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Pathological cardiac hypertrophy is considered one of the independent risk factors for heart failure, with a rather complex pathogenic machinery. Sorting nexins (SNXs), denoting a diverse family of cytoplasmic- and membrane-associated phosphoinositide-binding proteins, act as a pharmacological target against specific cardiovascular diseases including heart failure. Family member SNX5 was reported to play a pivotal role in a variety of biological processes. However, contribution of SNX5 to the development of cardiac hypertrophy, remains unclear. Mice underwent transverse aortic constriction (TAC) to induce cardiac hypertrophy and simulate pathological conditions. TAC model was validated using echocardiography and histological staining. Expression of SNX5 was assessed by western blotting. Then, SNX5 was delivered through intravenous administration of an adeno-associated virus serotype 9 carrying cTnT promoter (AAV9-cTnT-SNX5) to achieve SNX5 cardiac-specific overexpression. To assess the impact of SNX5, morphological analysis, echocardiography, histological staining, hypertrophic biomarkers, and cardiomyocyte contraction were evaluated. To unravel potential molecular events associated with SNX5, interactome analysis, fluorescence co-localization, and membrane protein profile were evaluated. Our results revealed significant downregulated protein level of SNX5 in TAC-induced hypertrophic hearts in mice. Interestingly, cardiac-specific overexpression of SNX5 improved cardiac function, with enhanced left ventricular ejection fraction, fraction shortening, as well as reduced cardiac fibrosis. Mechanistically, SNX5 directly bound to Rab11a, increasing membrane accumulation of Rab11a (a Rab GTPase). Afterwards, this intricate molecular interaction upregulated the membrane content of low-density lipoprotein receptor-related protein 6 (LRP6), a key regulator against cardiac hypertrophy. Our comprehensive assessment of siRab11a expression in HL-1 cells revealed its role in antagonism of LRP6 membrane accumulation under SNX5 overexpression. This study revealed that binding of SNX5 with LRP6 triggers their membrane translocation through Rab11a assisting, defending against cardiac remodeling and cardiac dysfunction under pressure overload. These findings provide new insights into the previously unrecognized role of SNX5 in the progression of cardiac hypertrophy. Cartoon model of cardiac protection mediated by SNX5 overexpression in heart tissues in response to pressure overload-induced cardiac hypertrophy. SNX5 overexpression promotes LRP6 membrane translocation and its activation through binding with Rab11a, to mitigate cardiac hypertrophy and cardiac dysfunction. [Display omitted] • The expression of sorting nexin 5 (SNX5) is down-regulated in hypertrophic heart induced by transverse aortic constriction (TAC); • Cardiomyocyte-specific overexpression of SNX5 attenuates TAC-induced cardiac hypertrophy and contractile dysfunction; • SNX5 interacts with LRP6 and modulates the subcellular distribution of LRP6; • SNX5 increases LRP6 membrane translocation through directly binding with Rab11a. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most occurring neurodegenerative disorder that destroys learning, memory, and thinking skills. Although the pathophysiology of the disease is least understood, the post-mortem brain of AD patients as well as animal models revealed the part of down regulated Wnt signalling in progression of the disease. The deficit in the Wnt signalling leads to the accumulation of amyloid beta peptides, phosphorylation of tau proteins, and synaptic dysfunctions, which are regarded as the major pathological features of AD. As the available drugs for AD are only able to mitigate the symptoms and are also associated with several side effects, the therapeutic potential of the bioactive compounds is being explored for their efficacies in managing the major pathologies. Consequently, a few bioactive compounds fundamentally isolated from Garcinia species are established as promising neuroprotective agents in AD, however; their potential to regulate the Wnt signalling pathway is yet to be discovered. Considering the neuroprotective properties, in the present study efficiency of six small bioactive compounds viz., amentoflavone, isovitexin, orientin, apigenin, kaempferol, and garcinol have been investigated in modulating the receptor proteins (LRP6, DKK1, WIF1 and GSK3β) of the Wnt signalling pathway by molecular docking technique. While all the bioactive compounds could efficiently interact with the target proteins, amentoflavone, orientin, and isovitexin interact with all the target proteins viz., LRP6, DKK1, WIF1, and GSK3β with higher free energy of binding, more number of interactions, and similar mode of binding in comparison to their known or reported modulators. Thus, the present study set forth the investigated small bioactive molecules as potential drug candidates in AD therapeutics. [ABSTRACT FROM AUTHOR]

Low‐density lipoprotein receptor‐related protein 6 (LRP6) is a co‐receptor of the Wnt signaling pathway, which plays an essential role in various biological activities during embryonic and postnatal development. LRP6 is exceptionally associated with rare diseases and always with autosomal dominant inheritance. Here we report a familial phenotype of high bone mass associated with skeletal anomalies and oligodontia but also persistent left superior vena cava, inguinal hernia, hepatic cysts, abnormal posterior fossa and genital malformations. Molecular analysis revealed a novel heterozygous variant, NM_002336.2: c.724T>C, p.(Trp242Arg), in affected individuals. This variant is located in the first β‐propellant motif of LRP6, to which sclerostin (SOST) and dickkopf1 (DKK1), two LRP6 co‐receptor inhibitors and various Wnt ligands bind. According to the literature and integrating data from structural analysis, this variant distorts the binding of SOST and DKK1, thus leading to overactivation of Wnt signaling pathways involved in osteoblast differentiation. This novel heterozygous variant in LRP6 underlies the role of LRP6 in skeletal and dental disorders as well as, probably, cardiac, cerebral and genital developments. [ABSTRACT FROM AUTHOR]

BackgroundLong non-coding RNAs (lncRNAs) have critical functions within esophageal squamous cell carcinoma (ESCC). However, the function and mechanism underlying ESCC-associated lncRNA-1 (ESCCAL-1) in ESCC tumorigenesis have not been well clarified.MethodsESCCAL-1, miR-590 and LRP6 were quantified using qRT-PCR. Cell viability, migration and invasion abilities were measured using CCK-8 assay and transwell assays. The protein pression was determined with western blot assay. The xenograft model assays were used to examine the impact of ESCCAL-1 on tumorigenic effect in vivo. Direct relationships among ESCCAL-1, miR-590 and LRP6 were confirmed using dual-luciferase reporter assays.ResultsThe present work discovered the ESCCAL-1 up-regulation within ESCC. Furthermore, ESCCAL-1 was found to interact with miR-590 and consequently restrict its expression. Functionally, knocking down ESCCAL-1 or over-expressing miR-590 hindered ESCC cell growth, invasion, and migration in vitro. Moreover, inhibition of miR-590 could reverse the effect of knockdown of ESCCAL-1 on cells. Importantly, it was confirmed that LRP6 was miR-590's downstream target and LRP6 over-expression also partly abolished the role of miR-590 overexpression in ESCC cells.ConclusionWe have uncovered a novel regulatory network comprising aberrant interaction of ESCCAL-1/miR-590/LRP6 participated in ESCC progression.

