FRZB Gene Research Articles

Altered expression of proteins involved in metabolism in LGMDR1 muscle is lost in cell culture conditions

BackgroundLimb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy due to mutations in the CAPN3 gene. While the pathophysiology of this disease has not been clearly established yet, Wnt and mTOR signaling pathways impairment in LGMDR1 muscles has been reported.ResultsA reduction in Akt phosphorylation ratio and upregulated expression of proteins implicated in glycolysis (HK-II) and in fructose and lactate transport (GLUT5 and MCT1) in LGMDR1 muscle was observed. In vitro analysis to establish mitochondrial and glycolytic functions of primary cultures were performed, however, no differences between control and patients were observed. Additionally, gene expression analysis showed a lack of correlation between primary myoblasts/myotubes and LGMDR1 muscle while skin fibroblasts and CD56− cells showed a slightly better correlation with muscle. FRZB gene was upregulated in all the analyzed cell types (except in myoblasts).ConclusionsProteins implicated in metabolism are deregulated in LGMDR1 patients’ muscle. Obtained results evidence the limited usefulness of primary myoblasts/myotubes for LGMDR1 gene expression and metabolic studies. However, since FRZB is the only gene that showed upregulation in all the analyzed cell types it is suggested its role as a key regulator of the pathophysiology of the LGMDR1 muscle fiber. The Wnt signaling pathway inactivation, secondary to FRZB upregulation, and GLUT5 overexpression may participate in the impaired adipogenesis in LGMD1R patients.

The FRZB gene regulates hair follicle development in rabbits via the Wnt/β-catenin signaling pathway

To explore the mechanism of the FRZB gene in hair follicle development by regulating the Wnt/β-catenin signalling pathway, Angora rabbits were selected to collect back skin samples for the experiment. The action mechanism is understood by cell culture and transfection, apoptosis and proliferation assays and TOP/FOP Flash Wnt Reporting System methods. The results showed that the interference and overexpression of the FRZB gene in rabbit dermal papilla cells indicated that overexpression could inhibit the expression of SFRP2, BMP4, and WNT2 genes (P<0.05). On the contrary, the expression of Wnt signalling pathway-related genes LEF1, CCND1, DKK1, and TCF7 was significantly up-regulated (P < 0.05). Further examination of the luciferase reporter system TOP/FOP revealed that pcDNA3.1-FRZB inhibits Wnt activity. PcDNA3.1-FRZB was found to promote the level of apoptosis in DP cells, whereas si-FRZB inhibited DP cell proliferation. Therefore, it is concluded that FRZB inhibits hair follicle development in long-haired rabbits by regulating the Wnt/β-catenin signalling pathway.

Identification MNS1, FRZB, OGN, LUM, SERP1NA3 and FCN3 as the potential immune-related key genes involved in ischaemic cardiomyopathy by random forest and nomogram.

The immune molecular mechanisms involved in ischaemic cardiomyopathy (ICM) have not been fully elucidated. The current study aimed to elucidate the immune cell infiltration pattern of the ICM and identify key immune-related genes that participate in the pathologic process of the ICM. The differentially expressed genes (DEGs) were identified from two datasets (GSE42955 combined with GSE57338) and the top 8 key DEGs related to ICM were screened using random forest and used to construct the nomogram model. Moreover, the "CIBERSORT" software package was used to determine the proportion of infiltrating immune cells in the ICM. A total of 39 DEGs (18 upregulated and 21 downregulated) were identified in the current study. Four upregulated DEGs, including MNS1, FRZB, OGN, and LUM, and four downregulated DEGs, SERP1NA3, RNASE2, FCN3 and SLCO4A1, were identified by the random forest model. The nomogram constructed based on the above 8 key genes suggested a diagnostic value of up to 99% to distinguish the ICM from healthy participants. Meanwhile, most of the key DEGs presented prominent interactions with immune cell infiltrates. The RT-qPCR results suggested that the expression levels of MNS1, FRZB, OGN, LUM, SERP1NA3 and FCN3 between the ICM and control groups were consistent with the bioinformatic analysis results. These results suggested that immune cell infiltration plays a critical role in the occurrence and progression of ICM. Several key immune-related genes, including the MNS1, FRZB, OGN, LUM, SERP1NA3 and FCN3 genes, are expected to be reliable serum markers for the diagnosis of ICM and potential molecular targets for ICM immunotherapy.

