A Mutation in the LDL Receptor–Related Protein 5 Gene Results in the Autosomal Dominant High–Bone-Mass Trait

Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high–bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted β-propeller module of the low-density lipoprotein receptor–related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.     

The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd

The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.     

Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake

LRP6,a co-receptor for the morphogen Wnt,aids endocytosis of anthrax complexes.Here we report that Dickkopf1(DKK1)protein,a secreted LRP6 ligand and antagonist,is also a modulator of anthrax toxin sensitivity.shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins.However,unlike the solely inhibitory effect on Wnt signaling,the effects of DKK1 overexpression on anthrax toxicity were bidirectional,depending on its endogenous expression and cell context.Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors,an event mediated by DKK1-LRP6-Kremen2 complex.Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin(LT).Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins.     

Increased Bone Mineral Density in Male Patients With Idiopathic Hypogonadotropic Hypogonadism Who Undergo Sex Hormone Therapy: Findings From Cross-Sectional and Longitudinal Studies

ObjectiveThis retrospective observational study assessed the long-term impact of pulsatile gonadotropin-releasing hormone, combined gonadotropin, or testosterone replacement therapy on total hip, femoral, and lumbar bone mineral density (BMD) and Z-scores in adult men with idiopathic hypogonadotropic hypogonadism (IHH).MethodsIn the cross-sectional study, 69 patients were allocated to untreated (n = 42) and treated (n = 27) groups. The untreated group included IHH patients without hormone therapy history, while the treated group included age- and body mass index-matched patients who had received hormone therapy for at least 5 years. The longitudinal study included 53 IHH patients, and their hip and lumbar BMDs were measured several times during hormone therapy. We then evaluated the changes in their BMD.ResultsOur cross-sectional study showed that the treated group had a significantly higher BMD and Z-score for total hip, femoral neck, and lumbar spine (P < 0.001 for all) than the untreated group, and the average bone mass even reached the age-matched normal range. The prevalence of low BMD was 80.95% and 11.11% in untreated and treated groups, respectively. In the longitudinal study (N = 53), the total hip, femoral neck, and lumbar spine BMD gradually increased during treatment. The lumbar spine showed a greater increment in BMD compared with the total hip and femoral neck (P < 0.05).ConclusionSex hormone therapy improved hip and lumbar spine BMD and Z-scores in patients with IHH. The lumbar spine showed a greater improvement in BMD compared with the total hip and femoral neck.     

Wnt antagonists bind through a short peptide to the first β-propeller domain of LRP5/6

The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.     

Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass-associated missense mutations in LRP5 affect canonical Wnt signaling

The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1.     

Impact of genetic variants of IL-6, IL6R, LRP5, ESR1 and SP7 genes on bone mineral density in postmenopausal Mexican-Mestizo women with obesity

Background

Since obesity and osteoporosis present a high genetic predisposition and polymorphisms of IL-6, IL6R, LRP5, ESR1 and SP7 may influence the risk of both diseases, the aim of this study was to analyze the possible association of polymorphisms in these genes, as well as their haplotypes, with BMD variations in postmenopausal Mexican-Mestizo women with grade 2 or grade 3 obesity.

Methods

One hundred eighty unrelated postmenopausal women with grade 2 or grade 3 obesity were included. BMD was measured in total hip and lumbar spine by dual-energy X-ray absorptiometry. DNA was obtained from blood leukocytes. Rs1800795 of IL-6, rs2228145 of IL6R, rs3736228 of LRP5, rs9340799 (XbaI) and rs2234693 (PvuII), of ESR1, rs10876432 and rs2016266, of SP7 (and their haplotypes), were studied by real-time PCR allelic discrimination. Deviations from Hardy–Weinberg equilibrium were tested. Pairwise linkage disequilibrium between single nucleotide polymorphisms was calculated by direct correlation r², and haplotype analysis was conducted.

Results

Using WHO criteria, 54.5% had grade 2 obesity, and 45.5% had grade 3 obesity. Regarding DXA results, 11.1% women had osteoporosis, 41.7% had osteopenia, and 47.2% had normal BMD. Genotype and haplotype analysis showed no significant differences with BMD variations at the lumbar spine, total hip or femoral neck.

