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Extraintestinal manifestations are common in inflammatory bowel disease and involve several organs, including the kidney. However, the mechanisms responsible for renal manifestation in inflammatory bowel disease are not known. In this study, we show that the Wnt–lipoprotein receptor-related proteins 5 and 6 (LRP5/6) signaling pathway in macrophages plays a critical role in regulating colitis-associated systemic inflammation and renal injury in a murine dextran sodium sulfate–induced colitis model. Conditional deletion of the Wnt coreceptors LRP5/6 in macrophages in mice results in enhanced susceptibility to dextran sodium sulfate colitis-induced systemic inflammation and acute kidney injury (AKI). Furthermore, our studies show that aggravated colitis-associated systemic inflammation and AKI observed in LRP5/6LysM mice are due to increased bacterial translocation to extraintestinal sites and microbiota-dependent increased proinflammatory cytokine levels in the kidney. Conversely, depletion of the gut microbiota mitigated colitis-associated systemic inflammation and AKI in LRP5/6LysM mice. Mechanistically, LRP5/6-deficient macrophages were hyperresponsive to TLR ligands and produced higher levels of proinflammatory cytokines, which are associated with increased activation of MAPKs. These results reveal how the Wnt–LRP5/6 signaling in macrophages controls colitis-induced systemic inflammation and AKI.

Podocyte injury is a hallmark of glomerular diseases; however, the underlying mechanisms remain unclear. B7-1 is increased in injured podocytes, but its intrinsic role is controversial. The clinical data here revealed the intimate correlation of urinary B7-1 with severity of glomerular injury. Through transcriptomic and biological assays in B7-1 transgenic and adriamycin nephropathy models, we identified B7-1 is a key mediator in podocyte injury and glomerulosclerosis through a series of signal transmission to β-catenin. Using LC-MS/MS, Hsp90ab1, a conserved molecular chaperone, was distinguished to be an anchor for transmitting signals from B7-1 to β-catenin. Molecular docking and subsequent mutant analysis further identified the residue K69 in the N terminal domain of Hsp90ab1 was the key binding site for B7-1 to activate LRP5/β-catenin pathway. The interaction and biological functions of B7-1-Hsp90ab1-LRP5 complex were further demonstrated in vitro and in vivo. We also found B7-1 is a novel downstream target of β-catenin. Our results indicate an intercrossed network of B7-1, which collectively induces podocyte injury and glomerulosclerosis. Our study provides an important clue to improve the therapeutic strategies to target B7-1.

Osteoporosis-pseudoglioma syndrome (OPPG; MIM #259770) is a rare autosomal recessively inherited disease, characterized by early-onset osteoporosis and congenital blindness, caused by loss-of-function mutations in the LRP5 gene. Beneficial effects of bisphosphonate treatment in patients with OPPG are well known, while follow-up data on growth and pubertal parameters are limited. This article provides clinical follow-up data and long-term bisphosphonate treatment results in four OPPG patients from three unrelated families, ranging between 2.5 and 7 years of age at presentation. Clinical diagnosis was molecularly confirmed in all patients, with four different germline biallelic LRP5 mutations including a novel nonsense variant c.3517CT (p.(Gln1173*)) in two siblings with marked phenotypic variability. Anthropometric and pubertal data and bone mineral density (BMD) measurements were evaluated retrospectively. Early puberty was observed in two patients. The bisphosphonate treatment duration of patients varied around 4-7 years and improvement in BMD z-scores with bisphosphonate treatment was demonstrated in all patients (z-score changes were +5.6, +4.0, +1.0, and +1.3). Although further research is needed to identify the possible association between early puberty and OPPG, all OPPG patients should be followed up with detailed endocrinological evaluation regarding pubertal status.

Objectives Childhood osteoporosis is an uncommon condition that usually develops secondary to underlying disease states. Idiopathic juvenile osteoporosis or early onset osteoporosis is a rare cause of primary osteoporosis in childhood associated with mutations in “bone fragility” genes. Case presentation The index case presented with upper back pain and was detected to have multiple vertebral fractures. Further workup for the cause revealed a homozygous benign mutation in low-density lipoprotein receptor-related protein 5, which was also detected in the mother who remained asymptomatic till presentation. The child was successfully treated with intravenous zoledronate. Conclusions The case report describes the management approach and four-year follow-up of the child.

Altres ajuts: Fundación Investigación Cardiovascular-Fundación Jesus Serra Altres ajuts: Sociedad Española de Cardiología FEC2019 Microvesicles (MV) contribute to cell-to-cell communication through their transported proteins and nucleic acids. MV, released into the extracellular space, exert paracrine regulation by modulating cellular responses after interaction with near and far target cells. MV are released at high concentrations by activated inflammatory cells. Different subtypes of human macrophages have been characterized based on surface epitopes being CD16 macrophages associated with anti-inflammatory phenotypes. We have previously shown that low-density lipoprotein receptor-related protein 5 (LRP5), a member of the LDLR family that participates in lipid homeostasis, is expressed in macrophage CD16 with repair and survival functions. The goal of our study was to characterize the cargo and tentative function of macrophage-derived MV, whether LRP5 is delivered into MV and whether these MV are able to induce inflammatory cell differentiation to a specific CD16 or CD16 phenotype. We show, for the first time, that lipid-loaded macrophages release MV containing LRP5. LDL loading induces increased expression of macrophage pro-inflammatory markers and increased release of MV containing pro-inflammatory markers. Conditioning of fresh macrophages with MV released by Lrp5-silenced macrophages induced the transcription of inflammatory genes and reduced the transcription of anti-inflammatory genes. Thus, MV containing LRP5 induce anti-inflammatory phenotypes in macrophages.