Abstract R-spondins (RSPOs) are secreted signaling molecules that potentiate the Wnt/β-catenin pathway by cooperating with Wnt ligands. RSPO1 is crucial in tissue development and tissue homeostasis. However, the molecular mechanism by which RSPOs activate Wnt/β-catenin signaling remains elusive. In this study, we found that RSPOs could mediate the degradation of Axin through the ubiquitin–proteasome pathway. The results of Co-IP showed that the recombinant RSPO1 protein promoted the interaction between Axin1 and CK1ε. Either knockout of the CK1ε gene or treatment with the CK1δ/CK1ε inhibitor SR3029 caused an increase in Axin1 protein levels and attenuated RSPO1-induced degradation of the Axin1 protein. Moreover, we observed an increase in the number of associations of LRP6 with CK1ε and Axin1 following RSPO1 stimulation. Overexpression of LRP6 further potentiated Axin1 degradation mediated by RSPO1 or CK1ε. In addition, recombinant RSPO1 and Wnt3A proteins synergistically downregulated the protein expression of Axin1 and enhanced the transcriptional activity of the SuperTOPFlash reporter. Taken together, these results uncover the novel mechanism by which RSPOs activate Wnt/β-catenin signaling through LRP6/CK1ε-mediated degradation of Axin. Graphical Abstract

R-spondins (RSPOs) are secreted signaling molecules that potentiate the Wnt/β-catenin pathway by cooperating with Wnt ligands. RSPO1 is crucial in tissue development and tissue homeostasis. However, the molecular mechanism by which RSPOs activate Wnt/β-catenin signaling remains elusive. In this study, we found that RSPOs could mediate the degradation of Axin through the ubiquitin–proteasome pathway. The results of Co-IP showed that the recombinant RSPO1 protein promoted the interaction between Axin1 and CK1ε. Either knockout of the CK1ε gene or treatment with the CK1δ/CK1ε inhibitor SR3029 caused an increase in Axin1 protein levels and attenuated RSPO1-induced degradation of the Axin1 protein. Moreover, we observed an increase in the number of associations of LRP6 with CK1ε and Axin1 following RSPO1 stimulation. Overexpression of LRP6 further potentiated Axin1 degradation mediated by RSPO1 or CK1ε. In addition, recombinant RSPO1 and Wnt3A proteins synergistically downregulated the protein expression of Axin1 and enhanced the transcriptional activity of the SuperTOPFlash reporter. Taken together, these results uncover the novel mechanism by which RSPOs activate Wnt/β-catenin signaling through LRP6/CK1ε-mediated degradation of Axin. [ABSTRACT FROM AUTHOR]

Hypertension is a heritable disease that affects one-fourth of the population and accounts for about 50% of cardiovascular deaths. The genetic basis of hypertension is multifaceted, involving both monogenic and most commonly complex polygenic forms. With the advent of the human genome project, genome-wide association studies (GWAS) have identified a plethora of loci linked to hypertension by examining common genetic variations. It's notable, however, that the majority of these genetic variants do not affect the protein-coding sequences, posing a considerable obstacle in pinpointing the actual genes responsible for hypertension. Despite these challenges, precise mapping of GWAS-identified loci is emerging as a promising strategy to reveal novel genes and potential targets for the pharmacological management of blood pressure. This review provides insight into the monogenic and polygenic causes of hypertension. Special attention is given to PRDM6, among the earliest functionally characterized GWAS-identified genes. Moreover, this review delves into the roles of genes contributing to renal and vascular forms of hypertension, offering insights into their genetic and epigenetic mechanisms of action. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a frequent malignancy with a poor prognosis and a high fatality rate, unlike other malignancies. Structural improvements have been developed to better comprehend the binding of small-molecule inhibitors, reducing the side effects of chemotherapy medications. A significant functional group extensively present in both pharmacological medicines and natural compounds is the nitrile group; consequently, novel treatment compounds are desperately needed. This study aimed to design and synthesize novel anticancer drug candidates and then examine their anticancer activity against human cancer cell lines. The anti-proliferation of human colon cancer cells (HCT116) was examined using the MMT assay and compared to the activity of doxorubicin as chemotherapy after characterizing novel derivatives of di (2-aryl hydrazonopropane) arene by elemental analyzer, FTIR, 1H, 13C NMR, and ESI-MS. Chemoinformatic tools predicted their targets, and then molecular docking was performed to predict the binding affinity of compounds to the main receptors expressed in colon cancer: the adenosine receptor (A2AR) and the low-density lipoprotein receptor-related protein (LRP6). To form the corresponding diaryl hydrazone coupling products from the di-(2-aryl hydrazonopropane) arene derivatives, equivalent amounts of enaminones were coupled with diazotized aniline derivatives. Compound 11b, containing a benzonitrile moiety, was the most potent against HCT116 cells (IC50 of 0.098 ± 0.03 μM) and had a greater binding affinity to both A2AR and LRP6 of −12.43 and −16.45 kcal/mol, respectively. Our results demonstrated that the dibenzonitrile moiety is a good part of compounds that are candidates as anticancer drugs against HCT116 cells. [ABSTRACT FROM AUTHOR]

Targeting specific molecular drivers of tumor growth is a key approach in cancer therapy. Among these targets, the low-density lipoprotein receptor-related protein 6 (LRP6), a vital component of the Wnt signaling pathway, has emerged as an intriguing candidate. As a cell-surface receptor and vital co-receptor, LRP6 is frequently overexpressed in various cancer types, implicating its pivotal role in driving tumor progression. The pursuit of LRP6 as a target for cancer treatment has gained substantial traction, offering a promising avenue for therapeutic intervention. Here, this comprehensive review explores recent breakthroughs in our understanding of LRP6's functions and underlying molecular mechanisms, providing a profound discussion of its involvement in cancer pathogenesis and drug resistance. Importantly, we go beyond discussing LRP6's role in cancer by discussing diverse potential therapeutic approaches targeting this enigmatic protein. These approaches encompass a wide spectrum, including pharmacological agents, natural compounds, non-coding RNAs, epigenetic factors, proteins, and peptides that modulate LRP6 expression or disrupt its interactions. In addition, also discussed the challenges associated with developing LRP6 inhibitors and their advantages over Wnt inhibitors, as well as the drugs that have entered phase II clinical trials. By shedding light on these innovative strategies, we aim to underscore LRP6’s significance as a valuable and multifaceted target for cancer treatment, igniting enthusiasm for further research and facilitating translation into clinical applications.

Abstract Metastasis has emerged as a major impediment to achieve successful therapeutic outcomes in hepatocellular carcinoma (HCC). Nonetheless, the intricate molecular mechanisms governing the progression of HCC remain elusive. Herein, we present evidence highlighting the influence exerted by insulin-like growth factor-binding protein 2 (IGFBP2) as a potent oncogene driving the malignant phenotype. Our investigation reveals a marked elevation of IGFBP2 expression in primary tumors, concomitant with the presence of mesenchymal biomarkers in HCC. Through in vitro and in vivo experimentation, we demonstrate that the overexpression of IGFBP2 expedites the progression of epithelial-mesenchymal transition (EMT) and facilitates the metastatic potential of HCC cells, chiefly mediated by the Wnt/β-catenin signaling pathway. Notably, knockdown of IGFBP2 significantly decreased the expression of total and nuclear β-catenin, N-cadherin and vimentin in the treatment of the specific activator of Wnt/β-catenin CHIR-99021. Collectively, our findings identify IGFBP2 as a pivotal regulator within the HCC EMT axis, whereby its overexpression confers the distinctly aggressive clinical features characteristic of the disease.

Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer. Circular RNAs (circRNAs) play a vital role in cancer development and progression. This study investigated the role and potential mechanism of circRNA filamin binding LIM protein 1 (circFBLIM1) in HCC. Methods: Exosomes were identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot assay. The levels of circFBLIM1, miR-338, and low-density lipoprotein receptor-related protein 6 (LRP6) were measured by quantitative real-time polymerase chain reaction or Western blot. Glycolysis was analyzed by detecting glucose consumption, lactate production, ATP level, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR). Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis was detected by flow cytometry. Xenograft assay was performed to analyze tumor growth in vivo. The interaction among circFBLIM1, miR-338, and LRP6 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. This study was approved by the Institutional Review Board of the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. Results: CircFBLIM1 was highly expressed in HCC serum exosomes and HCC cells. Inhibition of circFBLIM1 confined HCC glycolysis and progression. CircFBLIM1 knockdown blocked tumorigenesis in vivo. CircFBLIM1 was a sponge of miR-338 and promoted HCC progression and glycolysis by regulating miR-338. Moreover, miR-338 suppressed HCC progression and glycolysis via targeting LRP6. Mechanistically, circFBLIM1 functioned as an miR-338 sponge to upregulate LRP6. Conclusion: CircFBLIM1 facilitated HCC progression and glycolysis via modulating the miR-338/LRP6 axis, which may provide promising therapeutic targets for HCC. [ABSTRACT FROM AUTHOR]

Metastasis has emerged as a major impediment to achieve successful therapeutic outcomes in hepatocellular carcinoma (HCC). Nonetheless, the intricate molecular mechanisms governing the progression of HCC remain elusive. Herein, we present evidence highlighting the influence exerted by insulin-like growth factor-binding protein 2 (IGFBP2) as a potent oncogene driving the malignant phenotype. Our investigation reveals a marked elevation of IGFBP2 expression in primary tumors, concomitant with the presence of mesenchymal biomarkers in HCC. Through in vitro and in vivo experimentation, we demonstrate that the overexpression of IGFBP2 expedites the progression of epithelial-mesenchymal transition (EMT) and facilitates the metastatic potential of HCC cells, chiefly mediated by the Wnt/β-catenin signaling pathway. Notably, knockdown of IGFBP2 significantly decreased the expression of total and nuclear β-catenin, N-cadherin and vimentin in the treatment of the specific activator of Wnt/β-catenin CHIR-99021. Collectively, our findings identify IGFBP2 as a pivotal regulator within the HCC EMT axis, whereby its overexpression confers the distinctly aggressive clinical features characteristic of the disease. [ABSTRACT FROM AUTHOR]

Abstract Background Mechanotransduction mechanisms whereby periodontal ligament stem cells (PDLSCs) translate mechanical stress into biochemical signals and thereby trigger osteogenic programs necessary for alveolar bone remodeling are being deciphered. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt transmembrane receptor, has been qualified as a key monitor for mechanical cues. However, the role of LRP6 in the mechanotransduction of mechanically induced PDLSCs remains obscure. Methods The Tension System and tooth movement model were established to determine the expression profile of LRP6. The loss-of-function assay was used to investigate the role of LRP6 on force-regulated osteogenic commitment in PDLSCs. The ability of osteogenic differentiation and proliferation was estimated by alkaline phosphatase (ALP) staining, ALP activity assay, western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. Crystalline violet staining was used to visualize cell morphological change. Western blotting, qRT-PCR, and phalloidin staining were adopted to affirm filamentous actin (F-actin) alteration. YAP nucleoplasmic localization was assessed by immunofluorescence and western blotting. YAP transcriptional response was evaluated by qRT-PCR. Cytochalasin D was used to determine the effects of F-actin on osteogenic commitment and YAP switch behavior in mechanically induced PDLSCs. Results LRP6 was robustly activated in mechanically induced PDLSCs and PDL tissues. LRP6 deficiency impeded force-dependent osteogenic differentiation and proliferation in PDLSCs. Intriguingly, LRP6 loss caused cell morphological aberration, F-actin dynamics disruption, YAP nucleoplasmic relocation, and subsequent YAP inactivation. Moreover, disrupted F-actin dynamics inhibited osteogenic differentiation, proliferation, YAP nuclear translocation, and YAP activation in mechanically induced PDLSCs. Conclusions We identified that LRP6 in PDLSCs acted as the mechanosensor regulating mechanical stress-inducible osteogenic commitment via the F-actin/YAP cascade. Targeting LRP6 for controlling alveolar bone remodeling may be a prospective therapy to attenuate relapse of orthodontic treatment.

Background & Aims: Glycoprotein (GP)96 is an endoplasmic reticulum–resident master chaperone for cell surface receptors including the Wnt co-receptors low-density lipoprotein-receptor–related protein 5/6. Intestinal epithelial cell (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche is unknown. Here, we investigated how GP96 loss interferes with intestinal homeostasis by compromising viability, proliferation, and differentiation of IECs. Methods: Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 (Cre recombinase-Estrogen-Receptor Transgene 2) mice and intestinal organoids. With H&E and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC types. Real-time polymerase chain reaction and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. Results: IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by complete eradication of the epithelial layer and death within a few days. These effects were owing to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of endoplasmic reticulum stress. Although effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. Conclusions: Our work shows that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and therefore is indispensable for maintaining intestinal epithelial homeostasis.

Abstract Background Long non‐coding RNAs (lncRNAs) have critical functions within esophageal squamous cell carcinoma (ESCC). However, the function and mechanism underlying ESCC‐associated lncRNA‐1 (ESCCAL‐1) in ESCC tumorigenesis have not been well clarified. Methods ESCCAL‐1, miR‐590 and LRP6 were quantified using qRT‐PCR. Cell viability, migration and invasion abilities were measured using CCK‐8 assay and transwell assays. The protein pression was determined with western blot assay. The xenograft model assays were used to examine the impact of ESCCAL‐1 on tumorigenic effect in vivo. Direct relationships among ESCCAL‐1, miR‐590 and LRP6 were confirmed using dual‐luciferase reporter assays. Results The present work discovered the ESCCAL‐1 up‐regulation within ESCC. Furthermore, ESCCAL‐1 was found to interact with miR‐590 and consequently restrict its expression. Functionally, knocking down ESCCAL‐1 or over‐expressing miR‐590 hindered ESCC cell growth, invasion, and migration in vitro. Moreover, inhibition of miR‐590 could reverse the effect of knockdown of ESCCAL‐1 on cells. Importantly, it was confirmed that LRP6 was miR‐590’s downstream target and LRP6 over‐expression also partly abolished the role of miR‐590 overexpression in ESCC cells. Conclusion We have uncovered a novel regulatory network comprising aberrant interaction of ESCCAL‐1/miR‐590/LRP6 participated in ESCC progression.

The membrane‐tethered protease Tiki antagonizes Wnt3a signaling by cleaving and inactivating Wnt3a in Wnt‐producing cells. Tiki also functions in Wnt‐receiving cells to antagonize Wnt signaling by an unknown mechanism. Here, we demonstrate that Tiki inhibition of Wnt signaling at the cell surface requires Frizzled (FZD) receptors. Tiki associates with the Wnt‐FZD complex and cleaves the N‐terminus of Wnt3a or Wnt5a, preventing the Wnt‐FZD complex from recruiting and activating the coreceptor LRP6 or ROR1/2 without affecting Wnt‐FZD complex stability. Intriguingly, we demonstrate that the N‐terminus of Wnt3a is required for Wnt3a binding to LRP6 and activating β‐catenin signaling, while the N‐terminus of Wnt5a is dispensable for recruiting and phosphorylating ROR1/2. Both Tiki enzymatic activity and its association with the Wnt‐FZD complex contribute to its inhibitory function on Wnt5a. Our study uncovers the mechanism by which Tiki antagonizes Wnt signaling at the cell surface and reveals a negative role of FZDs in Wnt signaling by acting as Tiki cofactors. Our findings also reveal an unexpected role of the Wnt3a N‐terminus in the engagement of the coreceptor LRP6. Synopsis: Tiki associates with the Wnt‐FZD complex on the cell surface, cleaves the N‐terminus of FZD‐bound Wnt3a or Wnt5a, and prevents the Wnt‐FZD complex from binding to and activating coreceptors LRP6 or ROR1/2. This study uncovers the mechanism of Tiki function in Wnt‐receiving cells. Tiki interacts with the Wnt‐FZD complex on the cell surface.Tiki cleaves the N‐terminus of FZD‐bound Wnt3a or Wnt5a.Tiki prevents the Wnt‐FZD complex from binding to and activating coreceptors LRP6 or ROR1/2.The N‐terminus of Wnt3a is required for interacting with LRP6. [ABSTRACT FROM AUTHOR]

