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Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor–related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20–34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

Background: Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly because of impaired decidualization. Endocytic and transcytotic activity in the endometrium are crucial for impaired decidualization. The most representative endocytic genes is the C-terminal Eps15 homology domain-containing 1 (EHD1). Whether the EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains to be determined. Methods: Endometrial tissues were collected from RIF patients (n=27) and fertile controls (n=27) between July 2017 and December 2018. Meanwhile, endometrial biopsies were performed on tissues from 10 women with infertility secondary to tubal factor in the late proliferative and mid-secretory phases of the menstrual cycle. Primary HESCs isolated from eutopic endometrial tissues were used to investigate the role of EHD1 in decidualization. Findings: RNA-seq analysis demonstrated that EHD1 expression was significantly higher in the mid-secretory endometrium of the RIF group than in that of the fertile control group. During the menstrual cycle, the expression of EHD1 increased in the mid-proliferative phase and decreased periodically in the mid-secretory phase and decidual phase. Furthermore, EHD1 overexpression impaired decidualization by suppressing the expression of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) and the formation of the cytoskeleton. Mechanistically, Wingless-related MMTV integration site 4 (Wnt4) and its downregulated signaling showed significant changes following adenovirus-mediated overexpression of EHD1 in HESCs by RNA-seq analysis. The endocytosis pathway of the low-density-lipoprotein receptor-related proteins 5 and 6 (LRP5/6) co-receptor was decreased by EHD1 overexpression. Co-IP and confocal microscopy analysis showed that EHD1 bound to the Wnt4 protein, contributing to the decrease in Wnt4/β-catenin signaling during the decidualization of HESCs. Additionally, the Wnt4 agonist improved the impaired decidualization. Interpretation: Regulation of the EHD1-Wnt4 pathway may serve as a promising therapeutic strategy to improve endometrial receptivity in RIF women. Funding Statement: This work was supported by the National Key Research and Development Program of China (2018YFC1004401), the National Natural Science Foundation of China (81871165, 31872846, 81571402 and 31571189 ), a special grant for principal investigators from the Health Department of Jiangsu Province (ZDRCA2016070), and a special fund for the Jiangsu Province Social Development Project (NO.BE2018602). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: This study was carried out according to the Recommendations of Guidelines for Clinical Trials by the Ethics Committee of the Drum Tower Hospital. Informed consent was obtained from all participants before any study-related procedure was performed. This study was carried out according to the approval of Construction and Management of the Nanjing Multic-center Biobank Project (No. 2013-081-01).

Introduction & Objectives: Canonical Wnt signaling (Wnt/β-catenin signaling) maintains the bone homeostasis by promoting the osteoblastic activities. The inhibitory factor, Dickkopf (DKK)1, enhances the bone resorption, especially in malignancies. The low density lipoprotein related protein (LRP) 5 is a component of membranous co-receptor of Wnt/β-catenin signaling and is also involved in serum low density lipoprotein cholestrol (LDL-C) level regulation. The clear cell renal cell carcinoma bone metastasis (ccRCC-BM) is characterized by osteolytic bone resorption. Whether and how Wnt/β-catenin signaling plays roles in regulating the invasion, metastasis and osteolytic process of ccRCC to bone remain unclear. This study investigated the expression of DKK1, LRP5 proteins in primary and metastatic lesions of RCC-BM. The therapeutic potential of Wnt/β-catenin signaling target medication was also evaluated.Materials & Methods: ccRCC-BM patients with paired samples of primary and metastatic lesions were selected. ccRCC patients without any metastasis (ccRCC-only) were set as control. Slides of paraffin-embedded tissue underwent immunohistochemical staining with monoclonalanti-DKK1 antibody and polyclonal anti-LRP5 antibody. Semi-quantitatively scoring according to staining intensity was performed. The staining results in the renal tissue adjacent to RCC, the primary RCC lesions (with BM or without BM), and the RCC-BM lesions were recorded. The expression difference was analyzed by univariate analysis of variance (ANOVA).Results: The expression of DKK1 was significantly different amid renal tissue adjacent to RCC, primary RCC and RCC-BM tissues (p< 0.001). The expression of DKK1 in primary RCC was significantly lower than that in renal tissue adjacent to RCC (pConclusions: A "rebound" of DKK1 expression was found in bone metastasis lesions. Along with the decreasing LRP5 expression in primary lesions of RCC-BM patients, this suggests that the canonical Wnt signaling (Wnt/β-catenin signaling) is inhibited during the bone metastasis process in ccRCC. The overexpression of DKK1 and the down-regulation of LRP5 receptor are involved.