Frizzled related protein deficiency impairs muscle strength, gait and calpain 3 levels

BackgroundLimb-girdle muscular dystrophy recessive 1 calpain3-related (LGMDR1), previously known as LGMD2A, is a disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness and muscle degeneration. Frizzled related protein (FRZB), upregulated in LGMDR1, was identified as a key regulator of the crosstalk between Wnt and integrin signalling pathways. FRZB gene silencing showed a recovery in the expression of some of the costamere protein levels in myotubes.ResultsHere, we performed a comprehensive characterization of Frzb−/− mice muscles to study the absence of Frzb in skeletal muscle and eventual links with the molecular characteristics of LGMDR1 patient muscles. Frzb−/− mice showed reduced muscle size and strength. Gait analysis showed that Frzb−/− mice moved more slowly but no impaired regeneration capacity was observed after muscle injury. Additionally, Frzb−/− mice muscle showed an increased number of mesoangioblasts. Lack of Frzb gene in Frzb−/− mice and its increased expression in LGMDR1 patients, showed contrary regulation of Rora, Slc16a1, Tfrc and Capn3 genes. The reciprocal regulation of Frzb and Capn3 genes further supports this axis as a potential target for LGMDR1 patients.ConclusionsOur data confirm a role for Frzb in the regulation of Rora, Slc16a1, Tfrc, and Capn3 genes in muscle cells. In vivo, reduced muscle strength and gait in the Frzb−/− mice are intriguing features. The reciprocal relationship between FRZB and CAPN3 further supports a key role for this axis in patients with LGMDR1.

Risk of Wnt/β-catenin signalling pathway gene polymorphisms in primary Sjögren's syndrome.

To explore genetic polymorphisms of the Wnt/β-catenin signalling pathway in primary SS (PSS). We included 98 patients with PSS and 165 healthy volunteers. Genomic DNA was extracted from peripheral blood samples. Through an open-array platform of low density, we genotyped 25 polymorphisms from 14 genes (WISP1, DKK1, SOST, FRZB, LRP1, LRP4, LRP5, LRP6, GSKB, ADAMTS5, GDF5, FMN2, ADIPOQ and COL11A1) involved in the Wnt/β-catenin signalling pathway. We compared the allelic and genotypic frequencies with Fisher's exact test and logistic regression analysis adjusted by age, gender and individual admixture, as well as bootstrap-resampling analysis. We assessed the gene-gene interaction by the multifactor dimensionality reduction method. We found a positive significant association with four polymorphisms: LRP5 rs606989, FRZB rs409238, GSK3B rs2037547 and ADIPOQ rs2241766. All of them conferred risk for PSS, being the highest among subjects carrying three to four risk alleles (P < 0.001). According to a multifactor dimensionality reduction analysis, the best models included the LRP5 (rs606989), FRZB (rs409238) and ADIPOQ (rs2241766) polymorphisms. LRP5, FRZB and ADIPOQ genes related in the Wnt/β-catenin signalling pathway increased the risk of PSS. Further research is needed to establish their functional role in this clinical entity.

Polymorphisms in selected genes and analysis of their relationship with osteochondrosis in Polish sport horse breeds

The aim of this research was to evaluate the polymorphisms of selected genes and to find their potential effect on the occurrence of osteochondrosis in Polish Warmbloods (sport horse breeds). The study was conducted on a group of 198 horses subjected to official performance tests. Investigated joints-fetlock, hock and stifle-were X-rayed twice, once before and again at the end of the tests (first and second examination), and on this basis the degree of disease was evaluated. Based on the results of previous research, 13 candidate genes potentially associated with the occurrence of osteochondrosis were selected and, among them, 32 polymorphisms were tested. Seven SNPs located in the MATN1, CPVL, HYAL1, XIRP2, FRZB, COL5A2 and IGF1 genes were found to be associated with occurrence of osteochondrotic lesions in different joints. These intragenic polymorphisms seem to provide valuable information about the genetic basis of osteochondrosis in sport horse breeds.

