Unique and non-redundant function of csf1r paralogues in regulation and evolution of post-embryonic development of the zebrafish.

Evolution is replete with reuse of genes in different contexts, leading to multifunctional roles of signaling factors during development. Here, we explore osteoclast regulation during skeletal development through analysis of colony-stimulating factor 1 receptor (csf1r) function in the zebrafish. A primary role of Csf1r signaling is to regulate the proliferation, differentiation and function of myelomonocytic cells, including osteoclasts. We demonstrate the retention of two functional paralogues of csf1r in zebrafish. Mutant analysis indicates that the paralogues have shared, non-redundant roles in regulating osteoclast activity during the formation of the adult skeleton. csf1ra, however, has adopted unique roles in pigment cell patterning not seen in the second paralogue. We identify a unique noncoding element within csf1ra of fishes that is sufficient for controlling gene expression in pigment cells during development. As a role for Csf1r signaling in pigmentation is not observed in mammals or birds, it is likely that the overlapping roles of the two paralogues released functional constraints on csf1ra, allowing the signaling capacity of Csf1r to serve a novel function in the evolution of pigment pattern in fishes.

The skeletal phenotype of achondrogenesis type 1A is caused exclusively by cartilage defects.

Inactivating mutations in the ubiquitously expressed membrane trafficking component GMAP-210 (encoded by Trip11) cause achondrogenesis type 1A (ACG1A). ACG1A is surprisingly tissue specific, mainly affecting cartilage development. Bone development is also abnormal, but as chondrogenesis and osteogenesis are closely coupled, this could be a secondary consequence of the cartilage defect. A possible explanation for the tissue specificity of ACG1A is that cartilage and bone are highly secretory tissues with a high use of the membrane trafficking machinery. The perinatal lethality of ACG1A prevents investigating this hypothesis. We therefore generated mice with conditional Trip11 knockout alleles and inactivated Trip11 in chondrocytes, osteoblasts, osteoclasts and pancreas acinar cells, all highly secretory cell types. We discovered that the ACG1A skeletal phenotype is solely due to absence of GMAP-210 in chondrocytes. Mice lacking GMAP-210 in osteoblasts, osteoclasts and acinar cells were normal. When we inactivated Trip11 in primary chondrocyte cultures, GMAP-210 deficiency affected trafficking of a subset of chondrocyte-expressed proteins rather than globally impairing membrane trafficking. Thus, GMAP-210 is essential for trafficking specific cargoes in chondrocytes but is dispensable in other highly secretory cells.

Protective effect of an ERAP1 haplotype in ankylosing spondylitis: investigating non-MHC genes in HLA-B27-positive individuals.

OBJECTIVE: The association of non-MHC genes with AS has been recently suggested. We aimed to investigate the association of the ERAP1, IL23R and TNFSF15 regions and the susceptibility to and protection from AS in HLA-B27-positive individuals. METHODS: A total of 200 unrelated AS patients and 559 healthy unrelated subjects, all HLA-B27 positive, were tested. Twenty single nucleotide polymorphisms (SNPs) were investigated in and near IL23R (nine SNPs), in ERAP1 (five SNPs) and in TNFSF15 (six SNPs). RESULTS: ERAP1 rs30187 [odds ratio (OR) = 1.5, P = 4.7 × 10(-3)] had the strongest association with AS susceptibility. A protective effect was found in three of the ERAP1 SNPs: rs17482078 (OR = 0.7, P = 2.8 × 10(-2)), rs10050860 (OR = 0.7, P = 2.3 × 10(-2)), rs2287987 (OR = 0.6, P = 1.3 × 10(-2)). The ERAP1 haplotype rs17482078/rs10050860/rs30187/rs2287987-CCTT showed an association with AS susceptibility (P = 6.8 × 10(-3)) and a protective effect was identified in rs17482078/rs10050860/rs30187/rs2287987-TTCC (P = 3.1 × 10(-2)). Significant association with AS susceptibility was found in one IL23R marker (rs1004819, P = 4.3 × 10(-2), OR = 1.3). No associations were observed in the TNFSF15 region. CONCLUSION: The identification of a new protection haplotype in ERAP1 and the lack of association of the TNFSF15 region can provide new insights into the understanding of the mechanisms underlying the susceptibility to and protection from AS.

At the moment of occurrence of a fragility hip fracture, men have higher mechanical properties values in comparison with women.