Chronic psychological stress is closely correlated with breast cancer growth and metastasis. Sini San (SNS) formula is a classical prescription for relieving depression-related symptoms in traditional Chinese medicine (TCM). Current researches have suggested that chronic psychological stress is closely correlated with cancer stem cells (CSCs) and endoplasmic reticulum (ER) stress. This study aimed to investigate the effects of chronic psychological stress on ER stress-mediated breast cancer stemness and the therapeutic implication of SNS. Chronic psychological stress promoted lung metastasis in 4T1 breast tumor-bearing mice and increased the stem cell-like populations and stemness-related gene expression. Meanwhile, GRP78, a marker of ER stress, was significantly increased in the breast tumors and lung metastases under chronic psychological stress. As a biochemical hallmark of chronic psychological stress, cortisol dramatically enhanced the stem cell-like populations and mammospheres formation by activating GRP78 transcriptionally. However, GRP78 inhibitors or shRNA attenuated the stemness enhancement mediated by cortisol. Similarly, SNS inhibited chronic psychological stress-induced lung metastasis and stemness of breast cancer cells, as well as reversed cortisol-induced stem cell-like populations and mammospheres formation by attenuating GRP78 expression. Co-localization and co-immunoprecipitation experiments showed that SNS interrupted the interaction between GRP78 and LRP5 on the cell surface, thus inhibiting the Wnt/β-catenin signaling of breast CSCs. Altogether, this study not only uncovers the biological influence and molecular mechanism of chronic psychological stress on breast CSCs but also highlights SNS as a promising strategy for relieving GRP78-induced breast cancer stemness via inhibiting GRP78 activation.

Osteoporosis in premenopausal women with intact gonadal function and no known secondary cause of bone loss is termed idiopathic osteoporosis (IOP). Women with IOP diagnosed in adulthood have profound bone structural deficits and often report adult and childhood fractures, and family history of osteoporosis. Some have very low bone formation rates (BFR/BS) suggesting osteoblast dysfunction. These features led us to investigate potential genetic etiologies of bone fragility. In 75 IOP women (aged 20–49) with low trauma fractures and/or very low BMD who had undergone transiliac bone biopsies, we performed Whole Exome Sequencing (WES) using our variant analysis pipeline to select candidate rare and novel variants likely to affect known disease genes. We ran rare-variant burden analyses on all genes individually and on phenotypically-relevant gene sets. For particular genes implicated in osteoporosis, we also assessed the frequency of all (including common) variants in subjects versus 6540 non-comorbid female controls. The variant analysis pipeline identified 4 women with 4 heterozygous variants in LRP5 and PLS3 that were considered to contribute to osteoporosis. All 4 women had adult fractures, and 3 women also had multiple fractures, childhood fractures and a family history of osteoporosis. Two women presented during pregnancy/lactation. In an additional 4 subjects, 4 different relevant Variants of Uncertain Significance (VUS) were detected in the genes FKBP10, SLC34A3, and HGD. Of the subjects with VUS, 2 had multiple adult fractures, childhood fractures, and presented during pregnancy/lactation, and 2 had nephrolithiasis. BFR/BS varied among the 8 subjects with identified variants; BFR/BS was quite low in those with variants that are likely to have adverse effects on bone formation. The analysis pipeline did not discover candidate variants in COL1A1, COL1A2, WNT, or ALPL. Although we found several novel and rare variants in LRP5, cases did not have an increased burden of common LRP5 variants compared to controls. Cohort-wide collapsing analysis did not reveal any novel disease genes with genome-wide significance for qualifying variants between controls and our 75 cases. In summary, WES revealed likely pathogenic variants or relevant VUS in 8 (11%) of 75 women with IOP. Notably, the genetic variants identified were consistent with the affected women's diagnostic evaluations that revealed histological evidence of low BFR/BS or biochemical evidence of increased bone resorption and urinary calcium excretion. These results, and the fact that the majority of the women had no identifiable genetic etiology, also suggest that the pathogenesis of and mechanisms leading to osteoporosis in this cohort are heterogeneous Future research is necessary to identify both new genetic and non-genetic etiologies of early-onset osteoporosis.

Nutritional microenvironment determines the specification of progenitor cells, and lipid availability was found to modulate osteogenesis in skeletal progenitors. Here, we investigated the implications of lipid scarcity in the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) and the role of low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor transducing canonical Wnt/beta-catenin signals, in BMSC lipid uptake during osteogenesis. The osteogenic differentiation of murine BMSCs was suppressed by lipid scarcity and partially rescued by additional fatty acid treatment with oleate. The enhancement of osteogenesis by oleate was found to be dosage-dependent, along with the enhanced activation of beta-catenin and Wnt target genes. Conditional knockout (CKO) of Lrp5 gene in murine mesenchymal lineage using Lrp5fl/fl;Prrx1-cre mice led to decreased bone quality and altered fat distribution in vivo. After Lrp5 ablation using adenoviral Cre-recombinase, the accumulation of lipid droplets in BMSC cytoplasm was significantly reduced, and the osteogenesis of BMSCs was suppressed. Moreover, the impaired osteogenesis due to either lipid scarcity or Lrp5 ablation could be rescued by recombinant Wnt3a protein, indicating that the osteogenesis induced by Wnt/beta-catenin signaling was independent of LRP5-mediated lipid uptake. In conclusion, lipid scarcity suppresses BMSC osteogenic differentiation. LRP5 plays a role in the uptake of lipids in BMSCs and therefore mediates osteogenic specification.