Association study of candidate genes for susceptibility to Kashin-Beck disease in a Tibetan population

BackgroundMany osteoarthritis (OA) susceptibility genes have been identified in recent years. Given the overlap in the phenotype of joint inflammation between OA and Kashin-Beck disease (KBD), the aim of this study is to explore whether the reported OA susceptibility genes and two genes that may link to OA pathophysiology are associated with KBD in the Tibetan population.MethodFifteen single-nucleotide polymorphisms (SNPs) in 12 candidate genes previously reported as OA susceptibility loci were selected for investigation. Genotyping was performed using the SNaPshot method for these SNPs in a Tibetan population composed of 849 KBD patients and 565 normal controls. Meanwhile, the coding regions of two genes, COL10A1 and HABP2, which may involve in the pathological mechanism of OA/KBD, were sequenced by Sanger sequencing to identify susceptibility coding variants for KBD in the Tibetan population.ResultsThe two arthritis-susceptible candidate SNPs, rs7775 (p.Arg324Gly) in the FRZB gene and rs7033979 in the ASPN gene, showed associations with KBD (OR = 1.568, P = 4 × 10−3 and OR = 0.744, P = 8 × 10−3, respectively). The coding variants rs142463796 (p.Asp128Asn) and rs2228547 (p.Gly545Arg) in the COL10A1 gene (OR = 9.832 and P = 6 × 10−3 and OR = 1.242, P = 0.043, respectively) and rs548354451 (p.Asp272Glu) in the HABP2 gene (OR = 2.813, P = 0.010) were associated with KBD patients.ConclusionThese finding suggested that rs7775 in the FRZB gene may increase susceptibility to KBD, while rs7033979 in the ASPN gene may play a protective role in susceptibility to KBD in Tibetans. Moreover, genetic variants in chondrogenesis-related genes COL10A1 and HABP2 may play a role in the risk of developing KBD in the Tibetan population.

2-Methoxyestradiol-Mediated Induction of Frzb Contributes to Cell Death and Autophagy in MG63 Osteosarcoma Cells.

Osteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti-tumor compound, 2-methoxyestradiol (2-ME) on Wnt antagonist frizzled-related protein b (Frzb), also known as secreted frizzled-related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2-ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2-ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of β-catenin, and blocking β-catenin-mediated Wnt activation in osteosarcoma cells. 2-ME-mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2-ME-induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2-ME-mediated anti-tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497-1504, 2017. © 2016 Wiley Periodicals, Inc.

Polymorphisms of the COL9A2, AOAH and FRZB Genes in the Horse Genome and their Association with the Occurrence of Osteochondrosis

Abstract The aim of the study was to evaluate the chosen polymorphisms of the COL9A2, AOAH and FRZB genes and find their potential effect on the occurrence of osteochondrosis in Polish sport horses population. During two successive years, all 198 performance tested horses were checked for osteochondrosis. The health status of the horses was assessed based on 10 x-ray images of three joints: fetlock, hock and stifle, and scored on a 0-3 scale. The methodology of analysis of selected candidate genes using the PCR-RFLP technique was developed. The analysis of variance was performed to evaluate significance of the effect of the COL9A2, AOAH and FRZB genotype on the occurrence of osteochondrosis in individual joints. Fixed effects of breed, gender and training centre were taken into account in the analysis. The results showed a significant influence (P≤0.05) of the COL9A2 genotypes on the occurrence of osteochondrosis in fetlock and hock joints. Polymorphism of this gene, even not proved a causal mutation, appears to have effect on symptoms of the disease. In genes AOAH and FRZB there was no significant effect for investigated SNPs. Further analysis of the discussed genes/polymorphisms seems to be important.

FRZB and melusin, overexpressed in LGMD2A, regulate integrin β1D isoform replacement altering myoblast fusion and the integrin-signalling pathway.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is characterised by muscle wasting and progressive degeneration of proximal muscles because of mutations in the CAPN3 gene. However, the underlying pathophysiological mechanisms of muscle degeneration are still not well understood. The objective of this study was to assess the relevance of genes with differential expression in the muscle of LGMD2A patients. For this purpose, we analysed their in vitro expression in primary cultures of human myoblasts and myotubes. Abnormal fusion was observed in the myotubes of these patients, which may be explained by the lack of physiological replacement of integrin β1D. Owing to this observation, we focused on deregulated genes coding proteins that directly interact with integrin, ITGB1BP2 and CD9, as well as FRZB gene, because of its in vitro upregulation in myotubes. Silencing studies established that these genes are closely regulated, CD9 and FRZB being positive regulators of the expression of ITGB1BP2, and in turn, this gene being a negative regulator of the expression of FRZB. Interestingly, we observed that FRZB regulates integrin β1D expression, its silencing increasing integrin β1D expression to levels similar to those in controls. Finally, the administration of LiCl, an enhancer of the Wnt-signalling pathway showed similar experimentally beneficial effects, suggesting FRZB silencing or LiCl administration as potential therapeutic targets, though further studies are required.

