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We investigated whether the WNT/β-catenin signalling pathway is involved in paediatric Kashin–Beck disease (KBD) and T-2-toxin-induced cartilage injury in rats to better understand the mechanism of KBD. One hundred twenty-two children were selected and assigned to the case (31), internal control (41), and external control (50) groups. The serum β-catenin and bone morphogenetic protein 2（BMP2）levels in each group were measured and compared. Thirty-six rats were randomly assigned to three groups, which received no intervention, T-2 toxin, or solvent. After 18 weeks, the expression of LDL receptor related proteins 5 (LRP5), β-catenin, BMP2, BAX, BCL2, APAF1, and caspase 9 was measured and compared. The serum BMP2 levels were significantly elevated in the children with KBD and in the rats treated with T-2 toxin. In the T-2 toxin group, LRP5 and β-catenin expression was reduced, whereas BAX, APAF1, and caspase 9 expression was increased. In conclusion, the WNT/β-catenin signalling pathway is suppressed in KBD, which induces chondrocyte apoptosis, leading to cartilage injury. Therefore, BMP2 may play a role in the pathogenesis of KBD.

Canonical Wnt signaling plays an important role in skeletal development, homeostasis, and both endochondral and intramembranous repair. While studies have demonstrated that the inhibition of Wnt signaling impairs intramembranous bone regeneration, how its activation affects intramembranous bone regeneration has been underexplored. Therefore, we sought to determine the effects of activation of canonical Wnt signaling on intramembranous bone regeneration by using the well-established marrow ablation model. We hypothesized that mice with a mutation in the Wnt ligand coreceptor gene Lrp5 would have accelerated intramembranous bone regeneration. Male and female wild-type and Lrp5-mutant mice underwent unilateral femoral bone marrow ablation surgery in the right femur at 4 weeks of age. Both the left intact and right operated femurs were assessed at Days 3, 5, 7, 10, and 14. The intact femur of Lrp5 mutant mice of both sexes had higher bone mass than wild-type littermates, although to a greater degree in males than females. Overall, the regenerated bone volume in Lrp5 mutant male mice was 1.8-fold higher than that of littermate controls, whereas no changes were observed between female Lrp5 mutant and littermate control mice. In addition, the rate of intramembranous bone regeneration (from Day 3 to Day 7) was higher in Lrp5 mutant male mice compared to their same-sex littermate controls with no difference in the females. Thus, activation of canonical Wnt signaling increases bone mass in intact bones of both sexes, but accelerates intramembranous bone regeneration following an injury challenge only in male mice.

Various disease-related genes have recently been identified using single nucleotide polymorphisms (SNPs). This study identified disease-related genes by analyzing SNP using genomic DNA isolated from Japanese patients with periapical periodontitis. Results showed that the SNP in LRP5 demonstrated a significant genotypic association with periapical lesions (Fisher’s exact test, P Col1a1 and Runx2 increased in the LiCl application group compared to that in the control group. Furthermore, many CD45R-positive cells appeared in the periapical lesions in the LiCl application group. These results indicated that LiCl promoted the healing of periapical periodontitis by inducing bone formation and immune responses. Our findings suggest that the Wnt/β-catenin signaling pathway regulates the development of periapical periodontitis. We propose a bioactive next-generation root canal treatment agent for this dental lesion.

Masaki Nakano,1,* Haruka Yui,1,* Shingo Kikugawa,2 Ryosuke Tokida,3 Noriko Sakai,4 Naoki Kondo,5 Naoto Endo,5 Hirotaka Haro,6 Hiroki Shimodaira,1 Takako Suzuki,1,7 Hiroyuki Kato,1 Jun Takahashi,1 Yukio Nakamura1 1Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, 390-8621, Japan; 2DNA Chip Research Inc., Minato-ku, Tokyo, 105-0022, Japan; 3Rehabilitation Center, Shinshu University Hospital, Matsumoto, Nagano, 390-8621, Japan; 4Department of Orthopaedic Surgery, New Life Hospital, Obuse, Nagano, 381-0295, Japan; 5Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata, 951-8510, Japan; 6Department of Orthopaedic Surgery, University of Yamanashi Graduate School of Medicine, Chuo, Yamanashi, 409-3898, Japan; 7Department of Human Nutrition, Faculty of Human Nutrition, Tokyo Kasei Gakuin University, Chiyoda-ku, Tokyo, 102-8341, Japan*These authors contributed equally to this workCorrespondence: Yukio NakamuraDepartment of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, JapanTel +81-263-37-2659Fax +81-263-35-8844Email yxn14@aol.jpObjective: Osteoarthritis (OA) is a common and degenerative joint disorder in the elderly. A greater importance of understanding the relationship between genetic factors and OA prevalence has emerged with population aging. We therefore investigated the associations of several bone disease-related genetic variants with the prevalence of OA and osteoporosis in Japanese elderly women from the Obuse study cohort, which was randomly sampled from a basic town resident registry.Methods and Results: In total, 206 female participants (mean ± standard deviation age: 69.7 ± 11.0 years) who completed OA, bone mineral density, and genotype assessments were included. The number of patients diagnosed as having knee/hip OA and osteoporosis was 59 (28.6%) and 30 (14.6%), respectively. Fisher’s exact testing revealed significant relationships between the minor T allele of LDL receptor related protein 5 (LRP5) rs3736228 and the prevalence of knee/hip OA and osteoporosis. The respective odds ratios (ORs) of the TT genotype for knee/hip OA and osteoporosis were 7.28 (95% confidence interval [CI] 2.22– 28.08) and 5.24 (95% CI 0.95– 26.98). An additional subgroup analysis for knee OA revealed that the frequency of the common C allele of methylenetetrahydrofolate reductase (MTHFR) rs1801133 had a statistically significant protective association with the prevalence of knee OA (OR 0.58, 95% CI 0.35– 0.97).Conclusion: In sum, the present study demonstrated significant associations of LRP5 rs3736228 and MTHFR rs1801133 with knee/hip OA and osteoporosis prevalences and knee OA prevalence, respectively, in Japanese elderly women. These results will help further the understanding of OA pathogenesis and related genetic risk factors.Keywords: genetic variant, LRP5, MTHFR, osteoarthritis, osteoporosis