BACKGROUND The WNT signaling pathway is involved in the regulation of bone homeostasis, and the effect of WNT signaling pathway-related gene (WNT16 and LRP5) polymorphisms on osteoporosis risk among Chinese postmenopausal women is still unknown. Hence, we performed a case-control study to assess the association of WNT signaling pathway-related gene polymorphisms and osteoporosis risk. METHODS A total of 1026 women (515 osteoporosis patients and 511 controls) of postmenopausal age who were randomly sampled from Xi'an 630 Hospital (Shaanxi Province, China) were involved in this study. Seven genetic polymorphisms in WNT16 (rs3779381, rs3801387, rs917727 and rs7776725) and LRP5 (rs2291467, rs11228240 and rs12272917) were selected and genotyped using the Agena MassARRAY iPLEX system. The association of the genetic polymorphisms and osteoporosis risk was assessed by odds ratios and 95% confidence intervals. The multifactor dimensionality reduction (MDR) method was conducted to analyze single nucleotide polymorphism (SNP)-SNP interaction. RESULTS We found that LRP5 polymorphisms (rs2291467, rs11228240 and rs12272917) were significantly associated with a decreased risk of osteoporosis in homozygote, recessive and additive models (p 24 (p

To analyze the clinical features of LRP5 gene mutation-related familial exudative vitreoretinopathy and explore the potential phenotype-genotype correlation on LRP5 gene.Eighty-seven familial exudative vitreoretinopathy (FEVR) families with LRP5 mutations were selected from 722 FEVR patients, which were divided into 2 groups, including 22 autosomal-recessive FEVR (ar-FEVR) families and 65 autosomal-dominant FEVR (ad-FEVR) families. Clinical and genetic data were retrospectively analyzed. The potential phenotype-genotype correlation was explored from the mutation type and inheritance pattern.No significant difference between the LRP5 null mutation subgroup and the LRP5 missense mutation subgroup was observed in the proportion of FEVR stage and the ratio of ocular involvement. Instead, a significant difference between the LRP5 ar-FEVR subgroup and the LRP5 ad-FEVR subgroup was observed in the proportion of FEVR stage and the ratio of binocularly severe phenotype. The probands with LRP5 gene recessive mutation showed a higher incidence of severe phenotype. Moreover, the ratio of binocularly severe patients in ar-FEVR was nearly 3.5 times higher than that in ad-FEVR.The severity of phenotype was more likely to be related to the synergistic effect of the variants.

To expand the mutation spectrum of patients with familial exudative vitreoretinopathy (FEVR) disease.74 probands (53 families and 21 sporadic probands) with familial exudative vitreoretinopathy (FEVR) disease and their available family members (n = 188) were recruited for sequencing.Panel-based targeted screening was performed on all subjects. Before sanger sequencing, variants of LRP5, NDP, FZD4, TSPAN12, ZNF408, KIF11, RCBTB1, JAG1, and CTNNA1 genes were verified by a series of bioinformatics tools and genotype-phenotype co-segregation analysis.40.54% (30/74) of the probands were sighted to possess at least one etiological mutation of the nine FEVR-causative genes. The etiological mutation detection rate was 37.74% (20/53) in family-attainable probands while 47.62% (10/21) in sporadic cases. The diagnosis rate of patients in the early-onset subgroup (≤5 years old, 45.4%) is higher than that of the children or adolescence-onset subgroup (6-16 years old, 42.1%) and the late-onset subgroup (≥17 years old, 39.4%). A total of 36 etiological mutations were identified in this study, comprising 26 novel mutations and 10 reported mutations. LRP5 was the most prevalent mutant gene among the 36 mutation types with a percentage of 41.67% (15/36). Followed by FZD4 (10/36, 27.78%), TSPAN12 (5/36, 13.89%), NDP (4/36, 11.11%), KIF11 (1/36, 2.78%), and RCBTB1 (1/36, 2.78%). Among these mutations, 63.89% (23/36) were missense mutations, 25.00% (9/36) were frameshift mutations, 5.56% (2/36) were splicing mutations, 5.56% (2/36) were nonsense mutations. Moreover, the clinical pathogenicity of these variants was defined according to American College of Medical Genetics (ACMG) and genomics guidelines: 41.67% (15/36) were likely pathogenic variants, 27.78% (10/36) pathogenic variants, 30.55% (11/36) variants of uncertain significance. No etiological mutations discovered in the ZNF408, JAG1, and CTNNA1 genes in this FEVR cohort.We systematically screened nine FEVR disease-associated genes in a cohort of 74 Chinese probands with FEVR disease. With a detection rate of 40.54%, 36 etiological mutations of six genes were authenticated in 30 probands, including 26 novel mutations and 10 reported mutations. The most prevalent mutated gene is LRP5, followed by FZD4, TSPAN12, NDP, KIF11, and RCBTB1. In total, a de novo mutation was confirmed. Our study significantly clarified the mutation spectrum of variants bounded up to FEVR disease.

In order to investigate the effect of fluid shear stress on the proliferation of osteoblasts and the regulatory role of the Wnt/

Low-density Lipoprotein Receptor-related Protein 5 (LRP5) functions as a co-receptor for Wnt ligands, controlling expression of genes involved in osteogenesis. In humans, loss-of-function mutations in LRP5 cause Osteoporosis-Pseudoglioma syndrome, a low bone mass disorder, while gain-of-function missense mutations have been observed in individuals with high bone mass. Zebrafish (Danio rerio) is a popular model for human disease research, as genetic determinants that control bone formation are generally conserved between zebrafish and mammals. We generated lrp5- knock-out zebrafish to study its role in skeletogenesis and homeostasis. Loss of lrp5 in zebrafish leads to craniofacial deformities and low bone mineral density (total body and head) at adult ages. To understand the mechanism and consequences of the observed phenotypes, we performed transcriptome analysis of the cranium of adult lrp5 mutants and siblings. Enrichment analysis revealed upregulation of genes significantly associated with hydrolase activity: mmp9, mmp13a, acp5a. acp5a encodes Tartrate-resistant acid phosphatase (TRAP) which is commonly used as an osteoclast marker, while Matrix metalloprotease 9, Mmp9, is known to be secreted by osteoclasts and stimulate bone resorption. These genes point to changes in osteoclast differentiation regulated by lrp5. To analyze these changes functionally, we assessed osteoclast dynamics in mutants and observed increased TRAP staining, significantly larger resorption areas, and developmental skeletal dysmorphologies in the mutant, suggesting higher resorptive activity in the absence of Lrp5 signaling. Our findings support a conserved role of Lrp5 in maintaining bone mineral density and revealed unexpected insights into the function of Lrp5 in bone homeostasis through moderation of osteoclast function.

