The Role Of Fzd8 For Bone Development And Homeostasis In A Mouse Model Generated By CRISPR/Cas9 Genome Editing

The Role Of Fzd8 For Bone Development And Homeostasis In A Mouse Model Generated By CRISPR/Cas9 Genome Editing | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Confirmatory Results The Role Of Fzd8 For Bone Development And Homeostasis In A Mouse Model Generated By CRISPR/Cas9 Genome Editing Zhengkun Lin , Jianquan He , Hui Huang , Xiaomei Lin , Heqing Chen , Wen Zhang , Jian Chen doi: https://doi.org/10.1101/2025.01.19.633799 Zhengkun Lin a Department of Rehabilitation, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University , Xiamen, 361004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jianquan He a Department of Rehabilitation, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University , Xiamen, 361004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hui Huang a Department of Rehabilitation, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University , Xiamen, 361004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiaomei Lin a Department of Rehabilitation, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University , Xiamen, 361004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Heqing Chen a Department of Rehabilitation, Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University , Xiamen, 361004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wen Zhang b Icahn School of Medicine at Mount Sinai , New York, NY 10029, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jian Chen c Xiamen Humanity Rehabilitation Hospital , Xiamen, 361006, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: chenjiansci{at}163.com Abstract Full Text Info/History Metrics Preview PDF Abstract Background FZD8 could be a promising therapeutic target in osteoporosis (OP), although the signal transduction mechanism in OP regarding FZD8 has not been completely elucidated. Aims We used the CRISPR/Cas9 technique to develop an Fzd8 -knockout mouse model to study whether Fzd8 inactivation results in genetic changes with potential correlations to OP. Materials and Methods Genotypes of distinguished classified knockout mice, i.e., heterozygous, homozygous, and wild-type were identified through PCR. Applying the murine model, third generation mice were used for the downstream experiments. We investigated the potential relevance of differentially expressed genes (DEGs) in OP. Results We found that osteoclasts significantly increased in Fzd8 -knockout homozygous mice, compared to wild-type mice, while osteoblasts reduced significantly. Before transcription, heterozygous and homozygous mice possessed DEGs related to exons SNP, which are associated with exons CNV. After transcription, DEGs related to exons SNP in heterozygous and homozygous mice were observed, some of which are potentially associated with OP based on pathway and gene set enrichment analyses. Conclusions Our Fzd8- knockout murine model showed that there were significant alternations in Fzd10 and Lta gene expressions and Itgb3 and RANK protein expressions among the wild-type and homozygous mice, which are significantly associated with bone remodeling. Our results revealed that FZD8 could be a therapeutic target in OP. This study elucidates the molecular mechanisms in OP, providing evidence-based data for OP drug development and treatment. Introduction Osteoporosis (OP) is a complicated metabolic bone disorder characterized by decreased bone mass, compromised bone microarchitecture, and increased fracture risk. The fractures and other complications caused by OP seriously affect the patient’s quality of life, leading to a heavy burden on their families, as well as on society. Consequently, OP has emerged as one of the most urgent issues in global public health [ 1 ]. Former investigations by the Osteoporosis Foundation [ 2 ] summarized that OP is prevalent in 13% of the Chinese population. It is speculated that the number of patients with OP or low bone mineral density in China will reach 212 million by 2050. Studies suggest that the canonical