METTL21C Is a Potential Pleiotropic Gene for Osteoporosis and Sarcopenia Acting Through the Modulation of the NF‐κB Signaling Pathway

Sarcopenia and osteoporosis are important public health problems that occur concurrently. A bivariate genome‐wide association study (GWAS) identified METTL21c as a suggestive pleiotropic gene for both bone and muscle. The METTL21 family of proteins methylates chaperones involved in the etiology of both myopathy and inclusion body myositis with Paget's disease. To validate these GWAS results, Mettl21c mRNA expression was reduced with siRNA in a mouse myogenic C2C12 cell line and the mouse osteocyte‐like cell line MLO‐Y4. At day 3, as C2C12 myoblasts start to differentiate into myotubes, a significant reduction in the number of myocytes aligning/organizing for fusion was observed in the siRNA‐treated cells. At day 5, both fewer and smaller myotubes were observed in the siRNA‐treated cells as confirmed by histomorphometric analyses and immunostaining with myosin heavy chain (MHC) antibody, which only stains myocytes/myotubes but not myoblasts. Intracellular calcium (Ca²⁺) measurements of the siRNA‐treated myotubes showed a decrease in maximal amplitude peak response to caffeine, suggesting that less Ca²⁺ is available for release due to the partial silencing of Mettl21c, correlating with impaired myogenesis. In siRNA‐treated MLO‐Y4 cells, 48 hours after treatment with dexamethasone there was a significant increase in cell death, suggesting a role of Mettl21c in osteocyte survival. To investigate the molecular signaling machinery induced by the partial silencing of Mettl21c, we used a real‐time PCR gene array to monitor the activity of 10 signaling pathways. We discovered that Mettl21c knockdown modulated only the NF‐κB signaling pathway (ie, Birc3, Ccl5, and Tnf). These results suggest that Mettl21c might exert its bone‐muscle pleiotropic function via the regulation of the NF‐κB signaling pathway, which is critical for bone and muscle homeostasis. These studies also provide rationale for cellular and molecular validation of GWAS, and warrant additional in vitro and in vivo studies to advance our understanding of role of METTL21C in musculoskeletal biology. © 2014 American Society for Bone and Mineral Research     

Genetics of Bone Mass in Childhood and Adolescence: Effects of Sex and Maturation Interactions

We aimed to determine if adult bone mineral density (BMD) susceptibility loci were associated with pediatric bone mass and density, and if sex and pubertal stage influenced any association. We analyzed prospective areal BMD (aBMD) and bone mineral content (BMC) data from the Bone Mineral Density in Childhood Study (n = 603, European ancestry, 54% female). Linear mixed models were used to assess if 77 single‐nucleotide polymorphisms (SNPs) near known adult BMD susceptibility loci interacted with sex and pubertal stage to influence the aBMD/BMC; adjusting for age, BMI, physical activity, and dietary calcium. The strongest main association was observed between an SNP near C7orf58 and distal radius aBMD. However, this association had a significant sex?SNP interaction, revealing a significant association only in females (b = –0.32, p = 1.8 × 10^–6). Furthermore, the C12orf23 locus had significant interactions with both sex and pubertal stage, revealing associations in females during Tanner stage I for total hip aBMD (b = 0.24, p = 0.001) and femoral neck aBMD (b = 0.27, p = 3.0 × 10^–5). In contrast, the sex?SNP interactions for loci near LRP5 and WNT16 uncovered associations that were only in males for total body less head BMC (b = 0.22, p = 4.4 × 10^–4) and distal radius aBMD (b = 0.27, p = 0.001), respectively. Furthermore, the LRP5 locus interacted with both sex and pubertal stage, demonstrating associations that were exclusively in males during Tanner V for total hip aBMD (b = 0.29, p = 0.003). In total, significant sex?SNP interactions were found at 15 loci; pubertal stage?SNP interactions at 23 loci and 19 loci interacted with both sex and pubertal stage. In conclusion, variants originally associated with adult BMD influence bone mass in children of European ancestry, highlighting the fact that many of these loci operate early in life. However, the direction and magnitude of associations for a large number of SNPs only became evident when accounting for sex and maturation. © 2015 American Society for Bone and Mineral Research.     