WNT/β-catenin and BMP signaling pathways play important roles in the process of tooth development. Dysregulation of WNT/β-catenin and BMP signaling is implicated in a number of human malformations, including dental anomalies. Whole exome and Sanger sequencing identified seven patients with LRP5 mutations (p.Asn1121Asp, p.Asp856Asn, p.Val1433Met, and p.Val1245Met) and six patients with BMP4 mutations (p.Asn150Lys, p.Gly168Arg, p.Arg269Gln, and p.Ala42Glu). All patients were affected with isolated dental anomalies (dental anomalies with no other structural defects), including mesiodens, tooth agenesis, unseparated roots, narrow roots, shortened and tapered roots, and taurodontism. Five patients with LRP5 and one with BMP4 mutations had oral exostoses. Protein models of LRP5 mutations indicate the possible functional effects of the mutations. Here we report for the first time that mutations in LRP5 are associated with dental anomalies. LRP5 appears to be the first gene related to pathogenesis of mesiodens. We also show for the first time that in addition to tooth agenesis, mutations in BMP4 are also implicated in root maldevelopment and torus mandibularis. Sharing of the phenotypes of the patients with LRP5 and BMP4 mutations, which include root maldevelopment, tooth agenesis, and torus mandibularis, implicates cross talks between the WNT/β-catenin and BMP signaling pathways, especially during root development.

Wnt/β‐catenin signaling is indispensable for many biological processes, including embryonic development, cell cycle, inflammation, and carcinogenesis. Aberrant activation of the Wnt/β‐catenin signaling can promote tumorigenicity and enhance metastatic potential in hepatocellular carcinoma (HCC). Targeting this pathway is a new opportunity for precise medicine for HCC. However, inhibiting Wnt/β‐catenin signaling alone is unlikely to significantly improve HCC patient outcome due to the lack of specific inhibitors and the complexity of this pathway. Combination with other therapies will be an important next step in improving the efficacy of Wnt/β‐catenin signaling inhibitors. Protein kinases play a key and evolutionarily conserved role in the Wnt/β‐catenin signaling and have become one of the most important drug targets in cancer. Targeting Wnt/β‐catenin signaling and its regulatory kinase together will be a promising HCC management strategy. In this review, we summarize the kinases that modulate the Wnt/β‐catenin signaling in HCC and briefly discuss their molecular mechanisms. Furthermore, we list some small molecules that target the kinases and may inhibit Wnt/β‐catenin signaling, to offer new perspectives for preclinical and clinical HCC studies., In this review, we summarize the kinases that modulate the Wnt/β‐catenin signaling in HCC and briefly discuss their molecular mechanisms. Furthermore, we list some small molecules that target the kinases and may inhibit Wnt/β‐catenin signaling to offer new perspectives for preclinical and clinical HCC studies.

Objective To analyse the effect of gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (Lrp5) on orthodontic tooth movement (OTM). Setting and sample population A split-mouth study design was utilized. Thirty-two male Lrp5-high bone mass (HBM) knock-in mice including A214V and G171V mutants (n = 16/group) and sixteen C57BL/6 wild-type (WT) mice were included in the study. Materials and methods A mouse model of OTM was used for mesial movement of the maxillary first molar using a closed-coil nickel titanium (NiTi) spring attached between the molar and the incisors. After 21 days, the dissected maxillae were scanned for micro-computed tomography (micro-CT) analyses and embedded in methyl methacrylate and paraffin for histological staining and imaging. Histological analyses included immunohistochemistry for sclerostin (Sost), tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts and fluorescent imaging. Results OTM in the A214V and G171V groups was significantly less than the WT group. Bone volume (BV), per cent bone volume (BV/TV) and trabecular thickness (Tb.Th) were significantly increased in both A241V and G171V animals compared to the WT animals. On the compression side, decreased osteoclast activity was seen in both A214V and G171V groups compared to the WT group. Fluorescent labelling demonstrated that the pattern of bone deposition in the A214V animals was periosteal whereas the G171V animals added bone endocortically. Conclusion Gain-of-function mutations of Lrp5 decrease orthodontic tooth movement by increasing alveolar bone mass and reducing osteoclast-mediated bone resorption.

While Inherited Retinal Diseases (IRDs) are typically considered rare diseases, Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie Disease (ND) are more rare than retinitis pigmentosa. We wanted to determine if multigenic protein-altering variants are common in FEVR subjects within a set of FEVR-related genes. The potential occurrence of protein-altering variants in two different genes has been documented in a very small percentage of patients, but potential multigenic contributions to FEVR remain unclear. Genes involved in these orphan pediatric retinal diseases are not universally included in available IRD targeted-sequencing panels, and cost is also a factor limiting multigenic-sequence-based testing for these rare conditions. To provide an accurate solution at lower cost, we developed a targeted-sequencing protocol that includes seven genes involved in Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie disease. Seventy-six DNA samples from persons refered to clinic with possible FEVR and some close relatives were sequenced using a novel Oakland-ERI orphan pediatric retinal disease panel (version 2) providing 900 times average read coverage. The seven genes involved in FEVR/ND were: NDP (ChrX), CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11). A total of 33 variants were found that alter protein sequence, with the following relative distribution: LRP5 13/33 (40%), FZD4 9/33 (27%), ZNF408 6/33 (18%), (KIF11 3/33 (9%), NDP 1/33 (3%), CTNNB1 1/33 (3%). Most protein-altering variants, 85%, were found in three genes: FZD4, LRP5, and ZNF408. Four previously known pathogenic variants were detected in five families and two unrelated individuals. Two novel, likely pathogenic variants were detected in one family (FZD4: Cys450ter), and a likely pathogenic frame shift termination variant was detected in one unrelated individual (LRP5: Ala919CysfsTer67). The average number of genes with protein-altering variants was greater in subjects with confirmed FEVR (1.46, n = 30) compared to subjects confirmed unaffected by FEVR (0.95, n = 20), (p = 0.009). Thirty-four percent of persons sequenced had digenic and trigenic protein-altering variants within this set of FEVR genes, which was much greater than expected in the general population (3.6%), as derived from GnomAD data. While the potential contributions to FEVR are not known for most of the variants in a multigenic context, the high multigenic frequency suggests that potential multigenic contributions to FEVR severity warrant future investigation. The targeted-sequencing format developed will support such exploration by reducing the testing cost to $250 (US) for seven genes and facilitating greater access to genetic testing for families with this very rare inherited retinal disease.