BACKGROUND: It is well established that males have lower fracture risk in comparison with females, which suggests a higher bone resistance in men. The aim of our study was to find out if in older patients with hip fragility fractures, gender has also an impact on trabecular bone material behaviour, specifically to determine whether trabecular mechanical properties under compressive loading differ between men and women who suffered a fragility hip fracture. METHODS: Femoral epiphyses were consecutively collected during hip replacement surgery due to proximal femur fragility fracture. Trabecular bone cylinders were drilled and submitted to uniaxial compression tests and mechanical properties were assessed. RESULTS: Seventy-three patients, 55 women (mean age 81 years and standard deviation of 7 years) and 18 men (mean age 81 years and standard deviation of 8 years) were evaluated. The ultimate stress of trabecular bone was significantly higher in men than in women: the median values and the interquartile range (IQR) were respectively 8.04(5.35-10.90) MPa vs. 4.46(3.02-7.73) MPa, (p-value = 0.005). The same difference between male and female was observed in the Young's modulus: 293.68(166.67-538.18) MPa vs. 174.26(73.07-322.28) MPa, (p-value = 0.028), and also in the energy to failure: 0.25(0.07-0.42) MJ/m³ vs. 0.11(0.05-0.25) MJ/m³, (p-value = 0.058). These differences were also verified after adjusting the analysis for age in a multivariate model analysis. CONCLUSIONS: Our observations demonstrated that, even in a population who suffered a fragility hip fracture, men still have higher trabecular bone mechanical properties in comparison with women.

Smoking is a predictor of worse trabecular mechanical performance in hip fragility fracture patients.

Clinical risk factors (CRFs) are established predictors of fracture events. However, the influence of individual CRFs on trabecular mechanical fragility is still a subject of debate. In this study, we aimed to assess differences, adjusted for CRFs, between bone macrostructural parameters measured in ex-vivo specimens from hip fragility fracture patients and osteoarthritis patients, and to determine whether individual CRFs could predict trabecular bone mechanical behavior in hip fragility fractures. Additionally, we also looked for associations between the 10-year risk of major and hip fracture calculated by FRAX and trabecular bone mechanical performance. In this case-control study, a group of fragility fracture patients were compared with a group of osteoarthritis patients, both having undergone hip replacement surgery. A clinical protocol was applied in order to collect CRFs [body mass index (BMI), prior fragility fracture, parental history of hip fracture, long-term use of oral glucocorticoids, rheumatoid arthritis, current smoking, alcohol consumption, age and gender]. The 10-year probability of fracture was calculated. Serum bone turnover markers were determined and dual X-ray absorptiometry performed. Femoral head diameter was evaluated and trabecular bone cylinders were drilled for mechanical testing to determine bone strength, stiffness and toughness. We evaluated 40 hip fragility fracture and 52 osteoarthritis patients. Trabecular bone stiffness was significantly lower (p = 0.042) in hip fragility fracture patients when compared to osteoarthritic individuals, adjusted for age, gender and BMI. No other macrostructural parameter was statistically different between the groups. In hip fragility fracture patients, smoking habits (ß = -0.403; p = 0.018) and female gender (ß = -0.416; p = 0.008) were independently associated with lower stiffness. In addition, smoking was also independently associated with worse trabecular strength (ß = -0.323; p = 0.045), and toughness (ß = -0.403; p = 0.018). In these patients, the 10-year risk of major (r = -0.550; p = 0.012) and hip fracture (r = -0.513; p = 0.021) calculated using only CRFs was strongly correlated with femoral neck bone mineral density but not with mechanical performance. Our data showed that among fragility fracture patients active smoking is a predictor of worse intrinsic trabecular mechanical performance, and female gender is also independently associated with lower stiffness. In this population, the 10-year risk of fracture using CRFs with different weights only reflects bone mass loss but not trabecular mechanical properties.

TNF promoter -308 G>A and LTA 252 A>G polymorphisms in Portuguese patients with systemic lupus erythematosus.

The polymorphism of the tumor necrosis factor (TNF) promoter gene at position -308 and that of the lymphotoxin alpha (LTA) gene at position 252 have been implicated as genetic risk factors for systemic lupus erythematosus (SLE) in some populations. In a nested case-control study, we investigated the possible association of these polymorphisms with susceptibility to SLE and with phenotypic disease features in Portuguese Caucasian patients. TNF-308 G>A and LTA 252 A>G polymorphisms were determined by restriction fragment length polymorphism analysis in a cohort of 115 SLE patients and 152 unrelated healthy controls, and the magnitude of the association between genotypes and SLE diagnosis was calculated. For SLE patients, we also tested the association between disease characteristics and genotypes. No significant differences in genotype or allele frequencies could be identified between SLE cases and controls. Lupus nephritis (OR = 2.84; 95%CI 1.14-7.03, P = 0.02) and the presence of anti-Sm antibodies (OR = 3.11; 95%CI 1.08-8.94; P = 0.03) were significantly more prevalent among lupus patients possessing the TNF-308 A allele. The occurrence of nephritis was also higher in LTA 252 G allele carriers (OR = 2.90; 95%CI 1.12-7.54; P = 0.02). Our results do not support a major role of either the TNF-308 G>A or the LTA 252 A>G polymorphisms as genetic risk factors for SLE. Nevertheless, these polymorphisms appear to associate with the risk of renal lupus and distinct immunological features.