An imbalance between bone resorption and bone formation underlies the devastating osteolytic lesions and subsequent fractures seen in more than 90% of multiple myeloma (MM) patients. Currently, Wnt-targeted therapeutic agents that prevent soluble antagonists of the Wnt signaling pathway, sclerostin (SOST) and dickkopf-1 (DKK1), have been shown to prevent bone loss and improve bone strength in preclinical models of MM. In this study, we show increasing Wnt signaling via a novel anti--low-density lipoprotein receptor-related protein 6 (LRP6) antibody, which potentiates Wnt1-class ligand signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. When combined with an agent targeting the soluble Wnt antagonist DKK1, we showed more robust improvements in bone structure than anti-LRP6 treatment alone. Micro--computed tomography (μCT) analysis demonstrated substantial increases in trabecular bone volume in naïve mice given the anti-LRP6/DKK1 combination treatment strategy compared to control agents. Mice injected with 5TGM1eGFP murine myeloma cells had significant reductions in trabecular bone volume compared to naïve controls. The anti-LRP6/ DKK1 combination strategy significantly improved bone volume in 5TGM1-bearing mice by 111%, which was also superior to anti- LRP6 single treatment; with similar bone structural changes observed within L4 lumbar vertebrae. Consequently, this combination strategy significantly improved resistance to fracture in lumbar vertebrae in 5TGM1-bearing mice compared to their controls, providing greater protection against fracture compared to anti-LRP6 antibody alone. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1-bearingmice treated with the anti-LRP6/DKK1 combination strategy. Importantly,Wnt stimulation with either single or combined Wnt-targeted agents did not exacerbate tumor activity. This work provides a novel approach of targeting both membrane-bound and soluble Wnt pathway components to provide superior skeletal outcomes in patients with multiple myeloma and other bone destructive cancers. [ABSTRACT FROM AUTHOR]

Low-density lipoprotein receptor-related protein 6 (LRP6), a member of the low-density lipoprotein receptor (LDLR) family, displays a unique structure and ligand-binding function. As a co-receptor of the Wnt/β-catenin signaling pathway, LRP6 is a novel therapeutic target that plays an important role in the regulation of cardiovascular disease, lipid metabolism, tumorigenesis, and some classical signals. By using capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE-SELEX), with recombinant human LRP-6 as the target, four candidate aptamers with a stem-loop structure were selected from an ssDNA library—AptLRP6-A1, AptLRP6-A2, AptLRP6-A3, and AptLRP6-A4. The equilibrium dissociation constant KD values between these aptamers and the LRP6 protein were in the range of 0.105 to 1.279 μmol/L, as determined by CE-LIF analysis. Their affinities and specificities were further determined by the gold nanoparticle (AuNP) colorimetric method. Among them, AptLRP6-A3 showed the highest affinity with LRP6-overexpressed human breast cancer cells. Therefore, the LRP6 aptamer identified in this study constitutes a promising modality for the rapid diagnosis and treatment of LRP6-related diseases. [ABSTRACT FROM AUTHOR]

Mammalian preimplantation development depends on the interaction between embryonic autocrine and maternal paracrine signaling. Despite the robust independence of preimplantation embryos, oviductal factors are thought to be critical to pregnancy success. However, how oviductal factors regulate embryonic development and the underlying mechanism remain unknown. In the present study, focusing on WNT signaling, which has been reported to be essential for developmental reprogramming after fertilization, we analyzed the receptor-ligand repertoire of preimplantation embryonic WNT signaling, and identified that the WNT co-receptor LRP6 is necessary for early cleavage and has a prolonged effect on preimplantation development. LRP6 inhibition significantly impeded zygotic genome activation and disrupted relevant epigenetic reprogramming. Focusing on the potential oviductal WNT ligands, we found WNT2 as the candidate interacting with embryonic LRP6. More importantly, we found that WNT2 supplementation in culture medium significantly promoted zygotic genome activation (ZGA) and improved blastocyst formation and quality following in vitro fertilization (IVF). In addition, WNT2 supplementation significantly improved implantation rate and pregnancy outcomes following embryo transfer. Collectively, our findings not only provide novel insight into how maternal factors regulate preimplantation development through maternal-embryonic communication, but they also propose a promising strategy for improving current IVF systems. [ABSTRACT FROM AUTHOR]

Osteoblast Wnt/β‐catenin signaling conditions skeletal development and health. Bone formation is stimulated when on the osteoblast surface a Wnt binds to low‐density lipoprotein receptor‐related protein 5 (LRP5) or 6 (LRP6), in turn coupled to a frizzled receptor. Sclerostin and dickkopf1 inhibit osteogenesis if either links selectively to the first β‐propeller of LRP5 or LRP6, thereby disassociating these cognate co‐receptors from the frizzled receptor. Sixteen heterozygous mutations identified since 2002 within LRP5 and three heterozygous mutations identified since 2019 within LRP6 prevent this binding of sclerostin or dickkopf1 and account for the exceptionally rare, but highly instructive, autosomal dominant disorders called LRP5 and LRP6 high bone mass (HBM). Herein, we characterize LRP6 HBM in the first large affected family. Their novel heterozygous LRP6 missense mutation (c.719C>T, p.Thr240Ile) was present in two middle‐aged sisters and three of their sons. They considered themselves healthy. Their broad jaw and torus palatinus developed during childhood and, contrary to the two previous reports of LRP6 HBM, the appearance of their adult dentition was unremarkable. Skeletal modeling, defined radiographically, supported classification as an endosteal hyperostosis. Areal bone mineral density (g/cm2) of the lumbar spine and total hip featured accelerated increases reaching Z‐scores of ~ +8 and +6, respectively, although biochemical markers of bone formation were normal. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Junyu Zheng, Cong Li, Zining Zhu, Fengming Yang, Xiaoming Wang, Pan Jiang, Feng Yan Department of Clinical Laboratory, The Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research, Nanjing, Jiangsu, 210009, People’s Republic of ChinaCorrespondence: Feng Yan, Department of Clinical Laboratory, The Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research, 42 Baiziting Road, Nanjing, 210009, Jiangsu, People’s Republic of China, Tel +86-13851641895, Email yanfeng@jszlyy.com.cnBackground: Gastric cancer (GC) is a common type of gastrointestinal tumor in the world. Transfer RNA (tRNA) derived fragments (tsRNAs) implicate various cancers, but their roles in GC remain unclear. Our study aimed to investigate the potential biological functions and molecular mechanisms of tsRNAs in GC.Methods: Differentially expressed tsRNAs were identified using high-throughput sequencing. The expression levels of tsRNAs were validated in 62 paired GC tissues and adjacent normal tissues using RT-qPCR. In vitro functional assays were used to evaluate the influences of tsRNAs on GC cells. The potential mechanisms underlying tsRNAs were explored using bioinformatics analysis,RT-qPCR, RNA immunoprecipitation assays and Western blot.Results: We found that tiRNA-Val-CAC-001 was downregulated in GC tissues and cells, and demonstrated that tiRNA-Val-CAC-001 was a tsRNA sheared from mature tRNA-Val and mainly localized in the cytoplasm. tiRNA-Val-CAC-001 overexpression inhibited metastasis and proliferation but promoted apoptosis of GC cells; nevertheless, tiRNA-Val-CAC-001 knockdown increased metastasis and proliferation and reduced apoptosis (P< 0.05). GO and KEGG analyses indicated tiRNA-Val-CAC-001 may exert its effects via Wnt/β-catenin signaling pathway by targeting LRP6. Following experiments showed that tiRNA-Val-CAC-001 could downregulated the protein levels of LRP6 and β-catenin, but up-regulated p-β-catenin, which confirmed the findings in bioinformatics analysis.Conclusion: In conclusion, tiRNA-Val-CAC-001 works as a cancer suppressor in GC by targeting LRP6 via Wnt/β-catenin signaling pathway. tiRNA-Val-CAC-001 may serve as a therapy target and a biomarker of GC in the future.Key Points: tiRNA-Val-CAC-001 is downregulated in gastric cancer tissues and cell lines, tiRNA-Val-CAC-001 has potential to become a novel diagnostic biomarker in gastric cancer, and tiRNA-Val-CAC-001 regulates gastric cancer cells by targeting LRP6.Keywords: gastric cancer, tRNA-derived-fragments, LRP6, metastasis, proliferation, apoptosis