Wnt signaling pathway could enhance osteoclast (OC) differentiation and proliferation [ 3 ]. The non-canonical Wnt pathway affects bone remodeling as well. For instance, Wnt16 could suppress OC generation, which prevents fractures due to fragility [ 4 ]. The frizzled (Fzd) protein is a transmembrane receptor responsible for binding to the Wnt protein, which activates intracellular signal transduction molecules [ 5 ]. Bone mineral contents and secreted frizzled-related proteins (sFRPs) can compete with Fzd receptors on the cell membrane surface to suppress Wnt protein function in bone pathophysiology [ 6 ]. Fzd receptors lack the low-density lipoprotein receptor-related protein 5 (LRP5) and low-density lipoprotein receptor-related protein 6 (LRP6) . Secreted form with respect to FZD8, cysteine-rich domains would antagonize Wnt3a -induced accumulation of β -catenin in mouse fibroblasts [ 7 ]. Fzd8 is a Wnt receptor and is considered a β -catenin-independent pathway [ 8 ]. However, phenotype is not related to impaired Wnt signaling or osteoprotegerin (OPG) production through osteoblasts (OBs), which are irrelevant to bone formation [ 9 ]. Theoretical investigations have discovered that profiling multiple variants associated with bone phenotypes could improve fracture prediction accuracy [ 10 ]. Fzd8 might be a therapeutic target for OP, though its signal transduction mechanism has not been clarified. In recent years, gene editing techniques have developed swiftly [ 11 ], such as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique, which can target specific genes of high interest [ 12 ]. There are few studies on the effect of Fzd8 -knockout on OP, using whole genome sequencing (WGS). This study provides evidence-based medicine ideas for drug developments, which sheds new lights on OP rehabilitation diagnosis. Materials and methods Animals Our lab housed mice with temperature-/humidity-controlled environment (23℃ ± 3℃/ 70% ± 10%) on a 12-h light/12-h dark cycle. Technicians maintained mice on standard mouse chow with 18.0% protein, 4.5% fat, 58% carbohydrate with water in Zhongshan Hospital Xiamen University. All mice were sacrificed when they aged between 20 and 30 weeks. We purchased C57BL/6J mice from the Animal Experiment Center of Fujian Province. The Fzd8 knockout mice (Zhongshan Hospital Xiamen University) were generated and maintained with C57BL/6J background. All animal experiments were carried out in Zhongshan Hospital Xiamen University and approved by Animal Subjects Committee in Zhongshan Hospital Xiamen University (approval No. XMVLAC20120044). Fzd8 - knockout mouse model The Fzd8 -knockout mouse model was designed and developed by the Shanghai Model Organisms Center, Inc. (Shanghai, China). The Cas9 mRNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, TX, USA), following standard procedures ( Supplementary Figure S1 ). The 4 single guide RNAs (sgRNAs) targeted to delete the exon 3 of the Fzd8 gene were: 5’-AGGGAGTGGATCTCAAGCCTTGG-3’; 5’-TTACTCTGGGAGGTAGGGAGTGG-3’; 5’-CCGTAGTAAGAAGCTGAGTTAGG-3’; and, 5’-CCAAAGAGAAGGGTGCGGGCGGG-3’. We transcribed the sgRNAs in vitro using the MEGAshortscript Kit (Thermo Fisher Scientific, USA). We verified the first generation (F 0 ) mice through PCR, using the following primer pairs F-5’-CGAACTCTTGGCAGGTCTGT-3’ and R-5’-ATGCCCATTGGAGCCATGAA-3’. We selected positive Fzd8- knockout F 0 mice and mated them with the C57BL/6J mice to obtain the second generation (F 1 ) heterozygous Fzd8 -knockout mice. We intercrossed female and male F 1 heterozygous mice to to produce F2 homozygous (HO) Fzd8 -knockout mice. Micro-CT detection We used the SkyScan 1176 (Bruker, Aartselaar, Belgium) system to measure different bone phenotype parameters. The NRecon software was used for 3D image reconstruction and viewing. The CTan software (version 1.13) was applied for bone analysis. We conducted micro-CT examinations of the left mice femora frozen at -40 ℃ in a freezer. Hematoxylin and Eosin (HE) staining Mouse bones were fixed with 10% formalin and embedded in paraffin. Thereafter, slices in