The recent identification of homozygous WNT1 mutations in individuals with osteogenesis imperfecta type XV (OI-XV) has suggested that WNT1 is a key ligand promoting the differentiation and function of bone-forming osteoblasts. Although such an influence was supported by subsequent studies, a mouse model of OI-XV remained to be established. Therefore, we introduced a previously identified disease-causing mutation (G177C) into the murine Wnt1 gene. Homozygous Wnt1G177C/G177C mice were viable and did not display defects in brain development, but the majority of 24-week-old Wnt1G177C/G177C mice had skeletal fractures. This increased bone fragility was not fully explained by reduced bone mass but also by impaired bone matrix quality. Importantly, the homozygous presence of the G177C mutation did not interfere with the osteoanabolic influence of either parathyroid hormone injection or activating mutation of LRP5, the latter mimicking the effect of sclerostin neutralization. Finally, transcriptomic analyses revealed that short-term administration of WNT1 to osteogenic cells induced not only the expression of canonical WNT signaling targets but also the expression of genes encoding extracellular matrix modifiers. Taken together, our data demonstrate that regulating bone matrix quality is a primary function of WNT1. They further suggest that individuals with WNT1 mutations should profit from existing osteoanabolic therapies., Bone Research, 9 (1)

The Wnt/β-catenin pathway is upregulated in uterine leiomyomas, the most common benign tumors in the female reproductive tract. Simvastatin is an antihyperlipidemic drug, and previous in vitro and in vivo reports showed that it may have therapeutic effects in treating leiomyomas. The objective of this study was to examine the effects of simvastatin on the Wnt/β-catenin signaling pathway in leiomyoma. We treated primary and immortalized human leiomyoma cells with simvastatin and examined its effects using quantitative real-time polymerase chain reaction, Western blotting, and immunocytochemistry. We also examined the effects using human leiomyoma tissues from an ongoing randomized controlled trial in which women with symptomatic leiomyoma received simvastatin (40 mg) or placebo for 3 months prior to their surgery. The results of this study revealed that simvastatin significantly reduced the expression of Wnt4 and its co-receptor LRP5. After simvastatin treatment, levels of total β-catenin and its active form, nonphosphorylated β-catenin, were reduced in both cell types. Additionally, simvastatin reduced the expression of Wnt4 and total β-catenin, as well as nonphosphorylated β-catenin protein expression in response to estrogen and progesterone. Simvastatin also inhibited the expression of c-Myc, a downstream target of the Wnt/β-catenin pathway. The effect of simvastatin on nonphosphorylated-β-catenin, the key regulator of the Wnt/β-catenin pathway, was recapitulated in human leiomyoma tissue. These results suggest that simvastatin may have a beneficial effect on uterine leiomyoma through suppressing the overactive Wnt/β-catenin pathway.

Pathogenic variants in the Wnt-pathway co-receptor low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) cause high bone mass (LRP5-HBM) due to insensitivity to the endogenous antagonist of Wnt-signaling. Although indicating incessant progression of BMD and biomarkers reflecting bone formation, this has not been confirmed in individuals with LRP5-HBM. We investigated how the LRP5-HBM bone phenotype changes with age in adults and is associated with quantitative changes of bone turnover markers and bone-related microRNAs (miRNAs) in the circulation. Whole body, lumbar spine, total hip, and femoral neck areal BMD (aBMD) and radial and tibial bone microarchitecture and geometry were assessed using DXA and HR-pQCT scans of 15 individuals with LRP5-HBM T253I (11 women; median age 51 years; range, 19 to 85 years) with a time interval between scans of 5.8 years (range, 4.9 to 7.6 years). Fasting P1NP and CTX were measured in 14 LRP5-HBM T253I individuals and age-, sex-, and body mass index (BMI)-matched controls, and 187 preselected miRNAs were quantified using qPCR in 12 individuals and age-, sex-, and BMI-matched controls. DXA and HR-pQCT scans were assessed in subjects who had reached peak bone mass (aged >25 years, n = 12). Femoral neck aBMD decreased by 0.8%/year ( p = 0.01) and total hip by 0.3%/year, and radial volumetric BMD (vBMD) increased 0.3%/year ( p = 0.03). Differences in bone turnover markers at follow-up were not observed. Compared to controls, 11 of the 178 detectable miRNAs were downregulated and none upregulated in LRP5-HBM individuals, and five of the downregulated miRNAs are reported to be involved in Wnt-signaling. Bone loss at the hip in LRP5-HBM individuals demonstrates that the bone phenotype does not uniformly progress with age. Differentially expressed miRNAs may reflect changes in the regulation of bone turnover and balance in LRP5-HBM individuals. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

BackgroundThe healing environment within the posterolateral lumbar spine is one of the most clinically challenging bone healing environments in all of orthopaedics due to a lack of a contained space and the need to formde novobone in a non-bony environment. Our group has previously published data that suggests that sclerostin in expressed locally at high levels throughout the process of a spinal fusion mass maturing.MethodsWe computationally identified multiple FDA-approved drugs, as well as a novel drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of β-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion.ResultsWe show that by controlling GSK3b phosphorylation, these SMIs simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralizationin vivoin rats as well as significantly increase posterolateral spine fusion rates in rabbitsin vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft.ConclusionsFew if any osteogenic small molecules have been described that possess the osteoinductive potency of BMP itself – that is, the ability to formde novoectopic bone as a standalone agent, particularly in stringentin vivoenvironments. Herein, we describe two such SMIs that have this unique ability and thus may have potential application as novel cost-effective biologic bone graft substitutes for achieving consistent spinal fusion or even or critical-sized fracture defects.FundingThis work was supported by a Veteran Affairs Career Development Award (IK2-BX003845).