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events. [ABSTRACT FROM AUTHOR]

High expression of low-density lipoprotein receptor-related protein 6 (LRP6), a key component of the Wnt/β-catenin signaling pathway, is reported to be associated with malignant potential in some solid tumors including breast cancer and hepatocellular carcinoma. Few reports, however, have examined its function and clinical significance in colorectal cancers (CRC) demonstrating constitutive activation of Wnt signaling. Here, we compared the expression level and function of LRP6 in CRC with that of esophageal squamous cell carcinoma (ESCC) bearing few Wnt/β-catenin pathway mutations. On immunohistochemical staining, high LRP6 expression was noted in three of 68 cases (4.4%), and high β-catenin in 38 of 67 cases (56.7%) of CRC. High LRP6 expression was found in 21 of 82 cases (25.6%), and high β-catenin expression in 29 of 73 cases (39.7%) of ESCC. In our in vitro studies, LRP6 knockdown hardly changed Wnt signaling activity in CRC cell lines with mutations in Wnt signaling downstream genes. In contrast, in ESCC cell lines without Wnt signaling-related mutations, LRP6 knockdown significantly decreased Wnt signaling activity. LRP6 function may depend on constitutive activation of Wnt signaling. [ABSTRACT FROM AUTHOR]

Colorectal cancer mortality rate and highly altered proteins from the Wnt/β-catenin pathway increase the scientific community's interest in finding alternatives for prevention and treatment. This study aims to determine the biological effect of chlorogenic acid (CGA) on two colorectal cancer cell lines, HT-29 and SW480, and its interactions with β-catenin and LRP6 to elucidate a possible modulatory mechanism on the Wnt/β-catenin pathway. These effects were determined by propidium iodide and DiOC6 for mitochondrial membrane permeability, MitoTracker Red for mitochondrial ROS production, DNA content for cell distribution on cell cycle phases, and molecular docking for protein–ligand interactions and binding affinity. Here, it was found that CGA at 2000 µM significantly affects cell viability and causes DNA fragmentation in SW480 cells rather than in HT-29 cells, but in both cell lines, it induces ROS production. Additionally, CGA has similar affinity and interactions for LRP6 as niclosamide but has a higher affinity for both β-catenin sites than C2 and iCRT14. These results suggest a possible modulatory role of CGA over the Wnt/β-catenin pathway in colorectal cancer. [ABSTRACT FROM AUTHOR]

Background: Mechanotransduction mechanisms whereby periodontal ligament stem cells (PDLSCs) translate mechanical stress into biochemical signals and thereby trigger osteogenic programs necessary for alveolar bone remodeling are being deciphered. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt transmembrane receptor, has been qualified as a key monitor for mechanical cues. However, the role of LRP6 in the mechanotransduction of mechanically induced PDLSCs remains obscure. Methods: The Tension System and tooth movement model were established to determine the expression profile of LRP6. The loss-of-function assay was used to investigate the role of LRP6 on force-regulated osteogenic commitment in PDLSCs. The ability of osteogenic differentiation and proliferation was estimated by alkaline phosphatase (ALP) staining, ALP activity assay, western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. Crystalline violet staining was used to visualize cell morphological change. Western blotting, qRT-PCR, and phalloidin staining were adopted to affirm filamentous actin (F-actin) alteration. YAP nucleoplasmic localization was assessed by immunofluorescence and western blotting. YAP transcriptional response was evaluated by qRT-PCR. Cytochalasin D was used to determine the effects of F-actin on osteogenic commitment and YAP switch behavior in mechanically induced PDLSCs. Results: LRP6 was robustly activated in mechanically induced PDLSCs and PDL tissues. LRP6 deficiency impeded force-dependent osteogenic differentiation and proliferation in PDLSCs. Intriguingly, LRP6 loss caused cell morphological aberration, F-actin dynamics disruption, YAP nucleoplasmic relocation, and subsequent YAP inactivation. Moreover, disrupted F-actin dynamics inhibited osteogenic differentiation, proliferation, YAP nuclear translocation, and YAP activation in mechanically induced PDLSCs. Conclusions: We identified that LRP6 in PDLSCs acted as the mechanosensor regulating mechanical stress-inducible osteogenic commitment via the F-actin/YAP cascade. Targeting LRP6 for controlling alveolar bone remodeling may be a prospective therapy to attenuate relapse of orthodontic treatment. [ABSTRACT FROM AUTHOR]

The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling.

Abstract Background Emerging evidence has correlated the human antigen R (HuR) with the low-density lipoprotein receptor-related protein 6 (LRP6) gene, an important therapeutic target for osteoporosis. Herein, we sought to probe the regulatory role of HuR in the LRP6 gene and their interaction in the progression of osteoporosis. Methods HuR and downstream potential target genes were predicted by bioinformatics analysis to identify their potential functions in bone metabolism following osteoporosis. The effect of HuR on the osteoblastic differentiation and viability and apoptosis of mouse embryo osteoblast precursor cells (MC3T3-E1) was evaluated after artificial modulation of HuR expression. Results Bone phenotypes were observed in ovariectomized mice in response to adenovirus-mediated HuR overexpression. Poor expression of HuR was identified in the bone tissues of ovariectomized mice. Silencing of HuR inhibited the osteoblastic differentiation of MC3T3-E1 cells, as evidenced by decreased expression of Runx2 and Osterix along with reduced ALP activity. Mechanistically, HuR stabilized LRP6 mRNA and promoted its translation by binding to the 3'UTR of LRP6 mRNA, leading to activation of the downstream Wnt pathway. By this mechanism, osteoblastic differentiation of MC3T3-E1 cells was induced. In ovariectomized mice, overexpression of HuR alleviated osteoporosis-related phenotypes. Conclusion Overall, these data together support the promoting role of HuR in the osteoblastic differentiation, highlighting a potential novel strategy for osteoporosis treatment.

ABSTRACT Wild-type canine distemper virus (CDV) is an important pathogen of dogs as well as wildlife that can infect immune and epithelial cells through two known receptors: the signaling lymphocytic activation molecule (SLAM) and nectin-4, respectively. Conversely, the ferret and egg-adapted CDV-Onderstepoort strain (CDV-OP) is employed as an effective vaccine for dogs. CDV-OP also exhibits promising oncolytic properties, such as its abilities to infect and kill multiple cancer cells in vitro. Interestingly, several cancer cells do not express SLAM or nectin-4, suggesting the presence of a yet unknown entry factor for CDV-OP. By conducting a genome-wide CRISPR/Cas9 knockout (KO) screen in CDV-OP-susceptible canine mammary carcinoma P114 cells, which neither express SLAM nor nectin-4, we identified low-density lipoprotein receptor-related protein 6 (LRP6) as a host factor that promotes CDV-OP infectivity. Whereas the genetic ablation of LRP6 rendered cells resistant to infection, ectopic expression in resistant LRP6KO cells restored susceptibility. Furthermore, multiple functional studies revealed that (i) the overexpression of LRP6 leads to increased cell-cell fusion, (ii) a soluble construct of the viral receptor-binding protein (solHOP) interacts with a soluble form of LRP6 (solLRP6), (iii) an H-OP point mutant that prevents interaction with solLRP6 abrogates cell entry in multiple cell lines once transferred into recombinant viral particles, and (iv) vesicular stomatitis virus (VSV) pseudotyped with CDV-OP envelope glycoproteins loses its infectivity in LRP6KO cells. Collectively, our study identified LRP6 as the long sought-after cell entry receptor of CDV-OP in multiple cell lines, which set the molecular bases to refine our understanding of viral-cell adaptation and to further investigate its oncolytic properties. IMPORTANCE Oncolytic viruses (OV) have gathered increasing interest in recent years as an alternative option to treat cancers. The Onderstepoort strain of canine distemper virus (CDV-OP), an enveloped RNA virus belonging to the genus Morbillivirus, is employed as a safe and efficient vaccine for dogs against distemper disease. Importantly, although CDV-OP can infect and kill multiple cancer cell lines, the basic mechanisms of entry remain to be elucidated, as most of those transformed cells do not express natural receptors (i.e., SLAM and nectin-4). In this study, using a genome-wide CRISPR/Cas9 knockout screen, we describe the discovery of LRP6 as a novel functional entry receptor for CDV-OP in various cancer cell lines and thereby uncover a basic mechanism of cell culture adaptation. Since LRP6 is upregulated in various cancer types, our data provide important insights in order to further investigate the oncolytic properties of CDV-OP.