Osteoclast activity sculpts craniofacial form to permit sensorineural patterning in the zebrafish skull.

Efforts to understand the morphogenesis of complex craniofacial structures have largely focused on the role of chondrocytes and osteoblasts. Along with these bone-creating cells, bone-resorbing osteoclasts are critical in homeostasis of adult skeletal structures, but there is currently limited information on their role in the complex morphogenetic events of craniofacial development. Fundamental aspects of skull formation and general skeletal development are conserved from zebrafish to mammals. Using a cathepsinK reporter, we documented osteoclast location in the developing zebrafish skull over several weeks, from 5.18 mm to 9.6 mm standard length (approximately 15 to 34 days post fertilization). While broad distribution of osteoclasts is consistent across individuals, they are sparse and the exact locations vary among fish and across developmental time points. Interestingly, we observed osteoclasts concentrating at areas associated with neuromasts and their associated nerves, in particular the hyomandibular foramina and around the supraorbital lateral line. These are areas of active remodeling. In contrast, other areas of rapid bone growth, such as the osteogenic fronts of the frontal and parietal bones, show no particular concentration of osteoclasts, suggesting that they play a special role in shaping bone near neuromasts and nerves. In csf1ra mutants lacking functional osteoclasts, the morphology of the cranial bone was disrupted in both areas. The hyomandibular foramen is present in the initial cartilage template, but after the initiation of ossification, the diameter of the canal is significantly smaller in the absence of osteoclasts. The diameter of the supraorbital lateral line canals was also reduced in the mutants, as was the number of pores associated with neuromasts, which allow for the passage of associated nerves through the bone. Our findings define important and previously unappreciated roles for osteoclast activity in shaping craniofacial skeletal structures with a particular role in bone modeling around peripheral cranial nerves, providing a scaffold for wiring the sensioneural system during craniofacial development. This has important implications for the formation of the evolutionarily diverse lateral line system, as well understanding the mechanism of neurologic sequelae of congenital osteoclast dysfunction in human craniofacial development.

Alterations on peripheral blood B-cell subpopulations in very early arthritis patients.

OBJECTIVE: To characterize circulating B-cell subpopulations of arthritis patients with <6 weeks of disease duration. METHODS: Peripheral blood samples were collected from very early untreated polyarthritis patients, with <6 weeks of disease duration, for flow cytometric evaluation of B-cell subpopulations. Samples from patients who were later diagnosed as RA [very early RA (VERA)] were also collected 4-6 weeks after starting a low dose of prednisone (5-10 mg) and 4 months after reaching the minimum effective dose of MTX. A matched healthy group was used as a control. RESULTS: VERA patients have a lower percentage of total peripheral blood memory B cells (CD19(+)CD27(+)) and a significant decrease in the frequency of circulating pre-switch memory B cells (CD19(+)IgD(+)CD27(+)) as compared with controls. Therapy with corticosteroids or MTX was unable to restore the normal frequencies of these B-cell subpopulations. A significant decrease in peripheral pre-switch memory B cells is equally observed in other early arthritis patients. Furthermore, no significant differences are found in the frequencies of CD4(+) and CD8(+) T cells in all patient groups. CONCLUSIONS: In very early polyarthritis patients, there is a reduction in circulating pre-switch memory B cells. The reasons that may account for this effect are still unknown. Short-term corticosteroids and MTX do not seem to have a direct effect on circulating B-cell subpopulations in VERA patients.

celsr1a is essential for tissue homeostasis and onset of aging phenotypes in the zebrafish.

