suppression of bone resorption.

Previously, we showed that AMP-activated protein kinase (AMPK) negatively regulates receptor activator of nuclear factor-κB ligand-induced osteoclast formation in vitro. The present study investigated the effect of (-)-epigallocathechin-3-gallate (EGCG), an AMPK activator, on ovariectomy (OVX)-induced bone loss in mice. Female mice subjected to OVX were administered EGCG for 8 weeks. We measured total-body bone mineral density (BMD) before and after the operation at an interval of 4 weeks. We performed micro-computed tomography (micro-CT) of the tibia and bone histomorphometric examination of the femur. Western blot analysis was additionally performed, to detect levels of the phosphorylated and total forms of AMPK-α in calvarial extracts. EGCG prevented OVX-induced body weight gain. The OVX control did not show a significant increase in BMD values at baseline and after treatment, unlike the sham control. EGCG attenuated OVX-induced bone loss. Micro-CT experiments revealed that EGCG induced a significant increase in trabecular bone volume and trabecular number and a decrease in trabecular spacing compared to the OVX control. Histomorphometric analyses further showed that EGCG suppressed osteoclast surface and number. Phosphorylated AMPK expression was significantly elevated in bone following EGCG treatment. Our findings collectively indicate that EGCG decreases OVX-induced bone loss via inhibition of osteoclasts.

Efficacy of intravenously administered ibandronate in postmenopausal Korean women with insufficient response to orally administered bisphosphonates.

We investigated rates of insufficient and over-responsiveness to orally administered bisphosphonates in postmenopausal women, and tested the efficacy of intravenous ibandronate in patients with insufficient response to orally administered bisphosphonates. Postmenopausal women were treated with either alendronate (70 mg/week; n = 88) or risedronate (35 mg/week; n = 84) for 1 year, and their response to orally administered bisphosphonates was assessed using serum C-telopeptide (CTX) levels. Insufficient responders were changed to once-quarterly intravenous ibandronate 3 mg injection (n = 13) or maintained on orally administered bisphosphonates (n = 19), according to patients' preference, for an additional 1 year. There was no significant difference in baseline characteristics between two orally administered bisphosphonate groups except the bone mineral density values at the lumbar spine. Insufficient rate was higher in the risedronate group (19.0 %) than in the alendronate group (8.0 %), using the premenopausal serum CTX median as a cut-off (P = 0.043). The over-response rate among the alendronate group (59.1 %) was significantly higher than that in the risedronate group (38.1 %), based on a serum CTX cut-off value of 0.100 ng/ml (P = 0.006). Intravenous ibandronate suppressed serum CTX levels to a significantly greater degree at 7 days after the second dosing (0.191 ± 0.110 ng/mL; P < 0.001) and 3 months after the fourth dosing (0.274 ± 0.159 ng/mL; P = 0.004) among insufficient responders, compared with post-oral/pre-intravenous levels (0.450 ± 0.134 ng/mL). Rates of insufficient and over-responsiveness to orally administered bisphosphonates were considerable, and a change to intravenous bisphosphonates may be considered in patients showing an insufficient response to orally administered bisphosphonates.

Homocysteine-lowering therapy or antioxidant therapy for bone loss in Parkinson's disease.

We investigated whether homocysteine (Hcy)- lowering therapy or an antioxidant prevented bone loss in Parkinson's disease (PD) patients taking levodopa. Forty-two PD patients with low bone mineral density (BMD) taking levodopa were randomly assigned to Hcy-lowering therapy (5 mg folate and 1500 microg vitamin B(12) daily), alpha-lipoic acid (alpha-LA) therapy (1200 mg daily), or control groups. Primary outcomes were BMD changes from baseline to 12 months. Secondary outcomes were changes in Hcy level, and C-telopeptide (CTX) levels at 12 months. Forty-one patients completed the study. Hcy-lowering therapy resulted in significantly greater BMD changes at the lumbar spine (4.4%), total femur (2.8%), and femur shaft (2.8%) than control (P = 0.005-0.023). BMD changes in the alpha-LA therapy group were similar to those of the control group, but changes at the trochanter (4.6%) were significantly greater in the alpha-LA therapy group than in the control group after adjustment for body mass index changes. Hcy concentrations decreased to 35.2% +/- 13.4% in the Hcy-lowering therapy group, but increased in other groups. Serum CTX levels at 12 months tended to be lower in the Hcy-lowering group (0.442 +/- 0.024 ng/mL) than control group (0.628 +/- 0.039 ng/mL) (P = 0.159). This small trial suggests that Hcy-lowering therapy may prevent bone loss in PD patients taking levodopa.