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. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is a neurodegenerative disorder that causes age-dependent neurological and cognitive declines. The treatments for Alzheimer's disease pose a significant challenge, because the mechanisms of disease are not being fully understood. Malfunction of the blood–brain barrier is increasingly recognized as a major contributor to the pathophysiology of Alzheimer's disease, especially at the early stages of the disease. However, the underlying mechanisms remain poorly characterized, while few molecules can directly target and improve blood–brain barrier function in the context of Alzheimer's disease. Here, we showed dysfunctional blood–brain barrier in patients with Alzheimer's disease reflected by perivascular accumulation of blood-derived fibrinogen in the hippocampus and cortex, accompanied by decreased tight junction proteins Claudin-5 and glucose transporter Glut-1 in the brain endothelial cells. In the APPswe/PS1dE9 (APP/PS1) mouse model of Alzheimer's disease, blood–brain barrier dysfunction started at 4 months of age and became severe at 9 months of age. In the cerebral microvessels of APP/PS1 mice and amyloid-β-treated brain endothelial cells, we found suppressed Wnt/β-catenin signalling triggered by an increase of GSK3β activation, but not an inhibition of the AKT pathway or switching to the Wnt/planar cell polarity pathway. Furthermore, using our newly developed optogenetic tool for controlled regulation of LRP6 (upstream regulator of the Wnt signalling) to activate Wnt/β-catenin pathway, blood–brain barrier malfunction was restored by preventing amyloid-β-induced brain endothelial cells impairments and promoting the barrier repair. In conclusion, targeting LRP6 in the Wnt/β-catenin pathway in the brain endothelium can alleviate blood–brain barrier malfunction induced by amyloid-β, which may be a potential treatment strategy for Alzheimer's disease. [ABSTRACT FROM AUTHOR]

To keep pace with the rapid advancements in molecular genetics and rare diseases research, we have updated the list of ectodermal dysplasias based on the latest classification approach that was adopted in 2017 by an international panel of experts. For this purpose, we searched the databases PubMed and OMIM for the term "ectodermal dysplasia", referring mainly to changes in the last 5 years. We also tried to obtain information about those diseases on which the last scientific report appeared more than 15 years ago by contacting the authors of the most recent publication. A group of experts, composed of researchers who attended the 8th International Conference on Ectodermal Dysplasias and additional members of the previous classification panel, reviewed the proposed amendments and agreed on a final table listing all 49 currently known ectodermal dysplasias for which the molecular genetic basis has been clarified, including 15 new entities. A newly reported ectodermal dysplasia, linked to the gene LRP6, is described here in more detail. These ectodermal dysplasias, in the strict sense, should be distinguished from syndromes with features of ectodermal dysplasia that are related to genes extraneous to the currently known pathways involved in ectodermal development. The latter group consists of 34 syndromes which had been placed on the previous list of ectodermal dysplasias, but most if not all of them could actually be classified elsewhere. This update should streamline the classification of ectodermal dysplasias, provide guidance to the correct diagnosis of rare disease entities, and facilitate the identification of individuals who could benefit from novel treatment options. [ABSTRACT FROM AUTHOR]

Summary: We present a family with a rare mutation of the LRP6 gene and for the first time provide evidence for its association with low bone mineral density. Introduction: The Wnt pathway plays a critical role in bone homeostasis. Pathogenic variants of the Wnt co-receptor LRP6 have been associated with abnormal skeletal phenotypes or increased risk of cardiovascular events. Patient and methods: Here we report an index premenopausal patient and her family carrying a rare missense LRP6 pathogenic variant (rs141212743; 0.0002 frequency among Europeans). This variant has been previously associated with metabolic syndrome and atherosclerosis, in the presence of normal bone mineral density. However, the LRP6 variant was associated with low bone mineral density in this family, without evidence for association with serum lipid levels or cardiovascular events. Conclusion: Thus, this novel association shows that LRP6 pathogenic variants may be involved in some cases of early-onset osteoporosis, but the predominant effect, either skeletal or cardiovascular, may vary depending on the genetic background or other acquired factors. [ABSTRACT FROM AUTHOR]

Wnt signaling pathway is an evolutionarily conserved pathway responsible for neurogenesis, axon outgrowth, neuronal polarity, synapse formation, and maintenance. Downregulation of Wnt signaling has been found in patients with Alzheimer's disease (AD). Several experimental approaches to activate Wnt signaling pathway have proven to be beneficial in alleviating AD, which is one of the new therapeutic approaches for AD. The current study focuses on the computational structure-based virtual screening followed by the identification of potential phytomolecules targeting different markers of Wnt signaling like WIF1, DKK1, LRP6, GSK-3β, and acetylcholine esterase. Initially, screening of 1924 compounds from the plant-based library of Zinc database was done for the selected five proteins using docking approach followed by MM-GBSA calculations. The top five hit molecules were identified for each protein. Based on docking score, and binding interactions, the top two hit molecules for each protein were selected as promising molecules for the molecular dynamic (MD) simulation study with the five proteins. Therefore, from this in silico based study, we report that Mangiferin could be a potential molecule targeting Wnt signaling pathway modulating the LRP6 activity, Baicalin for AChE activity, Chebulic acid for DKK1, ZINC103539689 for WIF1, and Morin for GSk-3β protein. However, further validation of the activity is warranted based on in vivo and in vitro experiments for better understanding and strong claim. This study provides an in silico approach for the identification of modulators of the Wnt signaling pathway as a new therapeutic approach for AD. [ABSTRACT FROM AUTHOR]

Luqi Dai,1,2,&ast; Dan Xu,1,&ast; Chun Wan,1,2 Lian Liu,1 Fuqiang Wen1,2 1Division of Pulmonary Diseases, State Key Laboratory of Biotherapy, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Fuqiang Wen; Lian LiuDepartment of Respiratory and Critical Care Medicine, West China Hospital of Sichuan University, and Division of Pulmonary Diseases, State Key Laboratory of Biotherapy, Guoxuexiang 37 Road, Chengdu, Sichuan, 610041, People’s Republic of ChinaTel +86 28 8542 2350Fax +86 28 8558 2944Email wenfuqiang@scu.edu.cn; desertfoxliu@hotmail.comPurpose: WNT/β-catenin signal pathway is a potential hope for lung tissue repair. We investigated the levels of Dickkopf‐1 (DKK1), an endogenous inhibitor of WNT/β-catenin signal pathway, in chronic obstructive pulmonary disease (COPD) patients and airway inflammation.Patients and Methods: Collected the demographic and clinical characteristics of 36 healthy controls, 25 stable COPD patients and 10 acute exacerbation of COPD (AECOPD) patients, then performed pulmonary function and detected serum DKK1 levels. After over-expression of DKK1, detect the levels of DDK1, lipoprotein-related protein 6 (LRP6) and inflammatory factors in bronchial epithelial cells stimulated with cigarette smoke extract (CSE).Results: Serum DKK1 were reduced in stable COPD patients compared to healthy controls (3866.72 ± 775.33 pg/mL vs 5317.61 ± 1317.20 pg/mL, p< 0.0001), but there was no significant difference between stable and acutely exacerbated patients (3866.72 ± 775.33 pg/mL vs 3482.10 ± 841.25 pg/mL, p> 0.05). DKK1 was positively correlated with FEV1 (r = 0.570, p< 0.0001), FEV1/FVC (rho = 0.590, p< 0.0001), FEV1/Pre (r = 0.517, p< 0.0001). Multiple linear regression analysis also suggested that FEV1 levels were higher with increasing DKK1. In vitro, elevated IL-6, IL-8, TNF-α and decreased DKK1, LRP6 were found in Beas-2B cells after CSE treatments, and increased LRP6 and decreased inflammatory factors were found after overexpression of DKK1. Andrographolide restored the CSE-induced decrease in DKK1 and increase in IL-6 and IL-8.Conclusion: DKK1 levels were decreased in COPD patients and positively correlated with lung function, overexpression of DKK1 and andrographolide attenuated airway cell inflammation, both suggesting a potential role in pathophysiology and providing a disease-specific biomarker pattern.Keywords: LRP6, lung function, biomarker, andrographolide