The use of genetics has been invaluable in defining the complex mechanisms of aging and longevity. Zebrafish, while a prominent model for vertebrate development, have not been used systematically to address questions of how and why we age. In a mutagenesis screen focusing on late developmental phenotypes, we identified a new mutant that displays aging phenotypes at young adult stages. We find that the phenotypes are due to loss-of-function in the non-classical cadherin celsr1a. The premature aging is not associated with increased cellular senescence or telomere length but is a result of a failure to maintain progenitor cell populations. We show that celsr1a is essential for maintenance of stem cell progenitors in late stages. Caloric restriction can ameliorate celsr1a aging phenotypes. These data suggest that celsr1a function helps to mediate stem cell maintenance during maturation and homeostasis of tissues and thus regulates the onset or expressivity of aging phenotypes.

Low Serum Levels of DKK2 Predict Incident Low-Impact Fracture in Older Women.

There are currently no robust noninvasive markers of fragility fractures. Secreted frizzled related protein-1 (sFRP-1), dickkopf-related protein 1 (DKK1) and DKK2, and sclerostin (SOST) inhibit Wnt signaling and interfere with osteoblast-mediated bone formation. We evaluated associations of serum levels of sFRP-1, DKK1, DKK2, and SOST with incident low-impact fracture and BMD in 828 women aged ≥65 years from EpiDoC, a longitudinal population-based cohort. A structured questionnaire during a baseline clinical appointment assessed prevalent fragility fractures and clinical risk factors (CRFs) for fracture. Blood was collected to measure serum levels of bone turnover markers and Wnt regulators. Lumbar spine and hip BMD were determined by DXA scanning. Follow-up assessment was performed through a phone interview; incident fragility fracture was defined by any new self-reported low-impact fracture. Multivariate Cox proportional hazard models were used to analyze fracture risk adjusted for CRFs and BMD. During a mean follow-up of 2.3 ± 1.0 years, 62 low-impact fractures were sustained in 58 women. A low serum DKK2 level (per 1 SD decrease) was associated with a 1.5-fold increase in fracture risk independently of BMD and CRFs. Women in the two lowest DKK2 quartiles had a fracture incidence rate of 32 per 1000 person-years, whereas women in the two highest quartiles had 14 fragility fractures per 1000 person-years. A high serum sFRP1 level was associated with a 1.6-fold increase in fracture risk adjusted for CRFs, but not independently of BMD. Serum levels of SOST (r = 0.191; p = 0.0025) and DKK1(r = -0.1725; p = 0.011) were correlated with hip BMD, but not with incident fragility fracture. These results indicate that serum DKK2 and sFRP1 may predict low-impact fracture. The low number of incident fractures recorded is a limitation and serum levels of Wnt regulators should be further studied in other populations as potential noninvasive markers of fragility fractures. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Assessment of laboratory measurements and -308 TNFalpha gene promoter polymorphisms in normal bone mineral density.

The aim of this study was to identify and evaluate laboratory parameters associated with normal bone mineral density (BMD) and to test if -308 tumour necrosis factor (TNF) alpha gene promoter polymorphisms could influence BMD. We performed a comparative cross-sectional study of four main groups: young healthy individuals (20-30 years); subjects aged 50 years or over with normal BMD; osteoporotic subjects aged 50 years or over; osteoporotic women with active rheumatoid arthritis. Variables assessed included anthropometric features, diet intake, lifestyle, calcium-phosphorus balance, markers of bone turnover, sexual hormones, hormones related with body mass and growth, cytokines involved in inflammation and bone turnover, and -308 TNF alpha gene promoter polymorphisms. One hundred fifty-nine subjects were evaluated. Across the four groups, zinc serum levels were higher in men as compared to women. In addition, zinc serum levels were also higher in individuals with normal BMD as compared to osteoporotic subjects. Serum calcium levels were higher in normal BMD group. On the other hand, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were significantly higher in normal bone mass postmenopausal women and men as compared to age-matched osteoporotic groups. Finally, leptin was significantly lower in men, after correcting these results for body mass index values. The remaining variables assessed had a similar distribution among the different studied groups. In our population, low serum levels of leptin and high serum levels of zinc, calcium, FSH, and LH were associated with a higher BMD.

Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors.