Hyperhomocysteinemia due to levodopa treatment as a risk factor for osteoporosis in patients with Parkinson's disease.

Parkinson's disease (PD) patients have been reported to have lower bone mineral density (BMD) and higher fracture risk than individuals without PD. We assessed the association between hyperhomocysteinemia due to levodopa intake and BMD in PD patients. We measured serum homocysteine (Hcy) concentrations and BMD in the proximal femur and lumbar spine of PD patients aged 55 years or older (n = 95) and three age-/gender-matched control subjects (n = 285). The prevalence of osteoporosis was higher in both men (2.5-fold) and women (1.7-fold) with PD than in controls, and adjusted odds ratios for osteoporosis were 3.57 (95% confidence interval [CI], 1.25-10.20) for men and 2.54 for women (95% CI, 1.31-4.93) with PD. Serum Hcy concentrations were significantly higher in PD patients (median = 13.0 micromol/l) than controls (median = 11.5 micromol/l) (P = 0.005). Serum Hcy concentrations were independently associated with BMD values at all proximal femur sites in all subjects (P = 0.005 to 0.012). In PD patients, higher serum Hcy concentrations were independently associated with higher fracture risk (P = 0.029). PD patients taking higher doses of levodopa had significantly higher serum Hcy concentrations (P = 0.013), and greater levodopa intake was associated with lower BMD values in some areas (P = 0.008 to 0.029). In conclusion, these findings indicate that hyperhomocysteinemia due to levodopa intake may be one additional risk factor for osteoporosis and fracture in PD patients. Reducing Hcy may be a therapeutic modality for treating osteoporosis in PD patients taking levodopa.

Negative association between metabolic syndrome and bone mineral density in Koreans, especially in men.

Cardiovascular disease and osteoporosis are thought to share common risk factors, and metabolic syndrome (MS) is composed of major risk factors for cardiovascular disease. This study was performed to investigate the relationships between specific MS components and bone mineral density (BMD). BMD was measured at the femoral neck of Korean men aged 40 years or more (n = 1,780) and postmenopausal women (n = 1,108) using dual-energy X-ray absorptiometry. We identified subjects with MS as defined by two criteria, International Diabetes Federation (IDF) and American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI). Body fat and lean mass were measured via bioimpedance analysis. The prevalence of MS was 19.8% and 7.7% in men and 20.8% and 11.6% in postmenopausal women according to the AHA/NHLBI definition and the IDF definition, respectively. After multivariate adjustment, femoral neck BMD was significantly lower in subjects with MS regardless of diagnostic criteria. BMD decreased as the number of MS components increased (P < 0.001 for trends in both sexes). Among MS components, waist circumference was the most important factor in this negative association. When multiple linear regression models were applied to each 5-kg weight stratum to test for a linear trend, waist circumference and fat mass were negatively associated with BMD and lean mass was positively associated with BMD in men but not in women. MS was associated with a lower BMD in Korean men and postmenopausal women, suggesting that visceral fat may lead to bone loss, especially in men.

Association of a RUNX2 promoter polymorphism with bone mineral density in postmenopausal Korean women.

Osteoporosis is characterized by impaired osteoblastogenesis. Bone mineral density (BMD) is a major determinant of bone strength. RUNX2 is an osteoblast-specific transcription factor involved in osteoblast differentiation and ossification. To determine whether RUNX2 is associated with BMD in an ethnically distinct population, we investigated SNPs within the two RUNX2 promoters (P1 and P2) using the Illuminar GoldenGate system in 729 postmenopausal Korean women. Subjects bearing the minor homozygote genotype (CC) at the RUNX2 -1025 T > C SNP (rs7771980) located in P2 showed a significant association with reduced lumbar spine BMD (p = 0.02) and BMDs at proximal femur sites (trochanter, p = 0.05; total femur, p = 0.04) compared with subjects carrying the major homozygote genotype (TT) or the heterozygote genotype (TC), respectively. These results present an interesting genotype association complementary to the previously reported association of BMD with the RUNX2 -1025 T > C P2 SNP in Spanish and Australian cohorts. Therefore, we suggest that the RUNX2 P2 polymorphism (-1025 T > C) may be a useful genetic marker for bone metabolism and may play an important role in BMD in postmenopausal Korean women.