Methylation of Bone SOST,Its mRNA,and Serum Sclerostin Levels Correlate Strongly With Fracture Risk in Postmenopausal Women

Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T‐score > –1) and established OP (BMD T‐score < –2.5, with at least one low‐energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (n = 4) compared to age and body mass index (BMI) balanced controls (n = 4) (80.5% versus 63.2%, p = 0.0001) with replication in independent cohorts (n = 27 and n = 36, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age‐adjusted and BMI‐adjusted total hip BMD (r = 0.47 and r = 0.43, respectively; both p < 0.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population‐based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (p = 0.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation. © 2014 American Society for Bone and Mineral Research.     

Genetic Risk Scores Implicated in Adult Bone Fragility Associate With Pediatric Bone Density

Using adult identified bone mineral density (BMD) loci, we calculated genetic risk scores (GRS) to determine if they were associated with changes in BMD during childhood. Longitudinal data from the Bone Mineral Density in Childhood Study were analyzed (N = 798, 54% female, all European ancestry). Participants had up to 6 annual dual energy X‐ray scans, from which areal BMD (aBMD) Z‐scores for the spine, total hip, and femoral neck were estimated, as well as total body less head bone mineral content (TBLH‐BMC) Z‐scores. Sixty‐three single‐nucleotide polymorphisms (SNPs) were genotyped, and the percentage of BMD‐lowering alleles carried was calculated (overall adult GRS). Subtype GRS that include SNPs associated with fracture risk, pediatric BMD, WNT signaling, RANK‐RANKL‐OPG, and mesenchymal stem cell differentiation were also calculated. Linear mixed effects models were used to test associations between each GRS and bone Z‐scores, and if any association differed by sex and/or chronological age. The overall adult, fracture, and WNT signaling GRS were associated with lower Z‐scores (eg, spine aBMD Z‐score: β_adult = –0.04, p = 3.4 × 10^?7; β_fracture = –0.02, p = 8.9 × 10^?6; β_WNT = –0.01, p = 3.9 × 10^?4). The overall adult GRS was more strongly associated with lower Z‐scores in females (p‐interaction ≤ 0.05 for all sites). The fracture GRS was more strongly associated with lower Z‐scores with increasing age (p‐interaction ≤ 0.05 for all sites). The WNT GRS associations remained consistent for both sexes and all ages (p‐interaction > 0.05 for all sites). The RANK‐RANKL‐OPG GRS was more strongly associated in females with increasing age (p‐interaction < 0.05 for all sites). The mesenchymal stem cell GRS was associated with lower total hip and femoral neck Z‐scores, in both boys and girls, across all ages. No associations were observed between the pediatric GRS and bone Z‐scores. In conclusion, adult identified BMD loci associated with BMD and BMC in the pediatric setting, especially in females and in loci involved in fracture risk and WNT signaling. © 2015 American Society for Bone and Mineral Research.     

Longitudinal Effects of Single Hindlimb Radiation Therapy on Bone Strength and Morphology at Local and Contralateral Sites