Single nucleotide polymorphism scanning and expression of the FRZB gene in pig populations.

Secreted frizzled-related protein 3 (sFRP3), encoded by the gene FRZB, is a member of the sFRP family with important roles in inhibition of the Wnt signalling pathway through competitive binding of the Wnt receptor. Here, we investigated pig FRZB as a candidate gene for growth traits and identified three polymorphic sites, an insertion (A-532B) and two SNPs (G636A and C650T) in its 5'-UTR. The genotype distributions of G636A and C650T were significantly different among mini-type indigenous (Diannan Small-ear and Tibetan), normal indigenous (Laiwu and Huai), and introduced (Large Yorkshire and Landrace) breeds. In semi-quantitative PCR expression analysis, expression of FRZB mRNA was abundant in tissues of hypophysis, longissimus dorsi muscle, and adipose tissues, and low in the heart, hypothalamus, and brain. Quantitative determination of mRNA level and protein expression analysis were corresponding. The results demonstrated that FRZB gene expression in longissimus dorsi muscle and liver tissue was significantly higher in Diannan Small-ear and Tibetan pigs than in the Large Yorkshire breed (P<0.05); however, in back fat tissue, the expression was significantly higher in Diannan Small-ear pig than in Tibetan or Large Yorkshire breeds (P<0.05). Given the known growth and fat characteristics of the breeds, these results indicate that FRZB expression has a negative association with muscle growth and a positive association with fat deposition. In conclusion, FRZB may be a major candidate gene for growth traits in pigs.

GREM1, FRZB and DKK1 mRNA levels correlate with osteoarthritis and are regulated by osteoarthritis-associated factors

IntroductionOsteoarthritis is, at least in a subset of patients, associated with hypertrophic differentiation of articular chondrocytes. Recently, we identified the bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) signaling antagonists Gremlin 1 (GREM1), frizzled-related protein (FRZB) and dickkopf 1 homolog (Xenopus laevis) (DKK1) as articular cartilage’s natural brakes of hypertrophic differentiation. In this study, we investigated whether factors implicated in osteoarthritis or regulation of chondrocyte hypertrophy influence GREM1, FRZB and DKK1 expression levels.MethodsGREM1, FRZB and DKK1 mRNA levels were studied in articular cartilage from healthy preadolescents and healthy adults as well as in preserved and degrading osteoarthritic cartilage from the same osteoarthritic joint by quantitative PCR. Subsequently, we exposed human articular chondrocytes to WNT, BMP, IL-1β, Indian hedgehog, parathyroid hormone-related peptide, mechanical loading, different medium tonicities or distinct oxygen levels and investigated GREM1, FRZB and DKK1 expression levels using a time-course analysis.ResultsGREM1, FRZB and DKK1 mRNA expression were strongly decreased in osteoarthritis. Moreover, this downregulation is stronger in degrading cartilage compared with macroscopically preserved cartilage from the same osteoarthritic joint. WNT, BMP, IL-1β signaling and mechanical loading regulated GREM1, FRZB and DKK1 mRNA levels. Indian hedgehog, parathyroid hormone-related peptide and tonicity influenced the mRNA levels of at least one antagonist, while oxygen levels did not demonstrate any statistically significant effect. Interestingly, BMP and WNT signaling upregulated the expression of each other’s antagonists.ConclusionsTogether, the current study demonstrates an inverse correlation between osteoarthritis and GREM1, FRZB and DKK1 gene expression in cartilage and provides insight into the underlying transcriptional regulation. Furthermore, we show that BMP and WNT signaling are linked in a negative feedback loop, which might prove essential in articular cartilage homeostasis by balancing BMP and WNT activity.