Conclusions

We did not find a significant association between the polymorphisms analyzed or their haplotypes and BMD variations in postmenopausal women with obesity. The higher BMD observed in women with obesity could be the result of an adaptive response to the higher loading of the skeleton.     

Nuclear Isoforms of Fibroblast Growth Factor 2 Are Novel Inducers of Hypophosphatemia via Modulation of FGF23 and KLOTHO

FGF2 transgenic mice were developed in which type I collagen regulatory sequences drive the nuclear high molecular weight FGF2 isoforms in osteoblasts (TgHMW). The phenotype of TgHMW mice included dwarfism, decreased bone mineral density (BMD), osteomalacia, and decreased serum phosphate (P_i). When TgHMW mice were fed a high P_i diet, BMD was increased, and dwarfism was partially reversed. The TgHMW phenotype was similar to mice overexpressing FGF23. Serum FGF23 was increased in TgHMW mice. Fgf23 mRNA in bones and fibroblast growth factor receptors 1c and 3c and Klotho mRNAs in kidneys were increased in TgHMW mice, whereas the renal Na⁺/P_i co-transporter Npt2a mRNA was decreased. Immunohistochemistry and Western blot analyses of TgHMW kidneys showed increased KLOTHO and decreased NPT2a protein. The results suggest that overexpression of HMW FGF2 increases FGF23/FGFR/KLOTHO signaling to down-regulate NPT2a, causing P_i wasting, osteomalacia, and decreased BMD. We assessed whether HMW FGF2 expression was altered in the Hyp mouse, a mouse homolog of the human disease X-linked hypophosphatemic rickets/osteomalacia. Fgf2 mRNA was increased in bones, and Western blots showed increased FGF2 protein in nuclear fractions from osteoblasts of Hyp mice. In addition, immunohistochemistry demonstrated co-localization of FGF23 and HMW FGF2 protein in osteoblasts and osteocytes from Hyp mice. This study reveals a novel mechanism of regulation of the FGF23-P_i homeostatic axis.     

Hypoxia and vitamin D differently contribute to leptin and dickkopf-related protein 2 production in human osteoarthritic subchondral bone osteoblasts

Introduction

Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularization parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates because they are upregulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs.

Methods

Obs from the sclerotic portion of OA tibial plateaus were cultured under either 20% or 2% oxygen tension in the presence or not of 50 nM 1,25-dihydroxyvitamin D₃ (VitD₃). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factor 1α (Hif-1α) and Hif-2α was measured by real-time polymerase chain reaction and leptin production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing RNAs (siRNAs). The signalling pathway of hypoxia-induced leptin was investigated by Western blot analysis and with mitogen-activated protein kinase (MAPK) inhibitors.

Results

The expression of leptin and DKK2 in Obs was stimulated 7-fold and 1.8-fold, respectively (P <0.05) under hypoxia. Interestingly, whereas VitD₃ stimulated leptin and DKK2 expression 2- and 4.2-fold, respectively, under normoxia, it stimulated their expression by 28- and 6.2-fold, respectively, under hypoxia (P <0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in the presence of VitD₃ (P <0.02). Compared to Obs incubated in the presence of scramble siRNAs, siHif-2α inhibited VitD₃-stimulated leptin mRNA and protein levels by 70% (P =0.004) and 60% (P <0.02), respectively, whereas it failed to significantly alter the expression of DKK2. siHif-1α has no effect on these genes. Immunoblot analysis showed that VitD₃ greatly stabilized Hif-2α under hypoxic conditions. The increase in leptin expression under hypoxia was also regulated, by p38 MAPK (P <0.03) and phosphoinositide 3-kinase (P <0.05). We found that the expression of leptin and DKK2 were not related to each other under hypoxia.

Conclusions

Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin, particularly under VitD₃ stimulation, whereas DKK2 is regulated mainly by VitD₃ rather than hypoxia.     