Comparison of monthly ibandronate versus weekly risedronate in preference, convenience, and bone turnover markers in Korean postmenopausal osteoporotic women.

Patient preferences, convenience, and bone turnover markers were evaluated for the monthly ibandronate over the weekly risedronate regimen in Korean postmenopausal osteoporotic women. This was a 6-month, prospective, randomized, open-label, multicenter study with a two-period and two-sequence crossover treatment design. After a 30-day screening period, eligible participants with postmenopausal osteoporosis were randomized to receive either monthly oral ibandronate 150 mg for 3 months followed by weekly oral risedronate 35 mg for 12 weeks (sequence A) or the same regimen in reverse order (sequence B). Patient preference and convenience were evaluated by questionnaire. The changes in serum C-telopeptide after 3 months of treatment were analyzed. A total of 365 patients were enrolled in this study (sequence A 182, sequence B 183). Of patients expressing a preference (83.4%), 74.8% preferred the monthly ibandronate regimen over the weekly regimen (25.2%). More women stated that the monthly ibandronate regimen was more convenient (84.2%) than the weekly regimen (15.8%). There was no significant difference in the change in bone turnover marker between the two treatments. The two regimens were similarly tolerable. There were fewer adverse events in the monthly ibandronate group compared to the weekly risedronate group in terms of gastrointestinal side effects (nausea and abdominal distension). This study revealed a strong preference and convenience for monthly ibandronate over weekly risedronate in Korean postmenopausal osteoporotic women. There was no significant difference in change of bone turnover marker and safety profile between the two regimens.

Association of IL-15 polymorphisms with bone mineral density in postmenopausal Korean women.

Interleukin-15 (IL-15) has been suggested to participate in bone metabolism by stimulating osteoclast differentiation and mediating inflammatory bone loss. This study investigated the effect of IL-15 gene polymorphisms on the bone mineral density (BMD) and bone fracture rates of postmenopausal women. Sequencing of the IL-15 gene in 24 Koreans revealed 16 single-nucleotide polymorphisms (SNPs), of which five were selected for further study. Postmenopausal Korean women (n = 844) were genotyped for these SNPs, and their BMDs and risk of fractures were assessed. It was found that the +20A > G, +13467C > A, +13653A > T, and +13815A > T IL-15 gene polymorphisms were significantly associated with the BMD of the lumbar spine and femoral neck and that their effects were gene-dose dependent. BMD was reduced when the minor allele of +13467A and +13653T or the common allele of +20A and +13815A was present. Haplotype (ht) analyses revealed that ht1 (GCAT) and ht2 (AATA) were associated with BMD of the lumbar spine and femoral neck. However, there was no association between the risk of fracture and IL-15 SNPs or hts. These results suggest that the +20A > G, +13467C > A, +13653A > T, and +13815A > T SNPs in the IL-15 gene affect BMD and, thus, could be genetic markers of osteoporosis.

Associations of catalase gene polymorphisms with bone mineral density and bone turnover markers in postmenopausal women.

BACKGROUND: Oxidative stress has been recently suggested to play a part in the development of osteoporosis. Catalase is a major antioxidant enzyme that detoxifies hydrogen peroxide by converting it into water and oxygen, thereby preventing cellular injury by oxidative stress. AIMS: To examine the associations between the catalase gene (CAT) polymorphisms and bone mineral density (BMD) and bone turnover markers in postmenopausal Korean women. METHODS: All exons, their boundaries and the promoter region (approximately 1.5 kb) were directly sequenced in 24 individuals. Among 18 variants identified by a direct sequence method, four polymorphisms were selected and genotyped in all study participants (n = 560). BMD at the lumbar spine and proximal femur was measured using dual-energy x ray absorptiometry. Serum osteocalcin concentrations and bone-specific alkaline phosphatase activity were determined by an immunoradiometric assay and an immunoassay, respectively. RESULTS: The mean (standard deviation) age of the participants was 59.4 (7.2) years. Multivariate analysis showed an association of the +22348C-->T polymorphism with BMD at the lumbar spine (p = 0.01 in the dominant model) and at femur neck (p = 0.05 in the dominant model), and with serum osteocalcin level (p = 0.008 in the dominant model). Haplotype analyses showed that HT4 (-20T, +144C, +22348T, +33078A) was significantly associated with higher BMD at various sites (p<0.001-0.03) and with lower serum osteocalcin levels (p = 0.01 in the codominant model). CONCLUSIONS: These findings indicate that the +22348C-->T polymorphism and HT4 of CAT may be useful genetic markers for bone metabolism.