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low-density lipoprotein receptor-related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac-specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin-dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.

Objective The temporomandibular joint (TMJ) is a unique fibrocartilaginous joint that adapts to mechanical loading through cell signaling pathways such as the Wnt pathway. Increased expression of low-density lipoprotein receptor-related protein 5 (Lrp5), a co-receptor of the Wnt pathway, is associated with a high bone mass (HBM) phenotype. The objective of this study was to analyze the effect of overexpression of Lrp5 on the subchondral bone and cartilage of the TMJ in mice exhibiting the HBM phenotype. Design Sixteen-week-old Lrp5 knock-in transgenic mice carrying either the A214V (EXP-A) or G171V (EXP-G) missense mutations, and wildtype controls (CTRL) were included in this study. Fluorescent bone labels, calcein, alizarin complexone, and demeclocycline were injected at 3.5, 7.5, and 11.5 weeks of age, respectively. The left mandibular condyle was used to compare the subchondral bone micro-computed tomography parameters and the right TMJ was used for histological analyses. Cartilage thickness, matrix proteoglycan accumulation, and immunohistochemical localization of Lrp5 and sclerostin were compared between the groups. Results Subchondral bone volume (BV) and percent bone volume (BV/TV) were significantly increased in both EXP-A and EXP-G compared with CTRL ( P < 0.05) whereas trabecular spacing (Tb.Sp) was decreased. Cartilage thickness, extracellular matrix production, and expression of Lrp5 and Sost were all increased in the experimental groups. The separation between the fluorescent bone labels indicated increased endochondral maturation between 3.5 and 7.5 weeks. Conclusions These data demonstrate that Lrp5 overexpression leads to adaptation changes in the mandibular condylar cartilage of the TMJ to prevent cartilage degradation.

Significance Canonical Wnt signaling is one of the most important and widely distributed pathways in metazoan development. Dishevelled is thought to serve as an essential bridge between the membrane receptors and downstream signaling components, which has the tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo, when overexpressed. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems, we have expressed a fluorescent-tagged Dishevelled in cells at its physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step in signal transduction., Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/μm2 to 0.54/μm2. Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.

Bone erosion is a debilitating pathological process of osteopathic disorder like rheumatoid arthritis (RA). Current treatment strategies render low disease activity but with disease recurrence. To find an alternative, we designed this study with an aim to explore the underlying therapeutic effect of PEGylated liposomal BBR (PEG-BBR) against Wnt1/β-catenin mediated bone erosion in adjuvant-induced arthritic (AA) rat model and fibroblast-like synoviocytes (FLS) with reference to microRNA-23a (miR-23a) activity. Our initial studies using confocal microscopy and Near-Infrared Imaging (NIR) showed successful internalization of PEG-BBR and PEG-miR-23a in vitro and in vivo respectively and was retained till 48 h. The preferential internalization of PEG-BBR into the inflamed joint region significantly reduced the gene and protein level expression of major Wnt1 signaling mediators and reduced bone erosion in rats. Moreover, PEG-BBR treatment in FLS cells attenuated the gene and protein expression levels of FZD4, LRP5, β-catenin, and Dvl-1 through the induction of CYLD. Furthermore, inhibition of these factors resulted in reduced bone loss and increased calcium retainability by altering the RANKL/OPG axis. PEG-BBR treatment markedly inhibited the expression of LRP5 protein on par with the DKK-1 (LRP5/Wnt signaling inhibitor) and suppressed the transcriptional activation of β-catenin inside the cells. We further witnessed that miR-23a altered the expression levels of LRP5 through RNA interference. Overall, our findings endorsed that miR-23a possesses a multifaceted therapeutic efficiency like berberine in RA pathogenesis and can be considered as a potential candidate for therapeutic targeting of Wnt1/β-catenin signaling in RA disease condition.

Previous studies by us and others demonstrated that activation of Wnt/β-catenin signaling plays a pathogenic role in chronic kidney diseases (CKD). Wnt co-receptor LRP5 variants are reported to associate with autosomal dominant polycystic kidney disease; but their exact roles in this disease and renal fibrosis have not been explored. Here, we observed the upregulation of LRP5 in the renal tubules of both type 1 and type 2 diabetic models and of an obstructive nephropathy model. In the obstructed kidneys, Lrp5 knockout significantly ameliorated tubulointerstitial fibrosis and tubular injury without changing Wnt/β-catenin signaling. Instead, decreased levels of TGF-β1 and TGF-β receptors (TβRs) were detected in Lrp5 knockout kidneys, followed by attenuated activation and nuclear translocation of Smad2/3 in the renal tubules, suggesting a regulatory effect of LRP5 on TGF-β/Smad signaling. In consistent with this hypothesis, LRP5 overexpression resulted in enhanced TGF-β/Smad signaling activation in renal tubule epithelial cells. Furthermore, LRP5 was co-immunoprecipitated with TβRI and TβRII, and its extracellular domain was essential for interacting with TβRs and for its pro-fibrotic activity. In addition to stabilizing TβRs, LRP5 increased the basal membrane presentation and TGF-β1-induced internalization of these receptors. Notably, TGF-β1 also induced LRP5 internalization. These findings indicate that LRP5 promotes tubulointerstitial fibrosis, at least partially, via direct modulation of TGF-β/Smad signaling, a novel, Wnt-independent function.