Variant alleles of the Wnt antagonist FRZB are determinants of hip shape and modify the relationship between hip shape and osteoarthritis

To test whether single-nucleotide polymorphisms (SNPs) of the FRZB gene are associated with hip shape, and to determine whether FRZB variant alleles affect the relationship between hip shape and radiographic osteoarthritis (OA) of the hip. A nested case-control study of Caucasian women, age ≥65 years, from the Study of Osteoporotic Fractures cohort was performed. Cases (n = 451) were defined as subjects with radiographic evidence of incident hip OA during followup, while controls (n = 601) were subjects in whom no radiographic hip OA was identified at baseline or followup. Statistical shape modeling (SSM) of the digitized hip radiographs was performed to assess the shape of the proximal femur, using 10 independent modes of shape variation generated by principal components analysis. In addition, center-edge angle and acetabular depth were assessed as geometric measurements of acetabular shape. The association of the rs288326 and rs7775 FRZB variant alleles with hip shape was analyzed using linear regression. The effect of these alleles on the relationship between hip shape and radiographic hip OA was analyzed using a logistic regression model with or without inclusion of interaction terms. The rs288326 and rs7775 alleles were associated with the shape of the proximal femur (SSM mode 2). There was a significant interaction between the rs288326 SNP and proximal femur shape (SSM mode 2) in predicting radiographic hip OA (P for interaction = 0.022). Among subjects with the rs288326 variant allele, there was an increased likelihood of radiographic hip OA in association with increasing quartiles of proximal femur shape mode 2 (for the fourth quartile of mode 2, odds ratio 2.5, 95% confidence interval 1.15, 5.25; P for linear trend = 0.02). The rs288326 and rs7775 FRZB SNPs are associated with the shape of the proximal femur. The presence of the rs288326 SNP alters the relationship between proximal femur shape and incident radiographic hip OA. These findings suggest that FRZB may serve an important role in determining hip shape and may modify the relationship between hip shape and OA.

Relationship of sclerostin and secreted frizzled protein polymorphisms with bone mineral density

Secreted frizzled-related protein and sclerostin, encoded by FRZB and SOST genes, respectively, are extracellular Wnt inhibitors that tend to decrease bone formation. The purpose of this study was to explore the association of the sets of polymorphisms capturing common variations of these genes with bone mineral density (BMD). Twelve polymorphic loci of the FRZB gene and 7 of the SOST gene were genotyped in postmenopausal women from two Spanish regions (Cantabria, n = 1043, and Valencia, n = 342). The polymorphisms included tagging single nucleotide polymorphisms and single nucleotide polymorphisms with possible functional consequences assessed in silico. The rs4666865 polymorphism of the FRZB gene was associated with spine BMD in the Cantabria cohort in the single-locus (P = 0.008) and the haplotypic analysis. However, the results were not replicated in the Valencia cohort. Several polymorphisms at the 5' region of the SOST gene and, particularly, rs851056 were associated with BMD in women from both cohorts (P = 0.002 in Cantabria and P = 0.005 in Valencia). When the results of both cohorts were combined, the mean (SD) BMD difference across rs851056 genotypes was 47 (0.31) mg/cm(2) (P < 0.001). No differences in FRZB and SOST expression in femoral trabecular bone tissue were detected across genotypes. Polymorphisms in the 5' region of SOST gene are associated with BMD in postmenopausal women. They may contribute to explain, in part, the hereditary influence on bone mass.

FISH mapping of six genes responsible for development of the nervous and skeletal systems on donkey (Equus asinus) chromosomes

The results obtained in the present study made it possible to place selected markers responsible for development of the nervous and skeletal systems on the physical map of the donkey genome. Fluorescence in situ hybridization (FISH) was used to localize genes such as GDF5 (15q13), FRZB (4q23.1), TWIST (1q31), PAX6 (20q25), SALL1 (24q15) and SHH (1q35) on donkey chromosomes. The identification of their localization confirmed previously proposed homologies using ZOO-FISH technique, except for FRZB and SALL1 genes. This suggests that they were affected by rearrangements that changed their localization compared to horse, and in the case of the SALL1 gene also compared to human.