Masaki Nakano,1,* Haruka Yui,1,* Shingo Kikugawa,2 Ryosuke Tokida,3 Noriko Sakai,4 Naoki Kondo,5 Naoto Endo,5 Hirotaka Haro,6 Hiroki Shimodaira,1 Takako Suzuki,1,7 Hiroyuki Kato,1 Jun Takahashi,1 Yukio Nakamura1 1Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, 390-8621, Japan; 2DNA Chip Research Inc., Minato-ku, Tokyo, 105-0022, Japan; 3Rehabilitation Center, Shinshu University Hospital, Matsumoto, Nagano, 390-8621, Japan; 4Department of Orthopaedic Surgery, New Life Hospital, Obuse, Nagano, 381-0295, Japan; 5Division of Orthopedic Surgery, Department of Regenerative and Transplant Medicine, Niigata University Graduate School of Medical and Dental Sciences, Chuo-ku, Niigata, 951-8510, Japan; 6Department of Orthopaedic Surgery, University of Yamanashi Graduate School of Medicine, Chuo, Yamanashi, 409-3898, Japan; 7Department of Human Nutrition, Faculty of Human Nutrition, Tokyo Kasei Gakuin University, Chiyoda-ku, Tokyo, 102-8341, Japan*These authors contributed equally to this workCorrespondence: Yukio NakamuraDepartment of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, JapanTel +81-263-37-2659Fax +81-263-35-8844Email yxn14@aol.jpObjective: Osteoarthritis (OA) is a common and degenerative joint disorder in the elderly. A greater importance of understanding the relationship between genetic factors and OA prevalence has emerged with population aging. We therefore investigated the associations of several bone disease-related genetic variants with the prevalence of OA and osteoporosis in Japanese elderly women from the Obuse study cohort, which was randomly sampled from a basic town resident registry.Methods and Results: In total, 206 female participants (mean ± standard deviation age: 69.7 ± 11.0 years) who completed OA, bone mineral density, and genotype assessments were included. The number of patients diagnosed as having knee/hip OA and osteoporosis was 59 (28.6%) and 30 (14.6%), respectively. Fisher’s exact testing revealed significant relationships between the minor T allele of LDL receptor related protein 5 (LRP5) rs3736228 and the prevalence of knee/hip OA and osteoporosis. The respective odds ratios (ORs) of the TT genotype for knee/hip OA and osteoporosis were 7.28 (95% confidence interval [CI] 2.22– 28.08) and 5.24 (95% CI 0.95– 26.98). An additional subgroup analysis for knee OA revealed that the frequency of the common C allele of methylenetetrahydrofolate reductase (MTHFR) rs1801133 had a statistically significant protective association with the prevalence of knee OA (OR 0.58, 95% CI 0.35– 0.97).Conclusion: In sum, the present study demonstrated significant associations of LRP5 rs3736228 and MTHFR rs1801133 with knee/hip OA and osteoporosis prevalences and knee OA prevalence, respectively, in Japanese elderly women. These results will help further the understanding of OA pathogenesis and related genetic risk factors.Keywords: genetic variant, LRP5, MTHFR, osteoarthritis, osteoporosis