To explore the genetic basis for a Chinese pedigree with two individuals suffering from congenital blindness.Clinical data and peripheral blood samples of the pedigree were collected. Whole exome sequencing was carried out. Suspected variants were verified by Sanger sequencing. Pathogenicity of candidate variants was validated through searching of PubMed and related databases, and analyzed with bioinformatics software.Both patients had congenital blindness and a history of multiple fractures. Other features have included microphthalmia and cornea opacity. One patient had normal intelligence, whilst the other had a language deficit. Both patients were found to harbor compound heterozygous variants of the LRP5 gene, namely c.1007_1015delGTAAGGCAG (p.C336X), c.4400GA (p.R1467Q) and c.4600CT (p.R1534X). The first one was derived from their mother, whilst the latter two were derived from their father. None of the three variants was detected in their elder sister.The compound heterozygous variants of c.1007_1015delGTAAGGCAG (p.C336X) and c.4600CT (p.R1534X) of the LRP5 gene probably underlay the pathogenesis of the Osteoporosis-pseudoglioma syndrome in this pedigree. The clinical significance of the c.4400GA (p.R1467Q) variant has remained uncertain. Above finding has enriched the mutational spectrum of Osteoporosis-pseudoglioma syndrome.

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).

Background Multiple studies have demonstrated the involvement of low-density lipoprotein receptor-related protein 5 (LRP5) in metabolism-related diseases. This study explored the relationship between the LRP5 rs556442 gene polymorphism and the risks of non-alcoholic fatty liver disease (NAFLD) and coronary heart disease (CHD) in a Chinese Han population. Methods This retrospective case–control study included 247 patients with NAFLD, 200 patients with CHD, 118 patients with both NAFLD and CHD, and 339 healthy controls from June 2018 to June 2019 at Qingdao Municipal Hospital. Basic information and clinical characteristics were collected for all subjects. The genotype and allele frequency of LRP5 rs556442 were determined. Results The genotype distributions of LRP5 rs556442 differed significantly between the CHD and NAFLD + CHD groups (P LRP5 rs556442 GG genotype markedly promoted the risk of NAFLD in CHD patients [odds ratio (OR) = 2.857, 95% confidence interval (CI): 1.196–6.824, P = 0.018). After adjustment for sex, age, and body mass index (BMI), this association remained significant (OR = 3.252, 95% CI: 1.306–8.102, P = 0.011). In addition, the LRP5 rs556442 AA + AG genotype was associated with an increased BMI in obese NAFLD patients (OR = 1.526, 95% CI: 1.004–2.319, P = 0.048). However, after adjustment for sex and age, this association was no longer significant (OR = 1.504, 95% CI: 0.991–2.282, P = 0.055). Conclusions This study found that the LRP5 rs556442 GG genotype increased the risk of NAFLD in CHD patients and AA + AG genotype may be associated with an increased BMI in obese NAFLD patients among a Chinese Han population. Trial registration ChiCTR, ChiCTR1800015426. Registered 28 March 2018—Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=26239.

The overactivation of canonical Wnt/β-catenin pathway and the maintenance of cancer stem cells (CSCs) are essential for the onset and malignant progression of most human cancers. However, their regulatory mechanism in colorectal cancer (CRC) has not yet been well demonstrated. Low-density lipoprotein receptor-related protein 5 (LRP5) has been identified as an indispensable co-receptor with frizzled family members for the canonical Wnt/β-catenin signal transduction. Herein, we show that activation of LRP5 gene promotes CSCs-like phenotypes, including tumorigenicity and drug resistance in CRC cells, through activating the canonical Wnt/β-catenin and IL-6/STAT3 signalling pathways. Clinically, the expression of LRP5 is upregulated in human CRC tissues and closely associated with clinical stages of patients with CRC. Further analysis showed silencing of endogenous LRP5 gene is sufficient to suppress the CSCs-like phenotypes of CRC through inhibiting these two pathways. In conclusion, our findings not only reveal a regulatory cross-talk between canonical Wnt/β-catenin signalling pathway, IL-6/STAT3 signalling pathway and CD133-related stemness that promote the malignant behaviour of CRC, but also provide a valuable target for the diagnosis and treatment of CRC.

Canonical Wnt signaling is involved in skeletal muscle cell biology. The exact way in which this pathway exerts its contribution to myogenesis or neuromuscular junctions (NMJ) is a matter of debate. Next to the common co-receptors of canonical Wnt signaling, Lrp5 and Lrp6, the receptor tyrosine kinase MuSK was reported to bind at NMJs WNT glycoproteins by its extracellular cysteine-rich domain. Previously, we reported canonical Wnt signaling being active in fast muscle fiber types. Here, we used conditional Lrp5 or Lrp6 knockout mice to investigate the role of these receptors in muscle cells. Conditional double knockout mice died around E13 likely due to ectopic expression of the Cre recombinase. Phenotypes of single conditional knockout mice point to a very divergent role for the two receptors. First, muscle fiber type distribution and size were changed. Second, canonical Wnt signaling reporter mice suggested less signaling activity in the absence of Lrps. Third, expression of several myogenic marker genes was changed. Fourth, NMJs were of fragmented phenotype. Fifth, recordings revealed impaired neuromuscular transmission. In sum, our data show fundamental differences in absence of each of the Lrp co-receptors and suggest a differentiated view of canonical Wnt signaling pathway involvement in adult skeletal muscle cells.