INTRODUCTION: Ankylosing spondylitis (AS) is typically characterized by focal bone overgrowth and also by systemic bone loss. We hypothesize that the increased osteoproliferation found in AS might be partially due to reduced ability of osteoclast precursors (OCPs) to differentiate into osteoclasts (OCs). Therefore, our aim was to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes' phenotype, and in vitro osteoclast differentiation in AS patients. METHODS: Patients with active AS without any ongoing therapy and age- and gender-matched healthy donors were recruited. Receptor activator of nuclear factor-κß (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations were assessed. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and quantitative reverse transcription real-time PCR for OC-specific genes were performed. RESULTS: Pro- and anti-inflammatory cytokine serum levels were higher in AS patients than in controls. RANKL neutrophil expression was higher in AS patients when compared to healthy donors, but CD51/CD61 expression was lower in the classical monocyte subpopulation. Concerning osteoclastogenesis, we found no differences in the in vitro osteoclast differentiating potential of these cells when compared to healthy donors. However, we observed low expression of CSF1R, RANK, and NFATc1 in AS OCPs. CONCLUSION: Despite the high levels of pro-inflammatory cytokines present in AS patients, no differences in the number of OC or resorbed area were found between AS patients and healthy donors. Moreover, we observed that OCPs have low OC-specific gene expression. These findings support our hypothesis of an impaired response of OCPs to pro-osteoclastogenic stimuli in vivo in AS patients.

Methotrexate and low-dose prednisolone downregulate osteoclast function by decreasing receptor activator of nuclear factor-κß expression in monocytes from patients with early rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a systemic, immune-mediated inflammatory disease that ultimately leads to bone erosions and joint destruction. Methotrexate (MTX) slows bone damage but the mechanism by which it acts is still unknown. In this study, we aimed to assess the effect of MTX and low-dose prednisolone (PDN) on circulating osteoclast (OC) precursors and OC differentiation in patients with RA. METHODS: Patients with RA before and at least 6 months after MTX therapy were analysed and compared with healthy donors. A blood sample was collected in order to assess receptor activator of NF-κß (RANK) ligand surface expression on circulating leucocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines and OC differentiation assays were performed. RESULTS: Classical activation markers of monocytes and RANK increased in patients with RA at baseline, compared with control healthy donors, and after MTX and low-dose PDN (MTX+PDN) exposure they decreased to control levels. Although the number of OC was not different between groups, the percentage of resorbed area and the resorbed area per pit reduced after treatment. Serum soluble receptor activator of nuclear factor-kappa (RANKL) levels increased at baseline compared with healthy donors and normalised after therapy. CONCLUSION: Our results suggest that MTX+PDN play an important role in downregulating OC function, which we believe occurs through the decrease in RANK surface expression in monocytes.

Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.

We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.

Corrigendum: Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors.

[This corrects the article on p. 5 in vol. 4, PMID: 28191455.].

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis.

Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6.

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Ankylosing Spondylitis.

INTRODUCTION: Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. METHODS: 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. RESULTS: RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-ß levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. CONCLUSION: In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli.

Rheumatoid arthritis bone fragility is associated with upregulation of IL17 and DKK1 gene expression.

Our aim was to compare bone gene expression in rheumatoid arthritis (RA) and primary osteoporosis (OP) patients. Secondary aims were to determine the association of gene expression of the Wnt/ß-catenin signaling pathway with inflammatory cytokines in the bone microenvironment and to assess the serum levels of Wnt/ß-catenin proteins in both groups. RA patients referred for hip replacement surgery were recruited. Primary OP patients were used as controls. Gene expression of Wnt pathway mediators, matrix proteins, and pro-inflammatory cytokines were analyzed in bone samples. Bone turnover markers, inflammatory cytokines, and Wnt mediators were measured in serum. Twenty-two patients were included: 10 with RA and 12 with primary OP. The expressions of Wnt10b (p = 0.034), its co-receptor LRP6 (p = 0.041), and its negative regulator DKK1 (p = 0.008) were upregulated in RA bone. IL17 gene expression in bone was upregulated in RA patients (p = 0.031) and correlated positively with Wnt10b (r = 0.810, p = 0.015), DKK2 (r = 0.800, p = 0.010), and RANKL/OPG ratio (r = 0.762, p = 0.028). DKK2 (p = 0.04) was significantly decreased in RA serum compared with primary OP. In conclusion, bone fragility in RA patients is induced by an unbalanced bone microenvironment and is associated with a specific gene expression pattern, namely, the upregulation of IL17 and DKK1, suggesting that the modulation of these two pathways might prevent RA systemic bone loss.

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures.

INTRODUCTION: Osteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization. We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event. METHODS: In this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined. Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis. RESULTS: 64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p<0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~0; p=0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p=0.029). ucOC levels were higher in the fracture group although not significantly (p=0.058). No differences were found regarding total OC (p=0.602), apoE (p=0.467) and Vitamin K (p=0.371). In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (ß=0.607; p=0.013) and stiffness (ß=0.693; p=0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (ß=-0.411; p=0.030) and stiffness (ß=-0.487; p=0.009). CONCLUSION: We demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.