Intrauterine growth restriction combined with postnatal accelerated growth (CG-IUGR) could lead to long-term detrimental metabolic outcomes characte rized by insulin resistance. As an indispensable co-receptor of Wnt signaling, L RP6 plays a critical role in the susceptibility of metabolic disorders. However, whether LRP6 is involved in the metabolic programing is still unknown. We hypothesized that CG- IUGR programed impaired insulin sensitivity through the impaired LRP6-mediated Wnt signaling in skeletal muscle. A CG-IUGR rat model was employed. The transcriptional a nd translational alterations of the components of the Wnt and the insulin signaling in the skeletal muscle of the male CG-IUGR rats were determined. The role of LRP6 on t he insulin signaling was evaluated by shRNA knockdown or Wnt3a stimulation of LRP6. Comp ared with controls, the male CG-IUGR rats showed an insulin-resistant phenotype, wi th impaired insulin signaling and decreased expression of LRP6/β-catenin in skeletal muscle. LRP6 knockdown led to reduced expression of the IR-β/IRS-1 in C2C12 cell line, while Wnt3a-mediated LRP6 expression increased the expression of IRS-1 and IGF-1R but not IR-β in the primary muscle cells of male CG-IUGR rats. The impaired LRP6/β-catenin/IGF-1R/IRS-1 signaling is probably one of the critical mechanisms underlying the programed impaired insulin sensitivity in male CG-IUGR.

The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/β-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/β-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/β-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic β-catenin. Moreover, TPA increased the association of β-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/β-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/β-catenin signaling.

The process of tooth formation is a series of reciprocal interactions between the ectoderm and mesoderm, and it is believed that many genetic factors are involved in this complex process. More than a dozen genes have been identified in non-syndromic tooth agenesis; however, the genetic etiology underlying tooth agenesis is not fully understood yet. In this study, we identified two novel LRP6 mutations in two non-syndromic oligodontia families. Both probands had 16 and 17 missing teeth in their permanent dentition. Mutational analysis identified a de novo frameshift mutation by a 1-bp insertion in exon 9 (NM_002336.2: c.1870dupA, p.(Met624Asnfs*29)) and a splicing donor site mutation in intron 8 (c.1762+2T>C). An in vitro splicing assay confirmed the deletion of exon 8, and the deletion would result in a frameshift. Due to the premature termination codons introduced by the frameshift, both mutant transcripts would be degraded by nonsense-mediated mRNA decay, resulting in haploinsufficiency. [ABSTRACT FROM AUTHOR]

Background: Calcium oxalate (CaOx) is the most common type of kidney stone, but the mechanism of CaOx stones formation remains unclear. The injury of renal cells such as ferroptosis and autophagy has been considered a basis for stones formation. Methods: We conducted transmission electron microscope (TEM), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and C11-BODIPY analysis to explore whether CaOx could induce autophagy-dependent ferroptosis in vivo and in vitro. To explore the possible mechanism, we conducted bioinformatic analysis of patients with or without CaOx stones, Western blot and qPCR were used to identify the different genes we found in bioinformatic analysis. Results: In our study, we found that CaOx could induce autophagy-dependent ferroptosis no matter in vivo or in vitro, which might finally lead to urolithiasis. Bioinformatic analysis of the GSE73680 dataset indicated that the expression of caveolin-1 (CAV1) was higher in control patients than CaOx stone patients, the STRING database indicated that CAV1 might interact with low density lipoprotein receptro-related protein 6 (LRP6), Gene Set Enrichment Analysis (GSEA) showed that the WNT pathway positively associated with the control group while negatively related to the stone group, and LRP6 was the core gene of the WNT pathway. Western blot found that CAV1, LRP6, and Wnt/ß-Catenin were decreased in Human Kidney2 (HK2) cells stimulated with CaOx. Furthermore, the WNT pathway was considered to be involved in autophagy and ferroptosis. Conclusions: We presumed that CAV1 could ameliorate autophagy-dependent ferroptosis through the LRP6/Wnt/ß-Catenin axis, and finally alleviate CaOx stone formation. [ABSTRACT FROM AUTHOR]

Angiopoietin-like 4 (ANGPTL4) is a secreted signaling protein that is implicated in cardiovascular disease, metabolic disorder, and cancer. Outside of its role in lipid metabolism, ANGPTL4 signaling remains poorly understood. Here, we identify ANGPTL4 as a Wnt signaling antagonist that binds to syndecans and forms a ternary complex with the Wnt co-receptor Lipoprotein receptor-related protein 6 (LRP6). This protein complex is internalized via clathrin-mediated endocytosis and degraded in lysosomes, leading to attenuation of Wnt/β-catenin signaling. Angptl4 is expressed in the Spemann organizer of Xenopus embryos and acts as a Wnt antagonist to promote notochord formation and prevent muscle differentiation. This unexpected function of ANGPTL4 invites re-interpretation of its diverse physiological effects in light of Wnt signaling and may open therapeutic avenues for human disease.

Low-density lipoprotein receptor-related protein 6 (LRP6), a member of the low-density lipoprotein receptor (LDLR) family, displays a unique structure and ligand-binding function. As a co-receptor of the Wnt/β-catenin signaling pathway, LRP6 is a novel therapeutic target that plays an important role in the regulation of cardiovascular disease, lipid metabolism, tumorigenesis, and some classical signals. By using capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE-SELEX), with recombinant human LRP-6 as the target, four candidate aptamers with a stem-loop structure were selected from an ssDNA library—AptLRP6-A1, AptLRP6-A2, AptLRP6-A3, and AptLRP6-A4. The equilibrium dissociation constant KD values between these aptamers and the LRP6 protein were in the range of 0.105 to 1.279 μmol/L, as determined by CE-LIF analysis. Their affinities and specificities were further determined by the gold nanoparticle (AuNP) colorimetric method. Among them, AptLRP6-A3 showed the highest affinity with LRP6-overexpressed human breast cancer cells. Therefore, the LRP6 aptamer identified in this study constitutes a promising modality for the rapid diagnosis and treatment of LRP6-related diseases.