Background: Familial exudative vitreoretinopathy (FEVR) is a complex form of blindness-causing retinal degeneration. This study investigated the potential disease-causing variant and molecular basis in two Chinese families with FEVR. Methods: All family members underwent detailed ophthalmological examinations, including best-corrected visual acuity, fundus examination, and fluorescein fundus angiography. Whole exome sequencing, bioinformatics analysis, and Sanger sequencing of available family members were used to confirm the disease-causing gene variant. Results: The proband F1-II:1, his mother, and proband F2-II:1 were diagnosed with FEVR based on clinical symptoms, fundus manifestation, and fundus fluorescein angiography. Whole exome sequencing showed that the proband F1-II:1 had a novel heterozygous variant in the TSPAN12 gene, c.236T>G p. (Met79Arg), which may disturb the transmembrane domain of the TSPAN12 protein. The variant was cosegregated with the phenotypes of FEVR in the family. The proband F2-II:1 carried novel compound heterozygous variants, c.1210G>A p. (Gly404Arg) and c.1612C>T p. (Arg538Trp), in the LRP5 gene, which may disturb the second β-propeller motif of the LRP5 protein. These three variants were classified as likely pathogenic variants. The variants were predicted to be damaging or deleterious using multiple lines of prediction algorithms; they were not frequently found in multiple population databases. The residues affected by these variants are highly conserved among different species. The three novel variants may be potential disease-causing variants in the two families. Conclusions: These results expand the spectrum of variants in TSPAN12 and LRP5 genes and enrich the understanding of the molecular etiology of FEVR.

© 2023 Pan-Arab Association of GastroenterologyBackground and study aims: Intestinal metaplasia (IM), and Helicobacter pylori (HP) infection can be shown as risk factors in the development of gastric cancer (GC). WNT signaling pathway plays a critical role in carcinogenesis. However, the literature studies are limited on the significance of this pathway for the transition from IM to GC. Patients and methods: We aimed to investigate the importance of the genes of WNT signaling pathways diagnostic and prognostic markers in the presence and absence of HP in conversion from IM to GC. 104 patients, (GC group n = 35, IM group n = 45, control group n = 25) were included in this case-control study. Expression of genes in WNT signalling were searched in study groups with qRT-PCR array and qRT-PCR method. Data were analysed using PCR array data analysis software. Results: Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in the GC and IM groups compared to the control group (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was observed in patients with metastatic GC compared to patients with GC without metastasis (p < 0.05). It was found that the RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes were statistically significantly over-expressed in diffuse GC patients compared to non-diffuse GC patients (p < 0.05). Statistically significant overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes was detected in HP positive IM patients compared to HP negative IM patients (p < 0.05). Conclusion: Overexpression of RHOA, CSNK1A1, DVL2, FZD8 and LRP5 genes in IM may suggest that these genes are important markers in the development of IM and inflammation with HP. In addition, these genes are linked to tumor burden in the GC group. Consequently, we can conclude that these genes are poor prognosis biomarkers for GC and have the potential to be used as markers for future treatment monitoring.

The conversion of mechanical energy into biochemical changes within living cells is process known as mechanotransduction. Bone is a quintessential tissue for studying the molecular mechanisms of mechanotransduction, as the skeleton's mechanical competence is crucial for vertebrate movement. Bone cell mechanotransduction is facilitated by a number of cell biological pathways, one of the most prominent of which is the Wnt signaling cascade. The Wnt co-receptor Lrp5 has been identified as a crucial protein for mechanical signaling in bone, and modifiers of Lrp5 activity play important roles in mediating signaling efficiency through Lrp5, including sclerostin, Dkk1, and the co-receptor Lrp4. Mechanical regulation of sclerostin is mediated by certain members of the Hdac family. Other mechanisms that influence Wnt signaling-some of which are mechanoresponsive-are coming to light, including R-spondins and their role in organizing the Rnf43/Znrf3 and Lgr4/5/6 complex that liberates Lrp5. While the identity of the key Wnt proteins involved in bone cell mechanical signaling are elusive, the likely pool of key players is narrowing. Identification of Wnt-based molecular targets that can be modulated pharmacologically to make mechanical stimulation (e.g., exercise) more beneficial is an emerging approach to improving skeletal integrity and reducing fracture risk.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and characterized by accelerated extracellular matrix breakdown and IVD height loss but there is no approved pharmacological therapeutic. Deletion of Wnt signaling receptor Lrp5 induces IVD degeneration and suggests that Wnt signaling in the IVD may be responsive to inhibition of Wnt signaling inhibitors sost(gene)/sclerostin(protein) or dickkopf-1 (dkk1). Anti-sclerostin antibody (Scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We (1) determined if pharmacological neutralization of sclerostin, dkk1 or their combination stimulate Wnt signaling and promote IVD structure and (2) determined the extent of the response of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n=6-7/grp) were subcutaneously injected 2x/wk for 5.5 wk with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg) or vehicle (Veh). Separately, IVD of sost KO and WT (wildtype) mice (n=8, grp) were harvested at 16 weeks of age. First, compared to vehicle, scl-Ab, dkk1-Ab and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and β-catenin gene expression. Despite these similarities, scl-Ab decreased cellular stress-related heat shock protein gene expressions while neither dkk1-Ab nor scl-Ab/dkk1-Ab altered the same. Genetically and compared to WT, sost KO increased MRI-determined hydration and proteoglycan staining in the IVD. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling transcription factor β-catenin in whole IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and determined a reduction of cellular stress pathways. Together, suppression of sost/sclerostin or dkk1 each promote IVD structure by stimulating Wnt signaling, but sclerostin and dkk1 may differentially regulate cellular stress pathways. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health.Graphical abstractSuppression of Wnt signaling inhibitors by genetic or pharmacological approaches promotes intervertebral disc structure and hydration by Wnt signaling. However, persistent activation of Wnt signaling induces a compensatory reduction of Wnt signaling that shifts IVD cells toward a chondrocyte-like (CLC) phenotype. AF: annulus fibrosus, NC: notochordal cell, NP: nucleus pulposus, PG: proteoglycan