No association of the polymorphisms of the frizzled-related protein gene with peak bone mineral density in Chinese nuclear families

BackgroundThe Wnt/beta-catenin signaling pathway plays an important role in skeletal development. Polymorphisms of frizzled-related protein (FRZB), an antagonist of this pathway, may generate variations in bone mineral density (BMD). In this study, we analyzed the association between FRZB genotypes and peak BMD variation in the spines and hips of two relatively large samples of Chinese female-offspring and male-offspring nuclear families.MethodsWe recruited 1,260 subjects from 401 female-offspring nuclear families and 1,296 subjects from 427 male-offspring nuclear families and genotyped four tagging single nucleotide polymorphisms (tagSNPs) (rs6433993, rs409238, rs288324, and rs4666865) spanning the entire FRZB gene. The SNPs rs288326 and rs7775, which are associated with hip osteoarthritis, were not selected in this study because of their low minor allele frequencies (MAFs) in Chinese people. The quantitative transmission disequilibrium test (QTDT) was used to analyze the association between each SNP and haplotype with peak BMD in female- and male-offspring nuclear families.ResultsIn the female-offspring nuclear families, we found no evidence of an association between either single SNPs or haplotypes and peak BMD in the spine or hip. In the male-offspring nuclear families, no within-family association was observed for either SNPs or haplotypes, although a significant total association was found between rs4666865 and spine BMD (P = 0.0299).ConclusionOur results suggest that natural variation in FRZB is not a major contributor to the observed variability in peak BMD in either Chinese females or males. Because ethnic differences in the FRZB genotypes may exist, other studies in different population are required to confirm such results.

082 STREAMING POTENTIAL-BASED ARTHROSCOPIC DEVICE CAN DETECT CHANGES IMMEDIATELY FOLLOWING LOCALIZED IMPACT IN AN EQUINE IMPACT MODEL OF OSTEOARTHRITIS

Purpose: Frizzled related protein (FRZB/sFRP3) is a secreted WNT antagonist isolated from articular cartilage and expressed in developing skeletal elements. Polymorphisms in the human FRZB gene are associated with susceptibility for osteoarthritis. Induction of experimental osteoarthritis in Frzb–/– mice results in enhanced cartilage degradation associated with increased Wnt signalling, Mmp3 expression, Mmp activity and cortical bone thickness. We further studied the role of FRZB in osteoarthritis by FRZB adenoviral overexpression in the knee joint in the methylated bovine serum albumin (mBSA)-induced arthritis model. Methods: Adenovirus expressing FRZB or GFP (107 pfu/10 μl in PBS) was injected intra-articularly in the right knee joint of C57Bl/6 mice at day 0. At day 1 mBSA-induced arthritis was induced by intra-articular injection of 10 μl mBSA (20 mg/ml in PBS) followed by subcutaneous injections of IL-1β (250 ng) in the right footpad for 3 consecutive days. The left knee was used as a negative control and injected each time with 10 μl of PBS. At day 7, mice were sacrificed and the knee, synovium or articular cartilage were isolated for histology or mRNA analysis. Knee sections were stained with haematoxylin-eosin and safranin O and severity of arthritis was scored. Expression of inflammatory cytokines Il-1β, TNF-α and Il-6 and matrix-degrading enzymes Mmp3, Mmp9, Mmp13, Adamts4 and Adamts5 in synovium and articular cartilage was determined using quantitative RT-PCR. Results: Histomorphological analysis of the knee showed a significant increase in the score of inflammatory parameters such as infiltration, exsudate formation and pannus formation when FRZB was overexpressed. Cartilage damage was not different compared to GFP controls. Increased synovitis was confirmed by geneexpression analysis of inflammatory cytokines. Il-1β, TNF-α and Il-6 expression were increased in the synovium with FRZB overexpression. We also detected a decreased expression of Mmp3 in cartilage when FRZB was overexpressed and an increase of Adamts5 in synovium. Mmp9, Mmp13 and Adamts4 expression did not change in synovium and articular cartilage. The altered balance between Adamts5 and Mmp3 expression may explain the absence of changes in cartilage damage. Conclusions: In this study we demonstrate that FRZB overexpression leads to increased inflammation in mBSA-induced arthritis in mice. The increased inflammatory response appears to counteract potential chondroprotective effects of FRZB overexpression. These data further highlight the complex biology of FRZB.

Large‐scale analysis of association between GDF5 and FRZB variants and osteoarthritis of the hip, knee, and hand