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

In this study, fucoidans were extracted from the sea cucumber Thelenota ananas (Ta-FUCs) by enzymatic degradation. Four products with molecular weights of 1380.3, 524.0, 182.4, and 110.3 kDa were obtained, and the Ta-FUC showing optimal anti-adipogenic activities was determined. Results of MTT and Oil red O staining analyses showed that the Ta-FUCs inhibited the proliferation and differentiation of 3T3-L1 adipocytes. Futhermore, Ta-FUCs significantly downregulated the key transcriptional factors, such as SREBP-1c, PPARγ, and C/EBPα of adipocytes. The Ta-FUCs also activated Wnt/β-catenin pathway-related genes, such as β-catenin, LRP5, and FrZ. The Ta-FUCs suppressed lipid accumulation in 3T3-L1 adipocytes possibly by decreasing the expression of genes ACC, FAS, ME, GPAT, DGAT, and PILN, which are important in the synthesis of fatty acids and triglycerides; and by increasing the expression of genes PPARα, CPT-1α, and ACOX, which are crucial in fatty acid β-oxidation. The anti-adipogenic activities initially increased and then declined with decreasing molecular weight. Among the Ta-FUCs, the 182.4 kDa Ta-FUC exhibited optimal bioactivities. This study reports for the first time that Ta-FUCs can prevent obesity by regulating the differentiation and lipid accumulation of adipocytes.

Melittin is a major active peptide component of bee venom that has been demonstrated to show anti-tumor effects. Osteosarcoma is a type of bone tumor with a high degree of malignancy, and metastasis is the main challenge of osteosarcoma therapy. This study aimed to investigate the role of melittin in the lung metastasis of osteosarcoma. 143 B cells were treated with different concentrations of melittin in vitro. Wound-healing and transwell assays were performed to determine the cell migration and invasion potential. Quantitative real-time PCR and Western blot experiments were performed to evaluate the expression levels of Wnt/β-catenin signaling pathway-related factors after treatment with melittin. The orthotopic implantation model and hematoxylin-eosin staining were used to investigate the effect of melittin treatment on tumor formation and lung metastasis. Immunohistochemical staining and Western blot experiments were performed to indicate the melittin-mediated expression changes in Wnt/β-catenin signaling pathway-related factors. The cell migration and invasion potential were observed to be inhibited in a dose-dependent manner upon treatment with melittin. Treatment with medium and high concentrations of melittin attenuated the mRNA and protein expression of LRP5, β-catenin, MMP-2, cyclin D, c-Myc, survivin, MMP-9, and VEGF genes in vitro. Melittin significantly inhibited the growth of tibia xenografts in nude mice and decreased the number of lung metastatic nodules. Consistent with the results observed in vitro, treatment with melittin at medium and high concentrations attenuated the expression of Wnt/β-catenin signaling pathway-related factors in vivo. In vitro, Wnt/β-catenin signaling pathway was involved in Melittin-mediated -migration and invasion potential of 143 B cells. Similarly, as observed in the in vivo experiments, Wnt/β-catenin signaling pathway was also associated with the role of melittin on lung metastasis of osteosarcomas.

Wingless-integrated/β-catenin (Wnt/β-catenin) signaling pathway is an evolutionarily conserved signaling which is not only important for regulation of embryogenesis and organ development, but also for injury repair, homeostasis and tissue remodeling. Dysregulation of this pathway either by upregulation or even downregulation is highly implicated in various diseases such as; liver diseases, kidney diseases, lung fibrosis, heart failure, vascular calcification, osteoporosis, cellular senescence, neurodegeneration, Alzheimer's disease and cancers. Thus, targeting Wnt/β-catenin signaling has attracted scientific research as a good strategy ameliorating several diseases. In this review we discussed Wnt/β-catenin components, the Wnt-on state and the Wnt-off state. Furthermore, we summarized the effects of Wnt/β-catenin signaling in several distinct organs including brain, bone, liver, heart, lung and kidney. We also discussed various molecules that act as modulators of Wnt/β-catenin signaling pathway via targeting its components (Wnt, Frizzled, LRP5/6, Porcupine, Disheveled, β-catenin destruction complex and β-catenin activity) aiming to show their possible therapeutic potential in several diseases.

The incidence of nontuberculous Mycobacterium (NTM) lung disease is rapidly increasing, however, its diagnosis and prognosis remain unclear while selecting patients who will respond to appropriate treatment. Differences in DNA methylation patterns between NTM patients with good or poor prognosis could provide important therapeutic targets. We used the Illumina MethylationEPIC (850k) DNA methylation microarray to determine the pattern between differentially methylated regions (DMRs) in NTM patients with good or poor prognosis (n = 4/group). Moreover, we merged and compared 20 healthy controls from previous Illumina Methylation450k DNA methylation microarray data. We selected and visualized the DMRs in the form of heatmaps, and enriched terms associated with these DMRs were identified by functional annotation with the “pathfinder” package. In total, 461 and 293 DMRs (|Log2 fold change| &gt, 0.1 and p &lt, 0.03) were more methylated in patients with four poor and four good prognoses, respectively. Furthermore, 337 and 771 DMRs (|Log2 fold change| &gt, 0.08 and p &lt, 0.001) were more methylated in eight NTM patients and 20 healthy controls, respectively. TGFBr1 was significantly less methylated, whereas HLA-DR1 and HLA-DR5 were more methylated in patients with poor prognosis (compared to those with good prognosis). LRP5, E2F1, and ADCY3 were the top three less-methylated genes in NTM patients (compared with the controls). The mTOR and Wnt signaling pathway-related genes were less methylated in patients with NTM. Collectively, genes related to Th1-cell differentiation, such as TGFBr1 and HLA-DR, may be used as biomarkers for predicting the treatment response in patients with NTM lung disease.