Essential Hypertension (EH) results in the burden of cardiovascular disease (CVD) such as Heart Failure (HF) and Ischemic Stroke (IS). A rapidly emerging field involving the role of Wnt/β-catenin signaling pathway in cardiovascular development and dysfunction has recently drawn extensive attention. In the present study, we conducted a genetic association between genomic variants in Wnt/β-catenin signaling pathway and EH, HF, IS. A total of 95 SNPs in 12 Wnt signaling genes (WNT3A, WNT3, WNT4, DKK1, DKK2, LRP5, LRP6, CTNNB1, APC, FZD1, FRZB, SFRP1) were genotyped in 1,860 participants (440 patients with EH, 535 patients with HF, 421 patients with IS and 464 normal control subjects) using Sequenom MassArray technology. WNT3A rs752107(C > T) was strongly associated with an increased risk of EH, HF and IS. Compared with WNT3A rs752107 CC genotype, the CT genotype carriers had a 48% increased risk of EH (OR = 1.48, 95% CI = 1.12–1.96, P = 0.006), the TT genotype conferred a 139% increased risk of EH (OR = 2.39, 95% CI = 1.32–4.34, P = 0.003). Regarding HF and IS, the risk of HF in the T allele carriers (CT + TT) was nearly increased by 58% (OR = 1.58, 95% CI = 1.22–2.04, P = 4.40 × 10−4) and the risk of IS was increased by 37% (OR = 1.37, 95% CI = 1.04–1.79, P = 0.025). Expression quantitative trait loci (eQTL) analysis indicated that rs752107 C allele corresponded to a significant reduction of WNT3A expression. We described a genetic variant of WNT3A rs752107 in Wnt/β-catenin signaling strongly associated with the risk of EH, HF and IS for the first time.

Osteocytes are the most abundant cells in bone, which is a frequent site of breast cancer metastasis. Here, we focused on Wnt signaling and evaluated tumor-osteocyte interactions. In animal experiments, mammary tumor cells were inoculated into the mammary fat pad and tibia. The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model. The results revealed that administration of osteocytes or their conditioned medium (CM) inhibited tumor progression and osteolysis. Osteocytes overexpressing Lrp5 or β-catenin displayed strikingly elevated tumor-suppressive activity, accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53. The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound. Notably, silencing Lrp5 in tumors inhibited tumor progression, while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression. Osteocytes exhibited elevated Lrp5 expression in response to tumor cells, implying that osteocytes protect bone through canonical Wnt signaling. Thus, our results suggest that the Lrp5/β-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes. We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.

Job syndrome is a rare genetic disorder caused by STAT3 mutations and primarily characterized by immune dysfunction along with comorbid skeleton developmental abnormalities including osteopenia, recurrent fracture of long bones, and scoliosis. So far, there is no definitive cure for the skeletal defects in Job syndrome, and treatments are limited to management of clinical symptoms only. Here, we have investigated the molecular mechanism whereby Stat3 regulates skeletal development and osteoblast differentiation. We showed that removing Stat3 function in the developing limb mesenchyme or osteoprogenitor cells in mice resulted in shortened and bow limbs with multiple fractures in long bones that resembled the skeleton symptoms in the Job Syndrome. However, Stat3 loss did not alter chondrocyte differentiation and hypertrophy in embryonic development, while osteoblast differentiation was severely reduced. Genome-wide transcriptome analyses as well as biochemical and histological studies showed that Stat3 loss resulted in down-regulation of Wnt/β-catenin signaling. Restoration of Wnt/β-catenin signaling by injecting BIO, a small molecule inhibitor of GSK3, or crossing with a Lrp5 gain of function (GOF) allele, rescued the bone reduction phenotypes due to Stat3 loss to a great extent. These studies uncover the essential functions of Stat3 in maintaining Wnt/β-catenin signaling in early mesenchymal or osteoprogenitor cells and provide evidence that bone defects in the Job Syndrome are likely caused by Wnt/β-catenin signaling reduction due to reduced STAT3 activities in bone development. Enhancing Wnt/β-catenin signaling could be a therapeutic approach to reduce bone symptoms of Job syndrome patients.