GDF5 and FRZB have been proposed as genetic loci conferring susceptibility to osteoarthritis (OA); however, the results of several studies investigating the association of OA with the rs143383 polymorphism of the GDF5 gene or the rs7775 and rs288326 polymorphisms of the FRZB gene have been conflicting or inconclusive. To examine these associations, we performed a large-scale meta-analysis of individual-level data. Fourteen teams contributed data on polymorphisms and knee, hip, and hand OA. For rs143383, the total number of cases and controls, respectively, was 5,789 and 7,850 for hip OA, 5,085 and 8,135 for knee OA, and 4,040 and 4,792 for hand OA. For rs7775, the respective sample sizes were 4,352 and 10,843 for hip OA, 3,545 and 6,085 for knee OA, and 4,010 and 5,151 for hand OA, and for rs288326, they were 4,346 and 8,034 for hip OA, 3,595 and 6,106 for knee OA, and 3,982 and 5,152 for hand OA. For each individual study, sex-specific odds ratios (ORs) were calculated for each OA phenotype that had been investigated. The ORs for each phenotype were synthesized using both fixed-effects and random-effects models for allele-based effects, and also for haplotype effects for FRZB. A significant random-effects summary OR for knee OA was demonstrated for rs143383 (1.15 [95% confidence interval 1.09-1.22]) (P=9.4x10(-7)), with no significant between-study heterogeneity. Estimates of effect sizes for hip and hand OA were similar, but a large between-study heterogeneity was observed, and statistical significance was borderline (for OA of the hip [P=0.016]) or absent (for OA of the hand [P=0.19]). Analyses for FRZB polymorphisms and haplotypes did not reveal any statistically significant signals, except for a borderline association of rs288326 with hip OA (P=0.019). Evidence of an association between the GDF5 rs143383 polymorphism and OA is substantially strong, but the genetic effects are consistent across different populations only for knee OA. Findings of this collaborative analysis do not support the notion that FRZB rs7775 or rs288326 has any sizable genetic effect on OA phenotypes.

Wnt pathway genes in osteoporosis and osteoarthritis: differential expression and genetic association study

In comparison with hip fractures, increased expression of genes in the Wnt pathway and increased Wnt activity were found in bone samples and osteoblast cultures from patients with osteoarthritis, suggesting the involvement of this pathway in subchondral bone changes. No consistent differences were found in the genetic association study. This study aims to explore the allelic variations and expression of Wnt pathway genes in patients with osteoporosis and osteoarthritis. The expression of 86 genes was studied in bone samples and osteoblast primary cultures from patients with hip fractures and hip or knee osteoarthritis. The Wnt-related activity was assessed by measuring AXIN2 and in transfection experiments. Fifty-five SNPs of the LRP5, LRP6, FRZB, and SOST genes were analyzed in 1,128 patients. Several genes were differentially expressed in bone tissue, with the lowest values usually found in hip fracture and the highest in knee osteoarthritis. Overall, seven genes were consistently upregulated both in tissue samples and in cell cultures from patients with knee osteoarthritis (BCL9, FZD5, DVL2, EP300, FRZB, LRP5, and TCF7L1). The increased expression of AXIN2 and experiments of transient transfection of osteoblasts with the TOP-Flash construct confirmed the activation of Wnt signaling. Three SNPs of the LRP5 gene and one in the LRP6 gene showed marginally significant differences in allelic frequencies across the patient groups, but they did not resist multiple-test adjustment. Genes in the Wnt pathway are upregulated in the osteoarthritic bone, suggesting their involvement not only in cartilage distortion but also in subchondral bone changes.

Activation of β-Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis-Like Phenotype in Adult β-Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/beta-catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for beta-catenin in this disease has not been reported. Because tissue-specific activation of the beta-catenin gene (targeted by Col2a1-Cre) is embryonic lethal, we specifically activated the beta-catenin gene in articular chondrocytes in adult mice by generating beta-catenin conditional activation (cAct) mice through breeding of beta-catenin(fx(Ex3)/fx(Ex3)) mice with Col2a1-CreER(T2) transgenic mice. Deletion of exon 3 of the beta-catenin gene results in the production of a stabilized fusion beta-catenin protein that is resistant to phosphorylation by GSK-3beta. In this study, tamoxifen was administered to the 3- and 6-mo-old Col2a1-CreER(T2);beta-catenin(fx(Ex3)/wt) mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of beta-catenin protein was detected by immunostaining in articular cartilage tissues of beta-catenin cAct mice. In 5-mo-old beta-catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8-mo-old beta-catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in beta-catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp-9, Mmp-13, Alp, Oc, and colX, was significantly increased (3- to 6-fold) in articular chondrocytes derived from beta-catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6-fold increase) in these cells. In addition, we also observed overexpression of beta-catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of beta-catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA-like phenotype. This study provides direct and definitive evidence about the role of beta-catenin in the development of OA.