Osteoclast-secreted SLIT3 coordinates bone resorption and formation.

Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating ß-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.

Association of FLT3 polymorphisms with low BMD and risk of osteoporotic fracture in postmenopausal women.

UNLABELLED: The genetic effects of FLT3 polymorphisms on BMD and fracture risk in postmenopausal women were studied. We found that FLT3+13348C>T polymorphism and haplotype 2 were significantly associated with low BMD and high risk of fracture. INTRODUCTION: FMS-related tyrosine kinase 3 (FLT3) has been shown to play a critical role in the development of myelolymphoid progenitors and in the development of osteoclasts, but any possible genetic effect of FLT3 on bone metabolism has not been studied. MATERIALS AND METHODS: To study a possible genetic effect of FLT3, we directly sequenced the FLT3 gene in 24 Korean individuals and identified 23 sequence variants. Seven polymorphisms were selected and genotyped in Korean postmenopausal women (n = 946). RESULTS: We found that FLT3+13348C>T was associated with low BMD at the lumbar spine (p = 0.04) and femoral neck (p = 0.04). Haplotype analysis revealed that FLT3-ht2 (TTCTT) containing the rare allele in the +13348 position also showed significant association with low BMD in the lumbar spine (p = 0.04) and femoral neck (p = 0.05). Consistent with these results, the FLT3+13348C>T polymorphism and FLT3-ht2 were also significantly associated with high risk of fracture in the vertebrae (OR = 1.44-1.58; p = 0.03-0.04 and OR = 1.45-1.59; p = 0.02-0.03, respectively) and in any sites (OR = 1.34-1.81; p = 0.02-0.03 and OR = 1.34-1.81; p = 0.02-0.03, respectively). CONCLUSIONS: These results suggest that FLT3 polymorphisms play a role in determination of BMD and subsequent fractures in postmenopausal women.

Polymorphisms and haplotypes of integrinalpha1 (ITGA1) are associated with bone mineral density and fracture risk in postmenopausal Koreans.

INTRODUCTION: ITGA1 is involved in the early remodeling of osteoarthritic cartilage and plays an essential role in the regulation of mesenchymal stem cell proliferation and cartilage production. We investigated the association between bone parameters and ITGA1 polymorphisms and their haplotype linkage disequilibrium (LD) blocks (BL_hts). Genetic susceptibility to osteoporosis was studied in 946 postmenopausal Korean women. METHODS: We identified 67 genetic polymorphisms in ITGA1 region by direct sequencing (n = 114). Eight SNPs were genotyped to further investigate their potential involvement in osteoporosis in postmenopausal women (n = 946). Areal BMD of the lumbar spine and proximal femur was measured using dual-energy X-ray absorptiometry. RESULTS: The SNPs, +73187C>T (exon 3) and +76969T>G (intron 5), and their BL_hts were associated with bone mineral density (BMD) at various femur sites (p = 0.009-0.05). Moreover, +159174A>C (intron 28) and its haplotype BL3_ht1 showed a highly significant association with risk of non-vertebral fracture (p = 0.002-0.005) and the minor allele of +159174A>C showed a protective effect. CONCLUSIONS: These results are suggestive of the association of ITGA1 with osteoporosis and related risk in postmenopausal women.

Single nucleotide polymorphisms in bone turnover-related genes in Koreans: ethnic differences in linkage disequilibrium and haplotype.

BACKGROUND: Osteoporosis is defined as the loss of bone mineral density that leads to bone fragility with aging. Population-based case-control studies have identified polymorphisms in many candidate genes that have been associated with bone mass maintenance or osteoporotic fracture. To investigate single nucleotide polymorphisms (SNPs) that are associated with osteoporosis, we examined the genetic variation among Koreans by analyzing 81 genes according to their function in bone formation and resorption during bone remodeling. METHODS: We resequenced all the exons, splice junctions and promoter regions of candidate osteoporosis genes using 24 unrelated Korean individuals. Using the common SNPs from our study and the HapMap database, a statistical analysis of deviation in heterozygosity depicted. RESULTS: We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the SNPs, 557 (63%) had been previously identified and 331 (37%) were newly discovered in the Korean population. When compared SNPs in the Korean population with those in HapMap database, 1% (or less) of SNPs in the Japanese and Chinese subpopulations and 20% of those in Caucasian and African subpopulations were significantly differentiated from the Hardy-Weinberg expectations. In addition, an analysis of the genetic diversity showed that there were no significant differences among Korean, Han Chinese and Japanese populations, but African and Caucasian populations were significantly differentiated in selected genes. Nevertheless, in the detailed analysis of genetic properties, the LD and Haplotype block patterns among the five sub-populations were substantially different from one another. CONCLUSION: Through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with a minor allele frequency (MAF) > 0.05 were discovered in the Korean population. In addition, using the common SNPs between our study and HapMap, an analysis of genetic diversity and deviation in heterozygosity was performed and the polymorphisms of the above genes among the five populations were substantially differentiated from one another. Further studies of osteoporosis could utilize the polymorphisms identified in our data since they may have important implications for the selection of highly informative SNPs for future association studies.