Genetic factors play a critical role in the pathogenesis of osteoporosis. The imbalance of WNT/β-catenin will cause the occurrence of osteoporosis. LRP5 and AXIN1 play an important role in the classical Wnt/β-catenin signaling pathway. Our study was aimed to determine the association between five candidate single nucleotide polymorphisms (SNPs) of LRP5 or AXIN1 and osteoporosis susceptibility in Chinese Han population. A total of 599 osteoporosis patients and 599 healthy individuals were recruited for this case-control study. Agena MassARRAY was used to genotype SNPs. The association between SNPs and osteoporosis susceptibility in different genetic models was analyzed by PLINK software. We used false-positive report probability (FPRP) analysis to detect whether the positive results were just chance or noteworthy observations. Multifactor dimension reduction (MDR) was used to analyze the interaction of SNP–SNP in the osteoporosis risk. Finally, haplotype analysis was performed by plink1.07 and Haploview software. We found that LRP5 rs11228240, AXIN1 rs2301522, and rs9921222 were significantly associated with the osteoporosis susceptibility. The results of subgroup analysis showed that LRP5 rs11228240 (protective factor) and AXIN1 rs2301522 (risk factor) were associated with the susceptibility of osteoporosis among participants who were age >60 years, female or BMI ≤ 24; AXIN1 rs9921222 significantly increased the risk of osteoporosis among participants with BMI ≤ 24. The genotype Ars2301522Crs9921222 could increase the susceptibility of osteoporosis (p = 0.026). The rs11228219LPR5, rs11228240 LPR5, rs2301522AXIN1, and rs9921222AXIN1 four-site model was the best model for predicting the osteoporosis risk (test accuracy = 0.541; CVC = 10/10). The LRP5-rs11228240, AXIN1-rs2301522, and AXIN1- rs9921222 were associated with osteoporosis susceptibility in Chinese Han population.

BackgroundTissue derived fibroblast-like synoviocytes (td-FLS) are key actors in pannus formation and contribute to joint destruction and inflammation during rheumatoid arthritis (RA). Several members of the Wnt family, including Wnt5a, may contribute to RA td-FLS activation and can potentially serve as therapeutic targets.ObjectiveThe present work aimed to investigate the expression of Wnt5a signaling elements in RA td-FLS and their potential precursors (fluid derived (fd) FLS and fibrocytes). We also studied the role of Wnt5a in RA td-FLS pro-inflammatory activity and whether the inhibitor SFRP5 could restore Wnt5a-induced synovial dysfunction in vitro.Materials and MethodsThe levels of Wnt5a, SFRP5, Wnt5a receptors/coreceptors and Wnt5a pro-inflammatory targets were determined in cultured RA td-FLS, fd-FLS and fibrocytes using qPCR under basal conditions. The expression of pro-inflammatory molecules was assessed after RA td-FLS stimulation with Wnt5a and SFRP5 at different time points.ResultsOur data showed that td-FLS, fd-FLS and fibrocytes from patients with RA expressed similar levels of Wnt5a and a set of Wnt5a receptors/coreceptors. We also demonstrated that Wnt5a stimulated the expression of the pro-inflammatory targets, especially IL1β, IL8 and IL6 in RA td-FLS. Wnt5a-induced inflammation was enhanced in the presence of SFRP5. Furthermore, Wnt5a alone and in conjunction with SFRP5 inhibited the gene expression of TCF4 and the protein levels of the canonical coreceptor LRP5.ConclusionWnt5a pro-inflammatory effect is not inhibited but enhanced by SFRP5 in RA td-FLS. This research highlights the importance of carefully evaluating changes in Wnt5a response in the presence of SFRP5.

Low-density lipoprotein receptor-related protein 5 (LRP5) has been studied as a co-receptor for Wnt/β-catenin signaling. However, its role in the ischemic myocardium is largely unknown. Here, we show that LRP5 may act as a negative regulator of ischemic heart injury via its interaction with prolyl hydroxylase 2 (PHD2), resulting in hypoxia-inducible factor-1α (HIF-1α) degradation. Overexpression of LRP5 in cardiomyocytes promoted hypoxia-induced apoptotic cell death, whereas LRP5-silenced cardiomyocytes were protected from hypoxic insult. Gene expression analysis (mRNA-seq) demonstrated that overexpression of LRP5 limited the expression of HIF-1α target genes. LRP5 promoted HIF-1α degradation, as evidenced by the increased hydroxylation and shorter stability of HIF-1α under hypoxic conditions through the interaction between LRP5 and PHD2. Moreover, the specific phosphorylation of LRP5 at T1492 and S1503 is responsible for enhancing the hydroxylation activity of PHD2, resulting in HIF-1α degradation, which is independent of Wnt/β-catenin signaling. Importantly, direct myocardial delivery of adenoviral constructs, silencing LRP5 in vivo, significantly improved cardiac function in infarcted rat hearts, suggesting the potential value of LRP5 as a new target for ischemic injury treatment.

Wnt signaling plays crucial roles in development and tissue homeostasis, and its dysregulation leads to various diseases, notably cancer. Wnt/β-catenin signaling is initiated when the glycoprotein Wnt binds to and forms a ternary complex with the Frizzled and low-density lipoprotein receptor-related protein 5/6 (LRP5/6). Despite being identified as a Wnt co-receptor over 20 years ago, the molecular mechanisms governing how LRP6 senses Wnt and transduces downstream signaling cascades are still being deciphered. Due to its role as one of the main Wnt signaling components, the dysregulation or mutation of LRP6 is implicated in several diseases such as cancer, neurodegeneration, metabolic syndrome and skeletal disease. Herein, we will review how LRP6 is activated by Wnt stimulation and explore the various regulatory mechanisms involved. The participation of LRP6 in other signaling pathways will also be discussed. Finally, the relationship between LRP6 dysregulation and disease will be examined in detail.