Mechanical loading to the bone is known to be beneficial for bone homeostasis and for suppressing tumor-induced osteolysis in the loaded bone. However, whether loading to a weight-bearing hind limb can inhibit distant tumor growth in the brain is unknown. We examined the possibility of bone-to-brain mechanotransduction using a mouse model of a brain tumor by focusing on the response to Lrp5-mediated Wnt signaling and dopamine in tumor cells. The results revealed that loading the tibia with elevated levels of tyrosine hydroxylase, a rate-limiting enzyme in dopamine synthesis, markedly reduced the progression of the brain tumors. The simultaneous application of fluphenazine (FP), an antipsychotic dopamine modulator, enhanced tumor suppression. Dopamine and FP exerted antitumor effects through the dopamine receptors DRD1 and DRD2, respectively. Notably, dopamine downregulated Lrp5 via DRD1 in tumor cells. A cytokine array analysis revealed that the reduction in CCN4 was critical for loading-driven, dopamine-mediated tumor suppression. The silencing of Lrp5 reduced CCN4, and the administration of CCN4 elevated oncogenic genes such as MMP9, Runx2, and Snail. In summary, this study demonstrates that mechanical loading regulates dopaminergic signaling and remotely suppresses brain tumors by inhibiting the Lrp5-CCN4 axis via DRD1, indicating the possibility of developing an adjuvant bone-mediated loading therapy.

Sclerostin (SOST), a regulator of bone formation in osteocytes, inhibits the canonical Wnt signaling by interacting with low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to prevent Wnt binding. Loss-of-function mutations of the SOST gene caused massive bone outgrowth and SOST-null mouse exhibited a high bone density phenotype. Therefore, SOST has been suggested as a promising therapeutic target for osteoporosis. A few previous studies with X-ray crystallography identified the binding interfaces between LRP6 and SOST, but there are limitations in these studies as they used truncated SOST protein or SOST peptide. Here, we analyzed the conformational dynamics of SOST-LRP6 E1E2 complex using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We examined the effect of the C-terminal tail of SOST on LRP6 conformation upon complex formation. HDX-MS analysis suggested a new potential binding interface for the C-terminal region of SOST that was missing from the previous crystal structure of the SOST-LRP6 E1E2 complex.

Diabetes mellitus is an ill-famed metabolic disorder with varied repercussions including delayed fracture healing. Wnt/β-catenin axis is known to play a tight pivotal role in the bone healing process. Substance P (SubP) is a neuropeptide with established positive modulatory functions in fracture healing and associated neuronal milieu. In this study, we performed local delivery of recombinant adenovirus of Dickkopf-1 (DKK1) into the fracture site to understand the antagonizing the role of DKK1 against substance P. Rats were segregated into 4 groups: (i) Fractured non-diabetic rats; (ii) Fractured T1D rats; T1D was provoked by using STZ 50 mg/kg for 5 consecutive days; (iii) Fractured T1D + SubP (50 mg/ml/Kg; i.p.; 30 min prior to fracture procedure); (iv) Fractured T1D + SubP + Ad-DKK1. Bone radiographs were taken using a Faxitron X-ray machine and the residual gap size was measured using an electric caliper. Western blotting was also performed to determine the protein expression levels of osteogenic markers (RUNX2, OSTX and OSTC) bone resorption markers (OPG, RANKL and RANK) and also Wnt-signalling markers (β-catenin, LRP5 and GSK-3β). We observed that SubP promoted osteogenesis (as indicated by RUNX2, OSTX and OSTC upregulation) and mitigated the bone resorption (as indicated by optimized OPG/RANKL/RANK axis) via activated Wnt signalling (manifested by upmodulated β-catenin and LRP5, with downmodulated GSK-3β levels. Activation of endogenous SubP or administration of exogenous mimics might counter-protect the fractured bone against the deforming effects of T1D.

The LRP5 gene encodes a Wnt signaling receptor to which Wnt binds directly. In humans, pathogenic monoallelic variants in LRP5 have been associated with increased bone density and exudative vitreoretinopathy. In mice, LRP5 plays a role in tooth development, including periodontal tissue stability and cementum formation. Here, we report a 14-year-old patient with a de novo non-synonymous variant, p.(Val1245Met), in LRP5 who exhibited mildly reduced bone density and mild exudative vitreoretinopathy together with a previously unreported phenotype consisting of dental abnormalities that included fork-like small incisors with short roots and an anterior open bite, molars with a single root, and severe taurodontism. In that exudative vitreoretinopathy has been reported to be associated with heterozygous loss-of-function variants of LRP5 and that our patient reported here with the p.(Val1245Met) variant had mild exudative vitreoretinopathy, the variant can be considered as an incomplete loss-of-function variant. Alternatively, the p.(Val1245Met) variant can be considered as exerting a dominant-negative effect, as no patients with truncating LRP5 variants and exudative vitreoretinopathy have been reported to exhibit dental anomalies. The documentation of dental anomalies in the presently reported patient strongly supports the notion that LRP5 plays a critical role in odontogenesis in humans, similar to its role in mice.