Familial exudative vitreoretinopathy (FEVR) is an inherited ocular disease with clinical manifestations of aberrant retinal vasculature. We aimed to identify novel causative variants responsible for FEVR and provided evidence for the genetic counselling of FEVR.We applied whole-exome sequencing (WES) on the genomic DNA samples from the probands and performed Sanger sequencing for variant validation. Western blot analysis and luciferase assays were performed to test the expression levels and the activity of mutant proteins.We identified one novel heterozygous nonsense variant, and three novel heterozygous frameshift variants including c.1801GT (p.G601*), c.1965delC (p.H656Tfs*41), c.4445delC (p.S1482Cfs*17), and c.4482delC (p.P1495Rfs*4), which disabled the function of LRP5 on the Norrin/β-catenin signalling. Overexpression of variant-carrying LRP5 proteins resulted in down regulation of the protein levels of β-catenin and the Norrin/β-catenin signalling target genes c-Myc and Glut1.Our study showed that four inherited LRP5 variants can cause autosomal dominant FEVR via down regulation of Norrin/β-catenin signalling and expanded the spectrum of FEVR-associated LRP5 variants.

This study describes the clinical, radiological, and molecular data of four new patients with osteoporosis-pseudoglioma syndrome and assesses their response to bisphosphonate therapy.Osteoporosis-pseudoglioma syndrome (OPPG) is a very rare disorder characterized mainly by severe juvenile osteoporosis and congenital blindness. OPPG is caused by biallelic mutations in the gene encoding low-density lipoprotein receptor-related protein 5 (LRP5).We present the clinical, radiological, and molecular findings of four new patients with OPPG from Egypt. We also assessed patients' response to oral and intravenous bisphosphonate therapy.All patients had reduced bone mineral density (BMD) with variable number of fractures per year, in addition to bone abnormalities and the characteristic eye phenotype associated with OPPG. Mutation analyses of LRP5 gene revealed three different homozygous variants including two novel ones, c.7delG (p.A3Qfs*80) and c.3280G A (p.E1094K). The c.3280G A (p.E1094K) was recurrent in two unrelated patients who shared a unique haplotype suggesting a possible founder effect. The use of bisphosphonate therapy was beneficial; however, intravenous bisphosphonate administration led to a more favorable response.Our study described the phenotypic and genetic features of four patients with OPPG and identified two new LRP5 variants, thus expanding the mutational spectrum of OPPG. In addition, our study reinforces the efficiency of using intravenous bisphosphonates in the management of patients with OPPG.

Serotonin (5HT) is a chemical messenger with biologic activities affecting multiple organs. Within the skeletal system, studies in mice and humans suggest blood 5HT levels affect bone, with elevations impairing and reductions enhancing bone accrual. Other studies, however, have not supported this hypothesis. Recently, administering 5HT to a Piezo1 mutant mouse strain with hyposerotonemia, intestinal dysmotility, and a doubling of femoral trabecular bone mass at 2 months of age, was reported to return the animals' intestinal motility and bone mass to normal. However, whether the 5HT directly affected bone metabolism or indirectly affected metabolism by improving intestinal function was not determined. Therefore, we administered 5HT to mice with normal intestinal function. We randomized female C57BL6/J mice and male and female mice that have increased bone mass due to a missense mutation in the WNT co-receptor LRP5 (Lrp5

The WNT/β-catenin signalling pathway is a rich and complex network of cellular proteins that orchestrates diverse short-range cell-to-cell communication in metazoans and is essential for both embryonic development and adult homeostasis. Due to its fundamental importance in controlling cell behaviour at multiple levels, its deregulation is associated with a wide range of diseases in humans and identification of drugs targeting the pathway has attracted strong interest in the pharmaceutical sector. Transduction of WNT signals across the plasma membrane of cells involves a staggering degree of complexity and variety with respect to ligand-receptor, receptor-receptor and receptor-co-receptor interactions (Niehrs, Nat Rev Mol Cell Biol 13:767-779, 2012). Although the low-density-lipoprotein-receptor-related-protein (LRP) family is best known for its role in binding and endocytosis of lipoproteins, specific members appear to have additional roles in cellular communication. Indeed, for WNT/β-catenin signalling one apparently universal requirement is the presence of either LRP5 or LRP6 in combination with one of the ten Frizzled (FZD) WNT receptors (FZD

Periodontal ligament stem cells (PDLSCs) plays an important role in tissue engineering. As the age increased, the cell viability and osteogenic differentiation of PDLSCs all decreased. Low density lipoprotein receptor related protein 5 (LRP5) was found to promote bone marrow mesenchymal stem cells osteogenic differentiation. Therefore, our study explored the effect of LRP5 on normal and aged PDLSCs and relative mechanism. Here, we found that the expression of LRP5 in PDLSCs of 24 week-old mice was decreased compared with PDLSCs of 5 week-old mice (n = 5). . LRP5 overexpression in PDLSCs increased the intensity of alkaline phosphatase and alizarin red staining, accompanied with upregulated the levels of RUNX family transcription factor 2, collagen type I, and β-Catenin. LRP5 knockdown displayed the opposite results in PDLSCs in vitro. LRP5 overexpression in aged PDLSCs restored part ability of osteogenic differentiation. Meantime, LRP5 increased the protein expression of phosphorylation of mammalian target of rapamycin (p-mTOR) in normal and aged PDLSCs. Immunofluorescence showed that LRP5 increased the accumulation of p-mTOR nucleus. The effect of LRP5 in promoting osteogenic differentiation of PDLSCs can be antagonized by mTOR inhibitor rapamycin. These findings suggest that LRP5 positively regulate osteogenic differentiation of normal and aged PDLSCs and may be a potential target for enlarging the application of PDLSCs in tissue regeneration.

There remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy.We developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage.SZN-413-p induced Wnt/β-catenin signaling and upregulated blood-brain barrier/blood-retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P0.001) and aflibercept groups (P0.05 and P0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P0.01).Reduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage.FZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.