distilled water were dyed in a hematoxylin aqueous solution, which we then separated into acid and ammonia water. Slices were cleaned under running water for 1 h, and were then immersed in distilled water and dehydrated in 90% alcohol for 10 min. The size and morphology of the stained osteocytes in one section were investigated. Immunohistochemical (IHC) staining Before dewaxing, we placed all tissue sections at room temperature (20-25℃) for 1 h, and then baked in a 60 ℃ thermostat for 20 min. The tissue sections were immersed in xylene for 10 min and soaked for another 10 min after altering xylene. The mouse bones were fixed in 10% formalin, embedded in paraffin, and sliced into 10 μm sections. The sections were mounted on slides, and antigen retrieval was conducted by incubating them with EDTA at 90 ℃ for 10 min. We then incubated the sections with 0.3% H 2 O 2 for 0.5 h, followed by applying a blocking solution buffer for 1 h, at room temperature (20-25℃). We added 50 μL of the Anti-Wnt3a (Abcam; No. ab219412), Anti-RANK (Abcam; No. ab13918), and Anti-Integrin beta 3 (Abcam; No. ab179473) antibodies. We left the sections at room temperature (20-25℃) for 1 h, and cleaned them with PBS for 5 min, thrice. Next, we added 40-50 μL Goat Anti-Rabbit IgG H&L (Abcam; No. ab150077), and Goat Anti-Rabbit IgG H&L (Abcam; No. ab97051). The Image-Pro Expess analysis system (Informer Technologies, Inc 6.0) was utilized to quantify grayscale. Enzyme-linked immunosorbent assay (ELISA) We placed the ELISA kit (Shanghai Pan Ke Industrial Co., LTD. No. 20200004) at room temperature (20-25℃), for 15-30 min. We erased the labeling plate to add 50 μL standard solution into the blank micropores. We added 10 μL biotin into the sample well, excluding the blank control well, and added 100 μL enzyme-labeled solution to each well. We sealed the enzyme label plate with a sealant and incubated it at 37 ℃ for 1 h. The absorbance (OD value) for each well was sequentially detected using a blank air conditioner at 450 nm wavelength. RNA-sequencing (RNA-seq) Tail tissues of 8 wild-type (WT) and HO mice each, were separated into two groups for RNA-seq. Total RNA was obtained using the TRIzol reagent (Sigma), following standard protocols. RNA integrity was tested using the Bioanalyzer 2100 (Agilent). The cDNA libraries were checked for quality, and were further quantified using the 2100 Bioanalyzer. Each library was sequenced with Illumina Sequencing Kit (Illumina/TruSeq RNA Library Preparation Kit v2, /RS-122-2001/1 Ea) on one lane of NovaSeq 6000 sequencing system to obtain 150 bp paired-end reads. Whole genome sequencing (WGS) Total genome DNA was extracted from mouse tails homogenized in 3 volumes of DNA extraction buffer using the Teflon homogenizer. The homogenized samples were incubated in a water bath at 55 ℃. Phenol and chloroform extraction, followed by isopropanol precipitation using 0.2× volume of 10 M ammonium acetate, was conducted at 7500 × g for 10 min. Two paired-end (PE) libraries were made for the WGS, which were sequenced on NovaSeq 6000 sequencing system (Illumina, San Diego, CA, USA). All sequencing-relevant procedures were carried out following protocols (Illumina, FC-121-4002 San Diego, CA, USA). Validation using PCR PCR was used to verify the assembly features ( Supplementary Table S1 ) developed using 1μL DNA template in each reaction and mixed with the Golden_Star_T6_Super_PCR_Mix (TSE101, Tsingke Biotechnology Co., Ltd.). PCR was run using the A300 PCR instrument (Hangzhou LongGene Scientific Instrument Co., LTD), applying a 2-step fast PCR with a 2 s denaturation step at 98 ℃, along with a 2 min annealing and extension step at 72 ℃ for 35 cycles. Validation using quantitative PCR (qPCR) The assembly features developed were validated using qPCR (detailed information on qPCR primers is provided in Supplementary Table S2 ), using 5 μL RNA template in each reaction, mixed with RT6 cDNA Synthesis Kit Ver. 2 (Tsingke Biotechnology Co., Ltd. (TSK302S)). We conducted a 3-step fast qPCR (BIOER FQD-96A) using a 2 s denaturation step at 95 ℃. The cDNA developed was added to 10 μL ddH 2 O. Statistical evaluation criteria All data analyses were performed using SPSS (version 22.0), and P < 0.05 was considered statistically significant. Each group of continuous variables was represented as mean ± standard deviation (SD). Median, interquartile range, and classified variable standard error were also calculated. Pearson’s Chi-square test or Fisher’s exact test were used for categorical variable analyses. Baseline characteristics and comparisons of major or minor results between the groups were also analyzed. To adjust confounding, a linear regression model was applied to the continuous variables. Bioinformatics data processing We identified up-regulated and down-regulated genes in accordance with the log 2 ( Fold Change ) and P < 0.05, indicating significance levels. Samples were clustered in terms of differentially expressed genes that were identified using the fragments per kilobase of exon per million mapped fragments. Correlations between samples were analyzed according to phenotypes. The Pearson correlation test was employed to obtain Pearson values regarding homogeneous mice. We performed gene ontology enrichment analysis; q values denoted significance, informing transcriptomic expressions of molecular processes. We performed gene set enrichment analysis, and the number of genes in each pathway pattern were analyzed. Standard procedures and pipelines were applied while performing these analyses[ 13 ]. Results Baseline comparison between WT and HO mice Statistical analyses suggested that there were no statistically significant differences in baseline indicators between the WT and HO mice, including their weights [mean ± standard deviation (SD): 22.40 ± 1.13 g (WT) vs. 21.70 ± 1.82 g (HO), P=0.403], blood glucose ( GLU ) (deviation: 6.20 ± 0.76 (WT) vs. 6.07 ± 1.41 (HO), P =0.749), N-terminal propeptide of type I procollagen ( P1NP) (deviation: 5.69 ± 0.83 (WT) vs. 5.80 ± 1.36 (HO), P =0.857), and C-terminal cross-linking telopeptide (CTX) (deviation: 120.68 ± 22.44 (WT) vs. 109.42 ± 18.61 (HO), P =0.327) levels. The N-terminal propeptide of type I procollagen ( P1NP2) levels in the HO mice were lower compared to WT mice, and the C-terminal cross-linking telopeptide (CTX2) levels in HO mice were higher ( Figure 1 ). Download figure Open in new tab Figure 1. Comparison of WT and HO mice. The comparison of the weight (a) of the two groups of mice. The comparison of the blood glucose (GLU) (b) , N-terminal propeptide of type I procollagen (P1NP) (c) , and S-CTX (d) levels between the WT and HO mice before they euthanization. The comparison of the P1NP2 (e) and S-CTX2 (f) levels between the WT and HO mice after they were euthanized. Micro-CT detection There were significant differences in the various bone imaging indices between the WT and HO groups ( Supplementary Figure S2 ), such as the bone mineral density (BMD) of the cortical and trabecular bones, the total volume (TV), and bone volume (BV) of the cortical bone, the BV/TV of the cortical bone, among others. The mean and SD of bone phenotypic indicators in the WT and HO mice are provided in ( Supplementary Table S3 ). Among these indicators, the mean of the phenotypic indicators in the HO mice is less than that in the WT mice. Trabeculae, BV, and BMD decreased after FZD8 -knockout. The bone trabeculae, bone cortex, and BV in the HO mice reduced significantly after Fzd8 -knockout ( Figure 2 ). Download figure Open in new tab Figure 2. The coronal, cross-sectional, and 3D view of the middle and upper parts of the left femur in WT and HO mice. Coronal view of the middle and upper parts of the left femur in WT (a) and HO (b) mice. Cortical and trabecular bones of the HO mice have lower bone density, thinner cortices, and greater trabecular spacing, compared to the WT mice, as denoted by arrows. Cross-sectional view of the middle and upper parts of the left femur in WT (c) and HO (d) mice. Cortical bones of the HO mice have a lower bone density compared to the WT mice, as denoted by arrows. 