Background Distraction osteogenesis (DO) is an effective treatment in craniomaxillofacial surgery. However, the issue of sufficient blood supply at the regeneration tissue has limited its wide application. Panax notoginseng saponins (PNS) is a Traditional Chinese Medicine that is commonly used to treat a range of angiogenic diseases. However, the mechanisms whereby PNS alters angiogenesis in endothelial progenitor cells (EPCs) have yet to be clarified. Methods EPCs were identified by immunofluorescence, confirmed by their uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1. EPCs were treated with different concentrations of PNS, and the effects of PNS on cell proliferation were measured on the optimal concentration of PNS determined. The effects of PNS on angiogenesis and migration, angiogenic cytokines mRNA expression and the proteins of the Wnt pathway were investigated. Then knocked down β-catenin in EPCs and treated with the optimum concentrational PNS, their angiogenic potential was evaluated in tube formation and migration assays. In addition, the expression of cytokines associated with angiogenesis and Wnt/β-catenin was then assessed via WB and RT-qPCR. Results We were able to determine the optimal concentration of PNS in the promotion of cell proliferation, tube formation, and migration to be 6.25 mg/L. PNS treatment increased the mRNA levels of VEGF, bFGF, VE-Cadherin, WNT3a, LRP5, β-catenin, and TCF4. After knocked down β-catenin expression, we found that PNS could sufficient to partially reverse the suppression of EPC angiogenesis. Conclusions Overall, 6.25 mg/L PNS can promote EPC angiogenesis via Wnt/β-catenin signaling pathway activation.

Fractures are the most common large-organ, traumatic injury in humans. The fracture healing stage includes the inflammatory stage (0-5d), cartilage callus stage (5-14d) and hard callus stage (14-21d). All mice underwent open tibial fracture surgery and were treated with saline, Glu or SCII for 21d. Calluses were harvested 5d, 10d and 21d after fracture. Compared with the model group, SCII significantly decreased TNF-α and increased aggrecan serum levels by 5d. H&E results showed that fibrous calluses were already formed in the SCII group and that chondrocytes had begun to proliferate. By 10d, the chondrocytes in the SCII group became hypertrophic and mineralized, and the serum TGF-β and Col-Iα levels were significantly increased, which indicated that the mice with SCII treatment rapidly passed the cartilage repair period and new bone formation was accelerated. Skeletal muscle repaired bones through muscle paracrine factors. IGF-1 and irisin are the two major secretory cytokines. The results showed that the content of muscle homogenate IGF-1 in the SCII group reached the peak at 10d, followed by the up-regulation of Ihh, Patched, Gli1 and Col10α in the callus through the bone surface receptor IGF-1R. Besides, SCII also significantly elevated the muscle irisin level (10 and 21d), and then increased Wnt10b, LRP5, β-catenin and Runx2 expression in the callus by receptor αVβ5. These results suggest that SCII can accelerate the process of endochondral osteogenesis and promote fracture healing through activating the Ihh/PThrp and Wnt/β-catenin pathways by regulating muscle paracrine factors. To our knowledge, this is the first study to investigate the effect of marine-derived collagen on fracture healing. This study may provide a theoretical basis for the high-value application of the laryngeal cartilage of squid in the future.

Background Ovarian cancer (OCa) is the most lethal gynecological malignant tumor, with few or no specific symptoms in its early stage. There are many signaling pathways involved in the process of OCa progression, among which the highly complex Wnt signaling pathway plays a unique role in the occurrence and development of OCa because of its functions of regulating gene expression, cell proliferation, migration, and invasion. Lipoprotein associated receptor protein 5/6 (LRP5/6) binds to activate this key pathway. Therefore, it is very important to study the mechanism of Wnt-LRP5 signaling pathway in the proliferation and migration of OCa. Methods In the present study, we have investigated the role of Wnt-LRP5 signaling pathway in OCa proliferation and migration for the first time using the dominant negative plasmid of LRP5 (DN-LRP5) and human OCa cells HO8910PM plus in a mouse model. Results Our data showed inactivation of LRP5 resulted in shift of epithelial-mesenchymal transition (EMT), rearrangement of the cytoskeleton, lowered activity of pro-proliferation and pro-migration cancer signaling pathways including Akt, p38 and NF-κB, eventually decreased proliferation and migration of OCa cells HO8910PM in vitro. Moreover, in vivo OCa-DN-LRP5 mouse model developed significantly smaller tumors as determined by inoculation of HO8910PM-DN-LRP5 cells into nude mice. Conclusions Collectively, our results demonstrate the dominant role of Wnt-LRP5 in OCa proliferation and migration and its potential as a valuable therapeutic target.