Radiation therapy (RTx) is associated with increased risk for late‐onset fragility fractures in bone tissue underlying the radiation field. Bone tissue outside the RTx field is often selected as a “normal” comparator tissue in clinical assessment of fragility fracture risk, but the robustness of this comparison is limited by an incomplete understanding of the systemic effects of local radiotherapy. In this study, a mouse model of limited field irradiation was used to quantify longitudinal changes in local (irradiated) and systemic (non‐irradiated) femurs with respect to bone density, morphology, and strength. BALB/cJ mice aged 12 weeks underwent unilateral hindlimb irradiation (4 × 5 Gy) or a sham procedure. Femurs were collected at endpoints of 4 days before treatment and at 0, 1, 2, 4, 8, 12, and 26 weeks post‐treatment. Irradiated (RTx), Contralateral (non‐RTx), and Sham (non‐RTx) femurs were imaged by micro‐computed tomography and mechanically tested in three‐point bending. In both the RTx and Contralateral non‐RTx groups, the longer‐term (12‐ to 26‐week) outcomes included trabecular resorption, loss of diaphyseal cortical bone, and decreased bending strength. Contralateral femurs generally followed an intermediate response compared with RTx femurs. Change also varied by anatomic compartment; post‐RTx loss of trabecular bone was more profound in the metaphyseal than the epiphyseal compartment, and cortical bone thickness decreased at the mid‐diaphysis but increased at the metaphysis. These data demonstrate that changes in bone quantity, density, and architecture occur both locally and systemically after limited field irradiation and vary by anatomic compartment. Furthermore, the severity and persistence of systemic bone damage after limited field irradiation suggest selection of control tissues for assessment of fracture risk or changes in bone density after radiotherapy may be challenging. © 2017 American Society for Bone and Mineral Research.     

A Novel,Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice

Most US Food and Drug Administration (FDA)‐approved treatments for osteoporosis target osteoclastic bone resorption. Only PTH derivatives improve bone formation, but they have drawbacks, and novel bone‐anabolic agents are needed. Nitrates, which generate NO, improved BMD in estrogen‐deficient rats and may improve bone formation markers and BMD in postmenopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long‐term use. Here we studied nitrosyl‐cobinamide (NO‐Cbi), a novel, direct NO‐releasing agent, in a mouse model of estrogen deficiency–induced osteoporosis. In murine primary osteoblasts, NO‐Cbi increased intracellular cGMP, Wnt/β‐catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in intact and ovariectomized (OVX) female C57Bl/6 mice, NO‐Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and in OVX mice, it prevented osteocyte apoptosis. NO‐Cbi reduced osteoclasts in intact mice and prevented the known increase in osteoclasts in OVX mice, partially through a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and partially through direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. The positive NO effects in osteoblasts were mediated by cGMP/protein kinase G (PKG), but some of the osteoclast‐inhibitory effects appeared to be cGMP‐independent. NO‐Cbi increased trabecular bone mass in both intact and OVX mice, consistent with its in vitro effects on osteoblasts and osteoclasts. NO‐Cbi is a novel direct NO‐releasing agent that, in contrast to nitrates, does not generate oxygen radicals, and combines anabolic and antiresorptive effects in bone, making it an excellent candidate for treating osteoporosis. © 2016 American Society for Bone and Mineral Research.     

术前区域动脉灌注化疗治疗乳癌

目的　评价术前动脉灌注化疗药物在乳癌治疗中的作用。方法　采用Ｓｅｌｄｉｎｇｅｒ 法经皮股动脉穿刺,将导管置于锁骨下动脉与胸廓内动脉开口交界处的近侧,用充气止血带高于患者收缩压阻断患侧肱动脉血流后,注入化疗药物。根据病人情况３ ～４ 周重复灌注化疗１ 次。化疗方案：吡喃阿霉素（ＴＨＰ） ,或阿霉素（ＡＤＲ） ,或表阿霉素（ ＥＡＤＲ）５０ ～７０ ｍｇ／ｍ２ ,卡铂（ＣＰＢ）３００ ～４００ ｍｇ／ｍ２ ,５氟脲嘧啶（５Ｆｕ）７５０ ～１ ０００ ｍｇ／ｍ２ ,甲酰四氢叶酸钙（ ＣＦ）１００ ～２００ ｍｇ 。结果　本组３２ 例,随访６ 个月～７ 年（ 平均３６ 个月） ,ＣＲ２ 例,ＰＲ２５ 例,ＭＲ５ 例,总缓解率（ＣＲ＋ ＰＲ）８４．４ ％ ;６ 例曾接受全身化疗者ＰＲ５ 例,ＭＲ１ 例;２ 例曾行局部放疗者均为ＰＲ。结论　动脉内灌注化疗药物对提高乳腺癌的生存率有积极意义,且对全身化疗或局部放疗无效者仍有可能通过动脉灌注取得较好效果。