3D view of the middle and upper parts of the left femur in WT (e) and HO (f) mice. Cortical and trabecular bones of the HO mice have lower bone density, thinner cortices, and greater trabecular spacing compared to the WT mice, as denoted by arrows. Differences in protein expression between WT and HO mice There were significant differences among the RANK , Wnt3a , and Itgb3 protein expressions in the WT and HO mice. Independent experienced pathologists evaluated the immunoreactivity of the RANK , Wnt3a , and Itgb3 proteins in the WT and HO mice ( Figure 3-1 ). RANK , Wnt3a , and Itgb3 positive cells are represented by arrows. With the increase in age, various organ functions exhibited physiological degeneration and a pro-inflammatory state; identified by the low immune functions and decreased motor functions, among others. The HE staining charts ( Figure 3-1 ) show that the HO mice have significantly fewer bone trabeculae than the WT mice. Download figure Open in new tab Figure 3-1. Immunoreactivity of the Wnt3a , Itgb3 , and RANK proteins, along with HE staining. Independent experienced pathologists evaluated the immunoreactivity of the RANK Wnt3a , and Itgb3 proteins, between the WT and HO mice. RANK , Wnt3a , and Itgb3 positive cells were detected in bone tissues, as denoted by arrows (In immunohistochemistry, the positive staining is brownish yellow or brown particles). Representative images of H&E staining in the femoral metaphysis of different groups of mice (X200) are also provided. (a-b) Itgb3 was expressed in WT and HO mice, and positive expression was confirmed by the brown-yellow color; Itgb3 expression in HO mice was significantly higher than that in WT mice. (c-d) RANK was expressed in both WT and HO mice, and positive expression was confirmed by the brown-yellow color; RANK expression in HO mice was significantly higher than that in WT mice. (e-f) Wnt3a was expressed in both WT and HO mice, and positive expression was confirmed by the brown-yellow color; Wnt3a expression in HO mice was significantly higher than that in WT mice. (g-h) Under high magnification microscope (X200), the Itgb3 , Wnt3a , and RANK protein expressions in HO mice were higher than that in WT mice. All factors transmit information through the stimulated OPG / RANK / RANKL signal transduction system, which could reduce OPG / RANKL ratios and suppress OB function. β -catenin is the key factor in the Wnt/β -catenin signaling pathway. RANK and Itgb3 expressions were different in the two mice groups (WT and HO) due to Fzd8 -knockout ( Figure 3-2 ). Combined with the influence of RANK on OP, Fzd8- knockout affects OP indirectly. Download figure Open in new tab Figure 3-2. Comparison of the differences in immunohistochemical protein expressions between WT and HO mice after Fzd8 -knockout. The P value represents the differences in the expression levels of each immunohistochemical protein in the WT and HO mice after Fzd8 knockout; P 0.05 indicates that the expression level of the immunohistochemical protein was not significantly different between the two groups. Gene expression differences between WT and HO mice Expression levels of Fzd10 and lipoteichoic acid ( Lta ) were statistically different in the WT and HO mice ( Figure 4 ). Fzd10 expression was significantly down-regulated, while Lta expression was noticeably up-regulated. In this study, several OP-related genes, including Fzd10 in the canonical Wnt signaling pathway, are predicted to be the target genes of differentially expressed miRNAs, and Fzd10 expression was down-regulated in OP. Download figure Open in new tab Figure 4. Comparison of differences in gene expression in WT and HO mice after Fzd8 -knockout. The P value represents the difference between the expression levels of each gene in WT and HO mice after Fzd8 -knockout; P 0.05 indicates that the expression level of the gene was not significantly different between the two groups. Bioinformatics analysis outputs We found that the genes Col1a1 , Col1a2 , Col3a1 , Ibsp , S100a8 , BC100530 , Myoc , Col6a2 , and Pcdh12 were significantly differentially expressed ( P < 