Background Familial exudative vitreoretinopathy (FEVR) is a complex form of blindness-causing retinal degeneration. This study investigated the potential disease-causing variants in 20 Chinese families with FEVR. Methods All available family members underwent detailed ophthalmological examinations, including best-corrected visual acuity and fundus examination. All probands and most family members underwent fluorescein fundus angiography. Twenty probands underwent whole exome sequencing; 16 of them also underwent copy number variant and mitochondrial genome analysis. Bioinformatics analysis and Sanger sequencing of available family members were used to confirm the disease-causing gene variant. Results Twenty families were diagnosed with FEVR based on clinical symptoms, fundus manifestations, and fundus fluorescein angiography. Whole exome sequencing revealed 14 variants in NDP, FZD4, LRP5, and TSPAN12 genes among the 13 families. These variants were predicted to be damaging or deleterious according to multiple lines of prediction algorithms; they were not frequently found in multiple population databases. Seven variants had not previously been reported to cause FEVR: c.1039T>G p.(Phe347Val) in the FZD4 gene; c.1612C>T p.(Arg538Trp) and c.3237-2A>C in the LRP5 gene; and c.77T>A p.(Ile26Asn), c.170dupT p.(Leu57Phe fsTer60), c.236T>G p.(Met79Arg) and c.550dupA p.(Arg184Lys fsTer16) in the TSPAN12 gene. We did not detect any variants in the remaining seven families. Conclusions These results expand the spectrum of variants in the NDP, FZD4, LRP5, and TSPAN12 genes and provide insights regarding accurate diagnosis, family genetic counseling, and future gene therapy for FEVR.

Purpose Familial exudative vitreoretinopathy (FEVR) is an inherited retinal vascular disease genetically heterogeneous with multiple causative genes. The aim of this study is to report five novel copy number variation (CNV) regions in FEVR patients and to investigate the possible contributions of novel CNVs to FEVR. Methods In this study, 824 FEVR families were collected. All cases were performed using the targeted next generation sequencing (NGS) assay, and families with no definite pathogenic mutations in FEVR genes were screened for CNVs according to the NGS results. Droplet digital polymerase chain reaction (ddPCR) testing was introduced to validate the screened CNV regions. We also reviewed the clinical presentations of the probands and affected family members associated with the novel CNVs and conducted segregation analysis. Results Five CNVs in five patients were detected in this study: heterozygous deletions of kinesin family member 11 (KIF11) exons 2–4, KIF11 exon 11, KIF11 exons 1–10, tetraspanin-12 (TSPAN12) exons 1–3, and low-density lipoprotein receptor-related protein 5 (LRP5) exons 19–21. Among the five affected families, TSPAN12 exons 1–3 heterozygous deletion and LRP5 exons 19–21 heterozygous deletion originate from the mother and the father of the proband, respectively. No other family members manifested as FEVR except for the probands. The correlation between disease severity and CNV loci seems uncertain. Conclusions Five novel CNV loci in FEVR patients were uncovered in this study, including one maternally-inherited and one paternally-inherited CNV region. Though there is no evidence of co-segregation between these CNVs and FEVR, our findings suggest novel genetic risk factors for FEVR.

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.

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.

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.

Cardiosphere-derived cells (CDCs) are therapeutic candidates with disease-modifying bioactivity, but their variable potency has complicated their clinical translation. Transcriptomic analyses of CDCs from human donors have revealed that the therapeutic potency of these cells correlates with Wnt/β-catenin signalling and with β-catenin protein levels. Here, we show that skin fibroblasts engineered to overexpress β-catenin and the transcription factor Gata4 become immortal and therapeutically potent. Transplantation of the engineered fibroblasts into a mouse model of acute myocardial infarction led to improved cardiac function and mouse survival. And in the mdx mouse model of Duchenne muscular dystrophy, exosomes secreted by the engineered fibroblasts improved exercise capacity and reduced skeletal-muscle fibrosis. We also demonstrate that exosomes from high-potency CDCs exhibit enhanced levels of miR-92a (a known potentiator of the Wnt/β-catenin pathway), and that they activate cardioprotective bone-morphogenetic-protein signalling in cardiomyocytes. Our findings show that the modulation of canonical Wnt signalling can turn therapeutically inert mammalian cells into immortal exosome factories for cell-free therapies., EDITORIAL SUMMARY Overexpression of β-catenin and the transcription factor Gata4 in skin fibroblasts converts them into therapeutically active cells that secrete reparative exosomes as shown in mice models of myocardial infarction and Duchenne muscular dystrophy.

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.

Aim The main of this study was to investigate the association between the rs566442 (V1119V) coding polymorphism of Low-density lipoprotein receptor-related protein 5 (LRP5) with obesity and basal metabolic rate in Iranian postmenopausal women. Methods This cross-sectional study was performed on 350 postmenopausal women with a mean age of 57.8 years (SD ± 6.14). Body composition was analyzed by bioelectrical impedance analysis (BIA) resistance. Obesity was defined based on Body mass index (BMI) ≥30 kg/m2. To determine the genotype of SNP (rs556442), PCR-RFLP assay was performed and confirmed by sequencing. DNA samples from participants were genotyped using the RFLP-PCR method. Results Among the study population 37.1% (130) were obese. G allele had minor-allele frequency of 0.38% in our population. The frequency of genotypes in our study population was 12.9% (45 person) GG, 35.7% (125 person) AA and 51.4% (180) GA. After adjusting age and menopausal age, only basal metabolic rate showed significantly higher in GG group compare to other groups (p = 0.02). Our data showed basal metabolic rate was higher in obese women with GG genotype in comparison to obese women with AG and AA genotypes. Discussion The findings of this study suggest that the GG genotype of SNP (rs556442) could protective role in obese women through the association with BMR.