Differential induction of cell-mediated mineralization in rat marrow stroma by sera from women of low and high risk for vertebral fracture

The purpose of this study was to analyze the ability of sera to reflect the state of bone metabolism by testing the osteogenic response of mesenchymal cells in culture. Sera of 20 peri- and postmenopausal women were tested before the initiation of hormone replacement therapy. The responding cells were osteoprogenitors (OPC) of rat marrow stroma which normally respond to dexamethasone (DEX) and β-glycerophosphate (βGP) by proliferation, differentiation, and mineralization in culture. Instead of DEX, diluted sera (1:50) were applied to rat stromal cell cultures for analysis of their ability to affect cell proliferation, specific alkaline phosphatase (ALP) activity, and cell-mediated mineralization. The results were compared individually with the respective values of vertebral bone mineral density (BMD), expressed as the number of standard deviations above or below the mean BMD of reference populations (positive or negative Z-score). Serum donors were divided in two; the group with positive Z-scores was considered to have a low risk, and that with negative Z-scores was considered to have a higher risk for vertebral fractures. No significant difference was found between the two groups in the ability of their sera to induce cell proliferation or specific ALP activity. However, sera representing negative Z-scores induced sixteenfold less mineralization than those of positive Z-scores. The scatter of individual mineralization values was highly discriminatory between the two groups (α < 0.00). These results indicate that the serum-induced, cell-mediated mineralization in culture might be suitable for initial evaluation of fracture risk and thus deserve further investigation. © 1996 Wiley-Liss, Inc.     

Association between Variants in Atopy-Related Immunologic Candidate Genes and Pancreatic Cancer Risk

BackgroundMany epidemiology studies report that atopic conditions such as allergies are associated with reduced pancreas cancer risk. The reason for this relationship is not yet understood. This is the first study to comprehensively evaluate the association between variants in atopy-related candidate genes and pancreatic cancer risk.MethodsA population-based case-control study of pancreas cancer cases diagnosed during 2011-2012 (via Ontario Cancer Registry), and controls recruited using random digit dialing utilized DNA from 179 cases and 566 controls. Following an exhaustive literature review, SNPs in 180 candidate genes were pre-screened using dbGaP pancreas cancer GWAS data; 147 SNPs in 56 allergy-related immunologic genes were retained and genotyped. Logistic regression was used to estimate age-adjusted odd ratio (AOR) for each variant and false discovery rate was used to adjust Wald p-values for multiple testing. Subsequently, a risk allele score was derived based on statistically significant variants.Results18 SNPs in 14 candidate genes (CSF2, DENND1B, DPP10, FLG, IL13, IL13RA2, LRP1B, NOD1, NPSR1, ORMDL3, RORA, STAT4, TLR6, TRA) were significantly associated with pancreas cancer risk. After adjustment for multiple comparisons, two LRP1B SNPs remained statistically significant; for example, LRP1B rs1449477 (AA vs. CC: AOR=0.37, 95% CI: 0.22-0.62; p (adjusted)=0.04). Furthermore, the risk allele score was associated with a significant reduction in pancreas cancer risk (p=0.0007).ConclusionsPreliminary findings suggest certain atopy-related variants may be associated with pancreas cancer risk. Further studies are needed to replicate this, and to elucidate the biology behind the growing body of epidemiologic evidence suggesting allergies may reduce pancreatic cancer risk.     

In silico studying of the whole protein structure and dynamics of Dickkopf family members showed that N-terminal domain of Dickkopf 2 in contrary to other Dickkopfs facilitates its interaction with low density lipoprotein receptor related protein 5/6

Wnt (Wingless Int) signaling pathway has been known to be dysregulated in several human cancers, especially colorectal cancer (CRC). The Dickkopf (DKK) family which consists of four secreted proteins in vertebrates (DKK 1, 2, 3, 4) is one of the most critical antagonist families for Wnt signaling pathway. They typically antagonize Wnt/β-catenin signaling by binding and inhibiting Wnt co-receptors, LRP5/6 (low density lipoprotein receptor related protein 5/6). However, except for DKK1 (Dickkopf 1), details about structure and function of the members of this family are poorly defined. In this study, main Dickkopf family members were analyzed structurally, using protein structure prediction tools, molecular dynamics (MD), molecular docking and energy analyses. Three dimensional structure of whole DKKs was predicted and their interaction with LRP6 was investigated in detail. The results indicated that in DKK family members, a considerable diversity, in the case of structure, activity and physicochemical properties was seen. This diversity was more profound in DKK3 (Dickkopf3). Interestingly, the interaction mode of DKK2 (Dickkopf2) with its receptor, LRP6, was shown to be substantially different from other Dickkopf family members while N-terminal region of this ligand was also involved in the binding to the LRP6-P3P4. Moreover, the cysteine-rich domain 2 (CRD2) of DKK1 and DKK3 had a higher binding affinity to LRP6 in comparison with the whole protein structures.