The clearance of low-density lipoprotein (LDL) particles from the circulation is regulated by the LDL receptor (LDLR) and proprotein convertase subtilisin/kexin 9 (PCSK9) interaction. Its disruption reduces blood cholesterol levels and delays atherosclerosis progression. Whether other members of the LDLR superfamily are in vivo targets of PCSK9 has been poorly explored. The aim of this work was to study the interaction between PCSK9 and members of the LDLR superfamily in the regulation of liver cholesterol homeostasis in an in vivo low-density lipoprotein receptor related protein 5 (LRP5) deficient mice model challenged with high-fat diet. Our results show that Wt and Lrp5(-/-) mice fed a hypercholesterolemic diet (HC) have increased cholesterol ester accumulation and decreased liver LDLR and LRP5 gene and protein expression. Very low-density lipoprotein receptor (VLDLR), LRP6, LRP2, and LRP1 expression levels were analyzed in liver samples and show that they do not participate in Lrp5(-/-) liver cholesterol uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in liver-specific fat-storing stellate cells but not in structural HepG2 cells. Hepatic stellate cells silenced for LRP5 and/or PCSK9 expression and challenged with lipids show reduced cholesterol ester accumulation, indicating that both proteins are involved in lipid processing in the liver. Our results indicate that cholesterol esters accumulate in livers of Wt mice in a LDLR-family-members dependent manner as VLDLR, LRP2, and LRP6 show increased expression in HC mice. However, this increase is lost in livers of Lrp5(-/-) mice, where scavenger receptors are involved in cholesterol uptake. PCSK9 expression is strongly downregulated in mice livers after HC feeding. However PCSK9 and LRP5 bind in the cytoplasm of fat storing liver cells, indicating that this PCSK9-LRP5 interaction is cell-type specific and that both proteins contribute to lipid uptake.

A loss-of-function mutation in the Lrp5 gene in mice leads to a low bone mass disorder due to the inhibition of the canonical Wnt signaling pathway; however, the role of bone marrow microenvironment in mice with this mutation remains unclear. In this study, we evaluated proliferation and osteogenic potential of mouse osteoblasts using the MTT assay and Alizarin red staining. The levels of alkaline phosphatase, tartrate-resistant acid phosphatase, and adiponectin in culture supernatants were measured using the enzyme-linked immunosorbent assay. Osteoclast bone resorbing activity was evaluated by toluidine staining and the number and area of bone resorption pits were determined. We observed increased osteogenesis in osteoblasts co-cultured with the BM-derived myeloid cells compared to the osteoblasts cultured alone. Mice with global Lrp5 deletion had a relatively higher bone density compared to the mice carrying osteoblast/osteocyte-specific Lrp5 deletion. An increased frequency of M2 macrophages and reduced expression of inflammatory cytokines were detected in the myeloid cells derived from the bone marrow of mice with global Lrp5 deletion. Higher adipogenic potential and elevated levels of adiponectin in the global Lrp5 deletion mice contributed to the preferential M2 macrophage polarization. Here, we identified a novel systemic regulatory mechanism of bone formation and degradation in mice with global Lrp5 deletion. This mechanism depends on a crosstalk between the adipocytes and M2 macrophages in the bone marrow and is responsible for partly rescuing osteopenia developed as a result of decreased Wnt signaling.

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.

Background Apert syndrome is an autosomal, dominant inherited disorder characterized by craniosynostosis and syndactyly caused by gain‐of‐function mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. Wnt/β‐catenin signaling plays critical roles in regulating the skeletal development. Here, we analyzed the role of this pathway in the developing coronal sutures (CS) of a murine Apert syndrome model (Fgfr2S252W/+). Results We observed aberrantly increased mRNA expression of Lrp5 and Lrp6 in CS of Fgfr2S252W/+ mice, whereas both wild type (WT) and Fgfr2S252W/+ mice showed similar expression of other Wnt/β‐catenin‐related genes, such as Wnt3, Wnt3a, Fzd4, Fzd6, Axin2, and Dkk1 as evidenced by in situ hybridization. Significantly increased Lrp5 and Lrp6 mRNA expression was observed by quantitative PCR analysis of cultured cells isolated from CS of Fgfr2S252W/+ mice. Phospho‐LRP5, phospho‐LRP6, and non‐phospho‐β‐catenin were upregulated in Fgfr2S252W/+ CS compared with that in WT CS. Short‐interfering RNA targeting Lrp5 and Lrp6 significantly reduced runt‐related transcription factor 2, collagen type 1 alpha 1, and osteocalcin mRNA expression, and alkaline phosphatase activity in cultured cells. Conclusions The Wnt/β‐catenin pathway was activated in the CS of Fgfr2S252W/+ mice during craniofacial development, suggesting the involvement of the Wnt/β‐catenin pathway in the pathogenesis of CS synostosis in Fgfr2S252W/+ mice. This article is protected by copyright. All rights reserved.