Mammalian preimplantation development depends on the interaction between embryonic autocrine and maternal paracrine signaling. Despite the robust independence of preimplantation embryos, oviductal factors are thought to be critical to pregnancy success. However, how oviductal factors regulate embryonic development and the underlying mechanism remain unknown. In the present study, focusing on WNT signaling, which has been reported to be essential for developmental reprogramming after fertilization, we analyzed the receptor-ligand repertoire of preimplantation embryonic WNT signaling, and identified that the WNT co-receptor LRP6 is necessary for early cleavage and has a prolonged effect on preimplantation development. LRP6 inhibition significantly impeded zygotic genome activation and disrupted relevant epigenetic reprogramming. Focusing on the potential oviductal WNT ligands, we found WNT2 as the candidate interacting with embryonic LRP6. More importantly, we found that WNT2 supplementation in culture medium significantly promoted zygotic genome activation (ZGA) and improved blastocyst formation and quality following in vitro fertilization (IVF). In addition, WNT2 supplementation significantly improved implantation rate and pregnancy outcomes following embryo transfer. Collectively, our findings not only provide novel insight into how maternal factors regulate preimplantation development through maternal-embryonic communication, but they also propose a promising strategy for improving current IVF systems.

Sclerostin, a glycoprotein encoded by the SOST gene, is mainly produced by mature osteocytes and is a critical regulator of bone formation through its inhibitory effect on Wnt signaling. Osteocytes are differentiated osteoblasts that form a vast and highly complex communication network and orchestrate osteogenesis in response to both mechanical and hormonal cues. The three most commonly described pathways of SOST gene regulation are mechanotransduction, Wnt/β-catenin, and steroid signaling. Downregulation of SOST and thereby upregulation of local Wnt signaling is required for the osteogenic response to mechanical loading. This review covers recent findings concerning the identification of SOST, in vitro regulation of SOST gene expression, structural and functional properties of sclerostin, pathophysiology, biological variability, and recent assay developments for measuring circulating sclerostin. The three-dimensional structure of human sclerostin was generated with the AlphaFold Protein Structure Database applying a novel deep learning algorithm based on the amino acid sequence. The functional properties of the 3-loop conformation within the tertiary structure of sclerostin and molecular interaction with low-density lipoprotein receptor-related protein 6 (LRP6) are also reviewed. Second-generation immunoassays for intact/biointact sclerostin have recently been developed, which might overcome some of the reported methodological obstacles. Sclerostin assay standardization would be a long-term objective to overcome some of the problems with assay discrepancies. Besides the use of age- and sex-specific reference intervals for sclerostin, it is also pivotal to use assay-specific reference intervals since available immunoassays vary widely in their methodological characteristics. [ABSTRACT FROM AUTHOR]

Prodigiosin, a natural red pigment produced by numerous bacterial species, has exhibited promising anticancer activity; however, the molecular mechanisms of action of prodigiosin on malignant cells remain unclear. Aberrant activation of the Wnt/β-catenin signaling cascade is associated with numerous human cancers. In this study, we identified prodigiosin as a potent inhibitor of the Wnt/β-catenin pathway. Prodigiosin blocked Wnt/β-catenin signaling by targeting multiple sites of this pathway, including the low-density lipoprotein-receptor-related protein (LRP) 6, Dishevelled (DVL), and glycogen synthase kinase-3β (GSK3β). In breast cancer MDA-MB-231 and MDA-MB-468 cells, nanomolar concentrations of prodigiosin decreased phosphorylation of LRP6, DVL2, and GSK3β and suppressed β-catenin-stimulated Wnt target gene expression, including expression of cyclin D1. In MDA-MB-231 breast cancer xenografts and MMTV-Wnt1 transgenic mice, administration of prodigiosin slowed tumor progression and reduced the expression of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, active β-catenin, and cyclin D1. Through its ability to inhibit Wnt/β-catenin signaling and reduce cyclin D1 levels, prodigiosin could have therapeutic activity in advanced breast cancers.

Neural tube defects (NTDs) are among the common and severe birth defects with poorly understood etiology. Mutations in the Wnt co-receptor LRP6 are associated with NTDs in humans. Either gain-of-function (GOF) or loss-of-function (LOF) mutations of Lrp6 can cause NTDs in mice. NTDs in Lrp6-GOF mutants may be attributed to altered β-catenin-independent noncanonical Wnt signaling. However, the mechanisms underlying NTDs in Lrp6-LOF mutants and the role of Lrp6-mediated canonical Wnt/β-catenin signaling in neural tube closure remain unresolved. We previously demonstrated that β-catenin signaling is required for posterior neuropore (PNP) closure. In the current study, conditional ablation of Lrp6 in dorsal PNP caused spinal NTDs with diminished activities of Wnt/β-catenin signaling and its downstream target gene Pax3, which is required for PNP closure. β-catenin-GOF rescued NTDs in Lrp6-LOF mutants. Moreover, maternal supplementation of a Wnt/β-catenin signaling agonist reduced the frequency and severity of spinal NTDs in Lrp6-LOF mutants by restoring Pax3 expression. Together, these results demonstrate the essential role of Lrp6-mediated Wnt/β-catenin signaling in PNP closure, which could also provide a therapeutic target for NTD intervention through manipulation of canonical Wnt/β-catenin signaling activities.

Targeting specific molecular drivers of tumor growth is a key approach in cancer therapy. Among these targets, the low-density lipoprotein receptor-related protein 6 (LRP6), a vital component of the Wnt signaling pathway, has emerged as an intriguing candidate. As a cell-surface receptor and vital co-receptor, LRP6 is frequently overexpressed in various cancer types, implicating its pivotal role in driving tumor progression. The pursuit of LRP6 as a target for cancer treatment has gained substantial traction, offering a promising avenue for therapeutic intervention. Here, this comprehensive review explores recent breakthroughs in our understanding of LRP6's functions and underlying molecular mechanisms, providing a profound discussion of its involvement in cancer pathogenesis and drug resistance. Importantly, we go beyond discussing LRP6's role in cancer by discussing diverse potential therapeutic approaches targeting this enigmatic protein. These approaches encompass a wide spectrum, including pharmacological agents, natural compounds, non-coding RNAs, epigenetic factors, proteins, and peptides that modulate LRP6 expression or disrupt its interactions. In addition, also discussed the challenges associated with developing LRP6 inhibitors and their advantages over Wnt inhibitors, as well as the drugs that have entered phase II clinical trials. By shedding light on these innovative strategies, we aim to underscore LRP6's significance as a valuable and multifaceted target for cancer treatment, igniting enthusiasm for further research and facilitating translation into clinical applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Obesity has long been thought to be a main cause of hyperlipidemia. As a systemic disease, the impact of obesity on organs, tissues and cells is almost entirely negative. However, the relationship between obesity and bone loss is highly controversial. On the one hand, obesity has long been thought to have a positive effect on bone due to increased mechanical loading on the skeleton, conducive to increasing bone mass to accommodate the extra weight. On the other hand, obesity-related metabolic oxidative modification of low-density lipoprotein (LDL) in vivo causes a gradual increase of oxidized LDL (ox-LDL) in the bone marrow microenvironment. We have reported that low-density lipoprotein receptor-related protein 6 (LRP6) acts as a receptor of ox-LDL and mediates the bone marrow stromal cells (BMSCs) uptake of ox-LDL. We detected elevated serum ox-LDL in obese mice. We found that ox-LDL uptake by LRP6 led to an increase of intracellular reactive oxygen species (ROS) in BMSCs, and N -acetyl-L-cysteine (NAC) alleviated the cellular senescence and impairment of osteogenesis induced by ox-LDL. Moreover, LRP6 is a co-receptor of Wnt signaling. We found that LRP6 preferentially binds to ox-LDL rather than dickkopf-related protein 1 (DKK1), both inhibiting Wnt signaling and promoting BMSCs senescence. Mesoderm development LRP chaperone (MESD) overexpression inhibits ox-LDL binding to LRP6, attenuating oxidative stress and BMSCs senescence, eventually rescuing bone phenotype. • Obesity induces BMSCs senescence via ox-LDL and decreases osteogenic differentiation. • LRP6 promotes BMSCs senescence by uptake of ox-LDL. • Excessive intracellular ox-LDL disrupts cellular redox homeostasis, leading to the senescence of BMSCs. [ABSTRACT FROM AUTHOR]

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events.