Communicated by Ramaswamy H. Sarma     

Genetic Analysis of the Relationship between Bone Mineral Density and Low-Density Lipoprotein Receptor-Related Protein 5 Gene Polymorphisms

Background

A number of studies have examined the association between the polymorphisms of the low-density lipoprotein receptor-related protein 5 gene (LRP5), but previous results have been inconclusive. Thus we performed a meta-analysis of studies on the association between the LRP5 polymorphisms and bone mineral density (BMD) to assess their pooled effects.

Methods

Published literature from PubMed, EMBASE and ISI web of science were searched for eligible publications. Weighted mean difference (WMD) and 95% confidence interval (CI) was calculated using fixed- or random-effects model.

Results

A total of 19 studies with 25773 subjects were considered in this meta-analysis. Of them, 17 examined the association between the A1330V polymorphism and BMD, 8 were focused on the V667M polymorphism, and 2 analyzed the Q89R polymorphism. Individuals with the A1330V AA genotype showed significantly higher BMD than those with the AV/VV genotypes [at lumbar spine (LS): WMD = 0.02g/cm², 95% CI = 0.01-0.03, P < 10^-4; at femur neck (FN): WMD = 0.01g/cm², 95% CI = 0.00-0.02, P = 0.01] or VV genotype (at LS: WMD = 0.02g/cm², 95% CI = 0.01-0.04, P = 0.01). Significant associations were also detected in the analysis for V667M (VV vs. VM/MM: WMD at LS = 0.02g/cm², 95% CI = 0.02-0.03, P < 10^-5; WMD at FN = 0.01g/cm², 95% CI = 0.01-0.02, P = 0.0002). As for Q89R, subjects with the QQ genotype tended to have higher BMD than those with the QR/RR genotypes at FN (WMD = 0.03g/cm², 95% CI = 0.01-0.05, P = 0.005).

Conclusion

This meta-analysis demonstrated that the LRP5 polymorphisms may be modestly associated with BMD of LS and FN.     

Harnessing expression data to identify novel candidate genes in polycystic ovary syndrome

Novel pathways in polycystic ovary syndrome (PCOS) are being identified in gene expression studies in PCOS tissues; such pathways may contain key genes in disease etiology. Previous expression studies identified both dickkopf homolog 1 (DKK1) and DnaJ (Hsp40) homolog, subfamily B, member 1 (DNAJB1) as differentially expressed in PCOS tissue, implicating them as candidates for PCOS susceptibility. To test this, we genotyped a discovery cohort of 335 PCOS cases and 198 healthy controls for three DKK1 single nucleotide polymorphisms (SNPs) and four DNAJB1 SNPs and a replication cohort of 396 PCOS cases and 306 healthy controls for 1 DKK1 SNP and 1 DNAJB1 SNP. SNPs and haplotypes were determined and tested for association with PCOS and component phenotypes. We found that no single nucleotide polymorphisms were associated with PCOS risk; however, the major allele of rs1569198 from DKK1 was associated with increased total testosterone (discovery cohort P = 0.0035) and dehydroepiandrosterone sulfate (replication cohort P = 0.05). Minor allele carriers at rs3962158 from DNAJB1 had increased fasting insulin (discovery cohort P = 0.003), increased HOMA-IR (discovery cohort P = 0.006; replication cohort P = 0.036), and increased HOMA-%B (discovery cohort P = 0.004). Carriers of haplotype 2 at DNAJB1 also had increased fasting insulin, HOMA-IR, and HOMA-%B. These findings suggest that genetic variation in DKK1 and DNAJB1 may have a role in the hyperandrogenic and metabolic dysfunction of PCOS, respectively. Our results also demonstrate the utility of gene expression data as a source of novel candidate genes in PCOS, a complex and still incompletely defined disease, for which alternative methods of gene identification are needed.     

The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1

The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.