0.01; Figure 5 ). These genes are closely related to OP, further elucidating the effects of Fzd8 -knockout in OP. Col1a1 is the most significant gene that was up-regulated in this study. Download figure Open in new tab Figure 5. Volcano plot of differentially expressed genes. Up-regulated and down-regulated genes are identified by log2 (Fold Change) with P < 0.05. The top 8 up-regulated genes ( Col1a1, Col1a2, Col3a1, Ibsp, BC100530, S100a8, Myoc, and Col6a2 ) are represented by red dots, and one significantly down-regulated gene ( Pcdh12 ) is represented by the blue dot. Discussion OP is characterized by the imbalance between OC bone resorption and OB bone formation, which leads to bone loss and structural decay, thereby reducing bone strength and increasing fragility. Hence, new therapeutic targets, possibly through the molecular understanding of the communication and command signaling networks between bone formation OB and bone absorption OC, are currently under intense exploration [ 1 ]. Recent studies offer new insights into the mechanisms regulating bone and cartilage growth, along with homeostasis [ 14 ]. The Wnt signaling pathway plays a crucial role in bone development and homeostasis [ 3 ]. Both the canonical and non-canonical Wnt signaling pathways are activated by the binding of the Wnt ligand to Fzd receptors, alone or in combination with specific co-receptors [ 15 ]. Studies have shown that FZD8 , which plays a significant role in bone remodeling, is highly expressed in OC and OB [ 9 ]. Hence, Fzd8 might be a potential therapeutic target for OP, although its signal transduction mechanism has not been elucidated [ 6 ]. Different variants contributing to heritability tend to spread across the whole genome, which might explain why some loci/genes characterized by genome-wide association studies for BMD are considered not to influence OP pathophysiology, while other genes tend to be core genes [ 16 ]. We discovered that in Fzd8- knockout mice, P1NP decreases, and CTX increases. The micro-CT results showed that the trabeculae, BV, and BMD decreased after Fzd8 -knockout. Statistically significant differences in Itgb3 and RANK protein expressions were observed in the WT and HO mice based on IHC results. Moreover, in vitro and transgenic animal studies have demonstrated that Itgb3 has important functions in OC resorptive mechanisms [ 17 ]. Fzd10 expression, which positively regulates the Wnt signaling pathway, is significantly down-regulated in OP [ 18 ]. In this study, the genes Col1a1 , Col1a2 , Col3a1 , Ibsp , S100a8 , BC100530 , Myoc , Col6a2 , and Pcdh12 were differentially expressed ( P < 0.01, Figure 5 ). These genes are potentially relevant to OP [ 19 ], which further elucidates the effects of Fzd8 -knockout on OP. To date, only a few SNPs/genes and their functional mechanisms have been successfully characterized in OP [ 20 ]. By knocking out the Fzd8 gene, we observed significant differences in the expressions of the Fzd10, Lta, Itgb3 , and RANK proteins in the WT and HO mice. At present, proteins of the frizzled family, including FZD10 , may act as Wnt co-receptors on the cell membrane [ 21 ]. The FZD protein activates intracellular signal transduction molecules and regulates the target gene expressions. Moreover, sFRPs could inhibit Wnt protein activity in bone pathophysiology regulation, bone mineral content, and immune/inflammatory responses [ 3 ]. To identify such receptors, previous studies assessed the expression of known Fzd genes in distinguished tissues and bone cells [ 9 ]. Moreover, a study found that Itgb3 is highly expressed in OC, which is the most important integrin regulating OC function [ 22 ]. TNF-α antibodies can decrease Itgb3 and V-ATPase expressions in OC. Therefore, TNF-α may enhance OC bone resorption by increasing the Itgb3 and V-ATPase expression in OC [ 23 ]. Inflammatory cytokines are highly active and multi-functional small molecule proteins that are mainly generated by immune cells [ 24 ]. In this study, with Fzd8 -knockout, significant differences in Lta gene expression were