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.

Long noncoding RNAs (lncRNAs) have been implicated in a range of developmental processes and diseases, but the roles and mechanisms by which they act in adipogenic differentiation and adipose tissue biology are still unknown. By comparing the different expression patterns of lncRNAs before and after the adipocyte differentiation of human adipose-derived mesenchymal stem cells (hADSCs), we characterized a novel lncRNA, AC092834.1, which is significantly increased in preadipocytes. By gain- and loss-of-function experiments, we demonstrated that lncRNA AC092834.1 potentiated adipogenic differentiation through directly increasing the level of expression of DKK1, which competitively binds with LRP5 to inhibit the Wnt-β-catenin pathway and reduce the inhibition of adipogenesis by Wnt signaling. This finding provides novel mechanistic insights into a critical role for lncRNA AC092834.1 as a regulator of adipogenic differentiation, which expands our knowledge about the molecular mechanisms of obesity and other adipogenic differentiation-related disorders.

Wnt/β-catenin signaling pathway modulates miscellaneous biological events in cells including gene expression, cell growth, apoptosis, metabolism and transition. The aim of this study was to investigate the effect of endostatin on Wnt signaling pathway of stem-like cells in bladder cancer in tumor microenvironment. The qRT-PCR assay and western blot were conducted to evaluate related factors expressions of Wnt signaling pathway in both bladder cancer 5637 cells and stem cells. Loss of function assays were carried out to detect the influence of endostatin on the proliferation, migration, cell proliferation and apoptosis of bladder cancer cells. We demonstrated that endostatin triggered the degradation of β-catenin, a key mediator of Wnt signaling. The activation of the endostatin blocked β-catenin function and inhibited cell growth and migration of bladder cancer. In order to verify that the Wnt/β-catenin signaling pathway was inhibited by endostain in 5637 bladder cancer cells and stem cells, the Wnt/β-catenin signaling pathway-associated molecules, including DKK1, LRP5, TCF4, β-catenin, cyclin D1, and c-Myc, were evaluated in 5637 bladder cancer cells and stem cells. The western blotting results showed that expressions of these molecules were remarkably increased in the 5637 bladder cancer cells and stem cells compared to the control group. In summary, our study demonstrated that endostatin inhibited angiogenesis. The downregulation of the Wnt/β-catenin pathway may be engaged in the suppression of angiogenesis by endostatin in bladder cancer cells and cancer stem cells.

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.

Aberrant activation of the Wnt signaling pathway is associated with tumorigenesis, relapse/chemoresistance, and distance metastasis in TNBC. SM08502 is a novel, oral, small-molecule pan-CLK inhibitor that has been shown to potently inhibit the Wnt signaling pathway in several preclinical cancer models. The purpose of these studies was to examine the antitumor activity of SM08502 as monotherapy and in combination with standard chemotherapy in preclinical models of TNBC. The effect of SM08502 on cell proliferation was tested 13 BC cell lines, 7 of which were TNBC derived. Cell proliferation was strongly impaired by SM08502 across all lines (average EC50=0.170 µM [0.055-0.510]). SM08502 demonstrated similar potency between HR+ and TNBC cell lines with average EC50 values of 0.202 µM (0.058-0.510) and 0.142 µM (0.055-0.240), respectively. Relative to DMSO, SM08502 (1 μM) potently inhibited Wnt pathway-related gene and protein expression (TCF7, DVL2, LRP5, and ERBB2) in TNBC cell lines. Notably, SM08502 showed little inhibitory effect on cell proliferation in normal breast cells (Hs578Bst) compared with their paired TNBC cells (Hs578T) with an EC50 of 1.517 μM and 0.080 μM, respectively. Wnt pathway-related proteins were also overexpressed in Hs578T cells relative to Hs578Bst, and SM08502 (1 µM) strongly reduced their expression. In vivo antitumor effects and tolerability of oral SM08502 (25 mg/kg QD for 20 days) were assessed in mice bearing orthotopically implanted, luciferase-expressing, TNBC (MDA-MB231)-derived xenografts (n=5 mice per group). Significant tumor growth inhibition (TGI) vs. vehicle occurred in mice treated with SM08502 (80%, p Citation Format: Heekyung Chung, Lauren Sitts, Emily Creger, John Duc Nguyen, Brian Eastman, Chi-Ching Mak, Sunil KC, Betty Tam, Carine Bossard, Timothy Phalen, Steven Cha. SM08502, a novel, small-molecule CDC-like kinase (CLK) inhibitor, demonstrates strong inhibition of the Wnt signaling pathway and antitumor effects as monotherapy and in combination with chemotherapy in triple-negative breast cancer (TNBC) models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-13-01.