Background/aim: Genetic aspects play a role in insulin resistance in children. In this study, for the first time, the association of LRP5 (rs556442) polymorphism and insulin resistance in Iranian children and adolescents was investigated.Materials and methods: The study population comprises children and adolescents aged 9-18 years. Anthropometric and biochemical parameters were assessed. Insulin resistance/sensitivity was determined by the quantitative insulin sensitivity check index (QUICKI), homeostasis model assessment-insulin resistance (HOMA-IR), insulin-to-glucose ratio, McAuley index, revised McAuley index, fasting insulin resistance index (FIRI), and Bennett's index. LRP5 (rs566442) single nucleotide polymorphism (SNP) was identified using restriction fragment length polymorphism (RFLP). Linear regression analysis was used to determine the association between the LRP5 polymorphism (rs556442) and insulin sensitivity indexes.Results: Significant differences were found between GG genotype vs. AG/AA genotypes for McAuley index (P = 0.049) and revised McAuley index (P = 0.044) when adjusted for interaction factors (age, sex, and puberty) in regression models. No significant association was found between LRP5 (rs566442) and other insulin resistance indexes. Also, LRP5 (rs566442) did not show a significant impact on biochemical parameters. Conclusion: This study showed that LRP5 polymorphism (rs556442) was associated with insulin resistance in Iranian children and adolescents.

Hematopoietic stem cells (HSCs) have the capacity for self-renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self-renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low-density lipoprotein receptor-related protein 5 (Lrp5) and low-density lipoprotein receptor-related protein 6 (Lrp6) mice in adult hematopoiesis via Vav-Cre Loxp system. Inactivation of Lrp5 and -6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and -6 double deficiency HSCs showed intrinsic defects in self-renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up-regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down-regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and -6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and -6 are required for the self-renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.-Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.-H., Yu, S. Lrp5 and Lrp6 are required for maintaining self-renewal and differentiation of hematopoietic stem cells.

Wnt-induced β-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced β-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third β-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors., Enhanced Wnt receptor activity is a major cause of cancer development. Here the authors identify camelid single-domain antibody fragments (VHHs) that bind to the Wnt receptor LRP5/6 ectodomain, determine the crystal structures and show that these VHHs selectively inhibit Wnt3- mediated cellular responses and block the growth of mutant Wnt-hypersensitive intestinal tumor organoids.

Background Low-density lipoprotein receptor-related protein 5 (LRP5) has been intensively studied as a co-receptor for β-catenin-dependent Wnt signaling. Emerging evidences have demonstrated β-catenin-independent functions of LRP5. However, the biological role of LRP5 in the mammalian heart is largely unknown. Methods and results Conditional cardiac-specific Lrp5 knockout (Lrp5-CKO) mice were generated by crossing Lrp5flox/flox mice with αMHC/MerCreMer mice. Lrp5-CKO mice consistently displayed normal cardiac structure and function. Telemetric electrocardiogram recordings revealed a short QT interval in Lrp5-CKO mice, which was tightly linked to the striking abbreviation of action potential duration (APD) in ventricular myocytes. The analysis of whole-cell currents indicated that a reduction in activity and protein expression of L-type calcium channel (LTCC), rather than other ion channels, contributed to the abnormality in APD. Furthermore, we showed that Lrp5 ablation induced a significant convergence of CaV1.2α1c proteins to the endoplasmic reticulum. Consequently, increased proteasomal degradation of these proteins was observed, which was independent of the Wnt/β-catenin signaling pathway. Conclusions LRP5 directly modulates the degradation of LTCC to control cardiac QT interval. These findings provide compelling evidence for the potential role of LRPs in cardiac electrophysiology.

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.

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).

The overactivation of canonical Wnt/β-catenin pathway is one of the main cascades for the initiation, progression, and recurrence of most human malignancies. As an indispensable coreceptor for the signaling transduction of the canonical Wnt/β-catenin pathway, LRP5 is up-regulated and exerts a carcinogenic role in most types of cancer. However, its expression level and role in gastric cancer (GC) has not been clearly elucidated. The current work showed that LRP5 was overexpressed in GC tissues and the expression of LRP5 was positively associated with the advanced clinical stages and poor prognosis. Ectopic expression of LRP5 enhanced the proliferation, invasiveness, and drug resistance of GC cells in vitro, and accelerated the tumor growth in nude mice, through activating the canonical Wnt/β-catenin signaling pathway and up-regulating aerobic glycolysis, thus increasing the energy supply for GC cells. Additionally, the expression of LRP5 and glycolysis-related genes showed an obviously positive correlation in GC tissues. By contrast, the exact opposite results were observed when the endogenous LRP5 was silenced in GC cells. Collectively, these results not only reveal the carcinogenic role of LRP5 during GC development through activating the canonical Wnt/β-catenin and glycolysis pathways, but also provide a valuable candidate for the diagnosis and treatment of human GC.

5-Fluorouracil (5-FU) resistance has been long considered as an obstacle to the efficacy of chemotherapy in colorectal cancer (CRC). In this study, we demonstrated the role of miR-20b-5p-regulated syndecan-2 (SDC2) in 5-FU resistance of CRC cells. 5-FU-resistant SW480 CRC cells were established by treatment of SW480 cells with stepwise increase of 5-FU concentration. The results showed that SDC2 was expressed significantly higher in SW480/5-FU cells than in SW480/WT cells as revealed by quantitative real-time polymerase chain reaction and western blot analysis. MTT assay and BrdU assay showed that SDC2 overexpression led to increased cell survival rate, while SDC2 knockdown reversed the drug resistance of SW480/5-FU cells. Wound healing and transwell invasion assays revealed that knockdown of SDC2 inhibited the migratory and invasive ability of SW480/5-FU cells. Moreover, animal experiments indicated that si-SDC2 plays a suppressive role in tumor growth in vivo. We also confirmed that miR-20b-5p interacted with SDC2, which reversed the effect of SDC2 in SW480/5-FU cells via the c-Jun N-terminal kinase (JNK)/extracellular regulated protein kinases (ERK) signaling pathway. These findings showed that JNK/ERK signaling pathway is involved in miR-20b-5p/SDC2 axis-mediated 5-FU resistance in SW480/5-FU cells, indicating that the miR-20b-5p/SDC2 axis is a potential target for reversing 5-FU resistance in CRC.