observed among the WT and HO mice. Previous investigations have shown that Lta activates immune responses via downstream signaling pathways, including the expression of NF-κB [ 25 ]. Osteogenic markers such as alkaline phosphatase, type-I collagen , and Runx2 are up-regulated in Lta -stimulated mesenchymal stem cells [ 26 ]. Our data showed that Lta expression in the HO Fzd8 -knockout mice was significantly lower than that in WT mice. Previous reports have shown that Lta could promote the growth of OB indirectly, verifying that Fzd8 -knockout could promote OP [ 26 ]. In contrast, TNF-α exerts its function through each of two receptors, i.e., TNFR1 and TNFR2 , that either contains [ 27 ] or lacks a death domain [ 28 ], respectively. TNFR1 regulates OC formation to function positively [ 29 ]. Although TNF-α is capable of activating the NF-κB , JNK , p38 , ERK, and Akt pathways in OC precursors and OCs, the signaling cascades leading to pathway activation have not been established for OC precursors [ 25 ]. Many inflammatory cytokines, including IL-6 and TNF-α, participate in OC differentiation [ 30 ]. Our data show that Fzd10 is significantly down-regulated, while Itgb3 and RANK are significantly up-regulated in OP. This suggests that the corresponding differentially expressed miRNAs might regulate OP by suppressing positive regulators, which is in agreement with the findings of Albers et al. [ 9 ]. In combination with the perspective of WGS data, it is found that Lta is significantly down-regulated in OP. As an important factor of inflammatory response, Lta may be involved in the immune regulation of TNF on OC and OB during OP, but the special mechanisms remain to be elucidated, which could provide us with new insights for further research and another promising direction for precise treatment of OP. Our Fzd8 -knockout murine model showed that there were significant alternations in Fzd10 and Lta gene expressions at RNA level and Itgb3 and RANK protein expressions between WT and HO mice, which are significantly associated with bone remodeling. Our results revealed that FZD8 could be a therapeutic target in OP. This study elucidates the molecular mechanisms in OP, providing evidence-based data for OP drug development and treatment. Author contributions Zhengkun Lin, Jianquan He, and Jian Chen conceived and designed all experiments; Zhengkun Lin, Hui Huang, Jianquan He, Xiaomei Lin, and Heqing Chen performed the experiments; Wen Zhang analyzed the data; Zhengkun Lin, Jianquan He, Wen Zhang and Jian Chen wrote the paper. All authors have read and approved the final manuscript. Availability of data and materials All data generated or analyzed in the study are included either in this article or in the supplementary files. Relevant data are available from the corresponding author, upon request. Compliance with Ethical Standards All animal experiments were carried out in Zhongshan Hospital Xiamen University following the Instructive Notions with Respect to Caring for Laboratory Animals issued by the Ministry of Science and Technology of People’s Republic of China. Funding This study was funded by the National Natural Science Foundation of China (grant No. 81272168), the Key Clinical Specialty Discipline Construction Program of Fujian, P.R. China and the Health Youth Research Project of Fujian Province (grant No. 2020QNB060). Competing interests The authors declare that they have no competing interests. Ethical approval The study was confirmed and approved by the Animal Subjects Committee of Zhongshan Hospital Xiamen University (approval no. XMVLAC20120044). Consent for publication Not applicable. Acknowledgments This study was funded by the National Natural Science Foundation of China (grant No. 81272168), the Key Clinical Specialty Discipline Construction Program of Fujian, P.R. China and the Health Youth Research Project of Fujian Province (grant No. 2020QNB060). We express gratitude to Chengqi He for providing guidance, and Hesong Qiu for processing the data and managing this project. 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