Intestinal epithelial self-renewal is tightly regulated by signaling pathways controlling stem cell proliferation, determination and differentiation. In particular, Wnt/β-catenin signaling controls crypt cell division and survival and is required for maintenance of the intestinal stem cell niche. Most colorectal cancers are also initiated by mutations activating the Wnt/β-catenin pathway. Wnt signals are transduced through Frizzled receptors and LRP5/LRP6 coreceptors to downregulate GSK3β activity, resulting in increased nuclear β-catenin. Herein, we explored if LRP6 expression is required for maintenance of intestinal homeostasis, regeneration and oncogenesis. Mice with an intestinal epithelial cell-specific deletion of Lrp6 (Lrp6IEC-KO) were generated and their phenotype analyzed. No difference in intestinal architecture or in proliferative and stem cell numbers was found in Lrp6IEC-KO mice in comparison to controls. Nevertheless, using ex vivo intestinal organoid cultures, we found that LRP6 expression was critical for crypt cell proliferation and stem cell maintenance. When exposed to dextran sodium sulfate, Lrp6IEC-KO mice developed more severe colitis than control mice. However, loss of LRP6 did not affect tumorigenesis in Apc Min/+ mice nor growth of human colorectal cancer cells. By contrast, Lrp6 silencing diminished anchorage-independent growth of BRafV600E-transformed IEC. Thus, LRP6 controls intestinal stem cell functionality and is necessary for BRAF-induced IEC oncogenesis.

Objective: To investigate the variations in the expressions of LRP5, Runx2, Osterix, and RANKL factors in bone tissues associated with postmenopausal osteoporotic fractures (PMOPF). Method: Postmenopausal patients with femur fractures were initially divided into control (31 cases) and PMOPF groups (83 cases). All control group patients were operated within 1 day after injury. The patients with PMOPF were operated based on the time after fracture in the respective groups (patients were divided into groups A, B, and C based on the time after fracture). Samples were collected from femurs at fracture sites during the operation. The expression level of each factor in bone tissues was detected using RT-qPCR, and the bone mass samples were decalcified and then histologically analyzed by immunohistochemistry. We subsequently analyzed significant differences in the expressions of factors (LRP5, Runx2, Osterix, and RANKL) between PMOPF and control groups. Results: (1) LRP5, β-catenin, Runx2, and Osterix were under-expressed in patients with PMOPF relative to the controls (P(2) the expressions of LRP5 and Runx2 were lowest in Group A patients (1–3 days after fracture). Osterix expression was lowest in Group C patients (8–14 days after fracture). Conversely, RANKL expression was highest in Group B patients (4–7 days after fracture). Conclusion: The inhibition or reduction in the expressions of osteogenic factors including LRP5, Runx2, and Osterix of the Wnt/β-catenin and BMP-2/Runx2/Osterix signaling pathways are associated with PMOPF incidence. Specifically, upregulation of RANKL in the RANKL/RANK signaling pathway is associated with the incidence of PMOPF. LRP5 and Runx2 expressions decreased considerably within 1-3 days after fracture; Osterix expression decreased considerably within 8-14 days after fracture; RANKL expression was highest within 4-7 days after fracture, which could be associated with bone repair in PMOPF. The expression level of the aforementioned factors affects the development and progression of PMOPF.

The E3 ubiquitin ligases RING finger protein 43 (RNF43) and zinc and RING finger 3 (ZNRF3) have received great attention for their critical role in regulating WNT signalling during adult stem cell homeostasis. By promoting the turnover of WNT receptors, Frizzled and LRP5/6, RNF43 and ZNRF3 ensure that proper levels of WNT activity are maintained in stem cells. The molecular mechanism of RNF43/ZNRF3 activity is beginning to emerge from several recent studies, yet little is known about the regulation of RNF43/ZNRF3 at the post-translational level. A study in this issue of EMBO Reports identifies the deubiquitinating enzyme USP42 as a key regulator of WNT signalling, which acts by antagonizing the ubiquitin-dependent clearance of RNF43/ZNRF3 induced by R-spondins (Giebel et al, 2021).

Background Early‐onset osteoporosis (EOOP) is defined by low bone mineral density (BMD), which increases the risk of fracture. Although the prevalence of osteoporosis at a young age is unknown, low BMD is highly linked to genetic background. Heterozygous pathogenic variants in low‐density lipoprotein receptor‐related protein 5 (LRP5) are associated with EOOP. This study aimed to investigate the genetic profile in patients with EOOP to better understand the variation in phenotype severity by using a targeted gene sequencing panel associated with bone fragility. Method and Results We used a sequencing panel with 17 genes reported to be related to bone fragility for analysis of 68 patients with EOOP. We found a high positivity rate of EOOP with LRP5 variants (14 patients, 20.6%). The remaining 79.4% of patients with EOOP but without LRP5 variants showed variable disease severity, as observed in patients with at least one variant in this gene. One patient, with multiple fractures and spine L1‐L4 BMD Z‐score −2.9, carried a novel pathogenic homozygous variant, c.2918T>C, p.(Leu973Pro), without any pseudoglioma. In addition to carrying the LRP5 variant, 2 other patients carried a heterozygous variant in Wnt signaling pathway genes: dickkopf WNT signaling pathway inhibitor 1 (DKK1) [NM_012242.4: c.359G>T, p.(Arg120Leu)] and Wnt family member 3A (WNT3A) [NM_033131.3: c.377G>A, p. (Arg126His)]. As compared with single‐variant LRP5 carriers, double‐variant carriers had a significantly lower BMD Z‐score (−4.1 ± 0.8) and higher mean number of fractures (6.0 ± 2.8 vs. 2.2 ± 1.9). Analysis of the family segregation suggests the inheritance of BMD trait. Conclusion Severe forms of EOOP may occur with carriage of 2 pathogenic variants in genes encoding regulators of the Wnt signaling pathway. Two‐variant carriers of Wnt pathway genes had severe EOOP. Moreover, DKK1 and WNT3A genes should be included in next‐generation sequence analyses of bone fragility., Gene association may occur in the same signaling pathway and can generate a severe bone phenotype in early‐onset osteoporosis. Recessive form associated with lipoprotein receptor‐related protein 5 could be responsible for a stronger phenotype. Interestingly this recessive form is not associated with ocular problems as observed in pseudoglioma osteoporosis or vitreoretinopathy. Assessment of genetics based on an next generation sequencing panel should include WNT3A and DKK1.

Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics. IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. (C) 2021 American Society for Bone and Mineral Research. (C) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Mendelian bone fragility disorders are caused by genetic variants that can be inherited in an autosomal dominant, autosomal recessive or X-linked manner and have a large detrimental effect on bone strength. As a rule, the more damaging the genetic defect is, the earlier the first fracture will occur, typically during bone development. This review focusses on conditions where bone fragility is the most conspicuous characteristic, of which osteogenesis imperfecta (OI) is the best-known disorder. The large majority of individuals with an OI phenotype have disease-causing dominant variants in COL1A1 or COL1A2, the genes coding for collagen type I. Interestingly, large sequencing databases indicate that there are about 10 times more carriers of COL1A1/COL1A2 variants that should lead to OI than there are individuals with a diagnosis of OI. It is possible that at least some of these variants lead to incomplete OI phenotypes and are diagnosed as osteoporosis during adulthood. Apart from mutations affecting collagen type I production, biallelic mutations in LRP5 and WNT1 can cause very rare and severe bone fragility disorders. Heterozygous pathogenic variants in these genes are much more common and can cause the clinical picture of primary osteoporosis. As sequencing studies are more widely performed in adults with bone fragility disorders, evidence is emerging that what appears as primary osteoporosis in fact can be due to mutations in bona fide OI genes. The distinction between OI and primary osteoporosis is therefore likely to blur in future.

Recently, the emerging role of microRNAs (miRNAs) has been identified in osteogenesis and the development of osteoporosis. Here, we found that miR-545-3p was decreased with the progression of osteogenic differentiation of MC3T3-E1 cells. Gain-of-function assay elucidated that ectopic expression of miR-545-3p led to abolishment on the levels of osteogenic differentiation markers including OC, ALP and Runx2, as well as increase on the expression of SOST, a negative regulator of osteogenic differentiation. Meanwhile, we explained that the inhibitory role of miR-545-3p in the proliferation of differentiated MC3T3-E1 cells was attributed to its induction on apoptosis. Furthermore, the mechanistic investigations validated that miR-545-3p inactivated Wnt/β-catenin signaling pathway by post-transcriptionally silencing LRP5. Importantly, we verified that miR-545-3p-confined osteogenic differentiation was mediated by the inhibition of LRP5-dependent Wnt/β-catenin pathway. Furthermore, it was identified that miR-545-3p downregulation in osteogenic differentiation was due to the positive transcriptional regulation by SP1, an osteoporosis-promoting transcription factor that was proved to be lessened along with osteoblastic differentiation. Jointly, this study elaborated that the SP1-modulated miR-545-3p functions as an osteogenesis-inhibitory factor through targeting LRP5 to inactivate Wnt/β-catenin signaling. Remarkably, strategies targeting miR-545-3p might be an innovative idea for the therapy of patients with osteoporosis.

Summary Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse., Graphical Abstract, Highlights • Missense mutation in the third beta-propeller of Lrp4 improve bone properties • The R1170W mutation in Lrp4 interferes with sclerostin inhibition in vivo • The R1170W Lrp4 mutation alters the bone wasting effects of mechanical disuse, Biological Sciences; Molecular Biology; Cell Biology

Wnt ligands signal through canonical or non-canonical signaling pathways. Although both routes share common elements, such as the Fz2 receptor, they differ in the co-receptor and in many of the final responses; for instance, whereas canonical Wnts increase β-catenin stability, non-canonical ligands downregulate it. However, both types of ligands stimulate tumor cell invasion. We show here that both the canonical Wnt3a and the non-canonical Wnt5a stimulate Fz2 tyrosine phosphorylation, Fyn binding to Fz2, Fyn activation and Fyn-dependent Stat3 phosphorylation. Wnt3a and Wnt5a require Src for Fz2 tyrosine phosphorylation; Src binds to canonical and non-canonical co-receptors (LRP5/6 and Ror2, respectively) and is activated by Wnt3a and Wnt5a. This Fz2/Fyn/Stat3 branch is incompatible with the classical Fz2/Dvl2 pathway as shown by experiments of over-expression or depletion. Fyn is necessary for transcription of genes associated with invasiveness, such as Snail1, and for activation of cell invasion by both Wnt ligands. Our results extend the knowledge about canonical Wnt pathways, demonstrating additional roles for Fyn in this pathway and describing how this protein kinase is activated by both canonical and non-canonical Wnts.