The objective of this study was to analyze the expression and clinical role of Wnt pathway molecules in metastatic high-grade serous carcinoma (HGSC). mRNA expression by qPCR of 20 molecules related to Wnt signaling (WNT1, WNT2, WNT3, WNT4, WNT5A, WNT6, WNT7, WNT11, FZD1, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, LRP5, LRP6, DKK, CCND, RUNX2) was analyzed in 87 HGSC effusions. Thirty-nine surgical specimens (19 ovarian, 20 from other intra-abdominal sites) were analyzed for comparative purposes. Protein expression of YAP and LRP and their phosphorylated forms by western blotting were analyzed in 52 tumors. Significant differences in mRNA expression as a function of the anatomic site were observed for WNT3 (p = 0.005), WNT5A (p = 0.008), WNT7 (p

This study was conducted to investigate the effect of Shenning II decoction on renal function, renal pathology, epithelial-to-mesenchymal transition (EMT), and Wnt/beta-catenin signaling in unilateral ureteral obstruction (UUO) renal fibrosis. Sprague-Dawley rats were randomly divided into blank control, sham-operated, UUO model (untreated), and UUO with Shenning decoction treatment (high, medium, and low dose) groups. Renal function was evaluated based on blood urea nitrogen (BUN) and serum creatinine (Scr) levels. Histopathological analysis of rat kidney tubular tissue was carried out and E-cadherin, fibronectin, vimentin, Wnt4, glycogen synthase kinase (GSK)β, low-density lipoprotein receptor-related protein (LRP)5, LRP6, β-catenin, Snail, and fibroblast-specific protein (FSP)1 expression was evaluated by immunohistochemistry, western blotting, and real-time PCR. BUN and Scr were found to be increased in UUO when compared with the sham rats (P 0.05). Shenning II decoction decreased histopathological scores relative to the UUO rats (P β-catenin, and fibronectin was decreased in Shenning I-treated rats, when compared with the untreated UUO rats, as determined by immunohistochemistry and western blotting (P β-catenin, GSK-3β, LRP5, LRP6, Snail, and FSP1 mRNA levels were also downregulated by Shenning II decoction treatment (P < 0.05). Shenning II decoction, therefore, protects against renal fibrosis by blocking renal tubular EMT via suppression of Wnt/beta-catenin signaling.

Purpose: The signals responsible for homeostasis in the periodontal complex are unclear. The purpose of this study was to evaluate the role of Low-density lipoprotein receptor-related protein 5(LRP5) in this process by removing LRP5, and observing the effects of LRP5 depletion on cells of the periodontal structures. Material and Methods: The function of this LRP5 was evaluated by conditional elimination of the LRP5 gene using an Osteocalcin Cre driver. The OCN-Cre; mice were examined using micro-CT and histology, immunohistochemistry to evaluate the periodontal complex. Results: Elimination of LRP5 in the periodontal complex of OCN-Cre; mice results in a different expression of Fibromodulin in the periodontal ligament space. A decrease in osteoclastic activity was found in the periodontal ligament. Conclusion: Osteoclastic activities are decreased and expression of fibromodulin is decreased, which implies the involvement of LRP5 in homeostasis of the periodontal ligament.

A young man was diagnosed with transient regional osteoporosis (TRO). The genetic analysis revealed a novel de novo likely pathogenic variant in COL1A2 gene. Our hypothesis is that TRO may be a possible clinical manifestation of osteogenesis imperfecta due to a reduced bone mass and an impaired trabecular mechanical competence. Transient regional osteoporosis (TRO) is a disease characterized by episodes of pain in the lower limbs involving the hip, knee, ankle or foot. Here, we present a clinical case of a Caucasian 25-year-old man exhibiting TRO. Based on few mild clinical findings suggestive of osteogenesis imperfecta (OI), but without a history of fragility fractures, we performed a genetic assessment to investigate this hypothesis. Medical history was obtained from the patient and family members, including biochemical, RMI and DXA assessments. Next-generation sequencing of COL1A1, COL1A2, COL2A1, CASR, CYP19A1, CUL7, CRTAP, KAL1, LEPRE1, LRP5, PPIB and SLC9A3R1, genes involved in juvenile osteoporosis, was performed. We identified a novel de novo heterozygous missense variant, c.488G > A, in exon 11 of the COL1A2 gene (NM_000089.3), resulting in the putative p.Gly163Asp substitution in the N-terminal part of the helical domain of type I collagen. The variant was predicted to be damaging by the in silico prediction tools and the mutation was therefore classified as likely pathogenic. This mutation can affect skeletal health impairing bone mass and trabecular mechanical competence, inducing a disease whose features strictly evoke a TRO. The present study describes a novel de novo heterozygous missense variant in COL1A2 gene, possibly inducing a propensity to trabecular microfractures. The recurrent symptomatic bone marrow oedema episodes could be the clinical picture consistent with the hypothesis of an inherited connective tissue disorder giving bone fragility.

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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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