Microphthalmia-associated transcription factor polymorphisms and association with bone mineral density of the proximal femur in postmenopausal women.

Osteoporosis is a common metabolic bone disease characterized by low bone mineral density (BMD) with an increased risk of fracture. Low bone mass results from an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts. Microphthalmia-associated transcription factor (MITF) plays a critical role in osteoclast development and thus is an important candidate gene affecting bone turnover and BMD. In order to investigate the genetic effects of MITF variations on osteoporosis, we directly sequenced the MITF gene in 24 Koreans, and identified fifteen sequence variants. Two polymorphisms (+227719C > T and +228953A > G) were selected based on their allele frequencies, and then genotyped in a larger number of postmenopausal women (n = 560). Areal BMD (g/cm2) of the anterior-posterior lumbar spine and the non-dominant proximal femur was measured by dual-energy X-ray absorptiometry. We found that the MITF + 227719C > T polymorphism was significantly associated with low BMD of the trochanter (p = 0.005-0.006) and total femur (p = 0.02-0.03) (codominant and dominant models), while there was no association with BMD of the lumbar spine. The MITF+228953A > G polymorphism was also associated with low BMD of the femoral shaft (p = 0.05) in the recessive model. Haplotype analysis showed that haplotype 3 of the MITF gene (MITF-ht3) was associated with low BMD of the trochanter (p = 0.03-0.05) and total femur (p = 0.05) (dominant and codominant models). Our results suggest that MITF variants may play a role in the decreased BMD of the proximal femur in postmenopausal women.

Homocysteine enhances bone resorption by stimulation of osteoclast formation and activity through increased intracellular ROS generation.

UNLABELLED: Hyperhomocystinemia is a modifiable risk factor for osteoporosis and fracture. Physiologic concentrations of Hcy directly activate osteoclast formation and activity through stimulation of p38 MAPK and integrin beta3. The effects of Hcy were mediated by generation of intracellular ROS. INTRODUCTION: Hyperhomocysteinemia is a modifiable risk factor for osteoporosis and its related bone fractures. It has been reported that bone resorption and turnover rate were increased in hyperhomocystinemia. Using mouse bone marrow cells, we examined the direct effects of homocysteine (Hcy) on osteoclast formation and activity. MATERIALS AND METHODS: Osteoclast formation was determined by TRACP staining and TRACP activity. Intracellular reactive oxygen species (ROS) generation was measured using a fluorescent probe, dichlorodihydrofluorescein diacetate. Intracellular signaling cascades of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-kappaB were measured by Western blotting. Integrin beta3 mRNA levels were measured by RT-PCR. Actin ring formation and bone resorption assays were also performed. RESULTS: Physiologic concentrations of Hcy upregulated TRACP+ multinucleated cells and TRACP activity, stimulated actin ring formation, and increased the number of nuclei per cell and the level of expression of integrin beta3 mRNA. In addition, Hcy increased bone resorption and stimulated p38 MAPK activity and intracellular reactive oxygen species (ROS) generation. All of these Hcy-induced changes were blocked by pretreatment with the antioxidant, N-acetyl cysteine. CONCLUSIONS: Hcy directly activates osteoclast formation and activity through increased generation of intracellular ROS. These findings suggest that, in individuals with mild to moderate hyperhomocystinemia, increased bone resorption by osteoclasts may contribute to osteoporosis and that an antioxidant may attenuate bone loss in these individuals.

Homocysteine enhances apoptosis in human bone marrow stromal cells.

INTRODUCTION: High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation. MATERIALS AND METHODS: Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively. RESULTS: Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis. CONCLUSION: These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.