Role of Wnt signaling and sclerostin in bone and as therapeutic targets in skeletal disorders.

Wnt signaling and its bone tissue-specific inhibitor sclerostin are key regulators of bone homeostasis. The therapeutic potential of anti-sclerostin antibodies (Scl-Abs), for bone mass recovery and fragility fracture prevention in low bone mass phenotypes, has been supported by animal studies. The Scl-Ab romosozumab is currently used for osteoporosis treatment. INTRODUCTION: Wnt signaling is a key regulator of skeletal development and homeostasis; germinal mutations affecting genes encoding components, inhibitors, and enhancers of the Wnt pathways were shown to be responsible for the development of rare congenital metabolic bone disorders. Sclerostin is a bone tissue-specific inhibitor of the Wnt/ß-catenin pathway, secreted by osteocytes, negatively regulating osteogenic differentiation and bone formation, and promoting osteoclastogenesis and bone resorption. PURPOSE AND METHODS: Here, we reviewed current knowledge on the role of sclerostin and Wnt pathways in bone metabolism and skeletal disorders, and on the state of the art of therapy with sclerostin-neutralizing antibodies in low-bone-mass diseases. RESULTS: Various in vivo studies on animal models of human low-bone-mass diseases showed that targeting sclerostin to recover bone mass, restore bone strength, and prevent fragility fracture was safe and effective in osteoporosis, osteogenesis imperfecta, and osteoporosis pseudoglioma. Currently, only treatment with romosozumab, a humanized monoclonal anti-sclerostin antibody, has been approved in human clinical practice for the treatment of osteoporosis, showing a valuable capability to increase BMD at various skeletal sites and reduce the occurrence of new vertebral, non-vertebral, and hip fragility fractures in treated male and female osteoporotic patients. CONCLUSIONS: Preclinical studies demonstrated safety and efficacy of therapy with anti-sclerostin monoclonal antibodies in the preservation/restoration of bone mass and prevention of fragility fractures in low-bone-mass clinical phenotypes, other than osteoporosis, to be validated by clinical studies for their approved translation into prevalent clinical practice.

The study on the repair effect of Dipsacus saponins Ⅵ on tibial fracture model rats based on Wnt/β-catenin pathway / 中国医师杂志

Objective:To observe the repair effect and possible mechanism of Dipsacus saponins Ⅵ on tibial fracture model rats.Methods:Thirty Sprague Dawley (SD) rats were randomly divided into model group, intervention group, and combination group, with 10 rats in each group, to establish a tibial fracture rat model using the sawing method. On the second day after surgery, the intervention group was intraperitoneally injected with 10 mg/kg of Chuanduduan saponin Ⅵ; The combination group received intraperitoneal injection of Dipsacus saponins Ⅵ 10 mg/kg and XAV939 1 mg/animal; The model group was intraperitoneally injected with 0.2 ml of physiological saline solution and 0.2 ml of dimethylsulfoxide (DMSO) solution; Once a day, continuous intervention for 14 days. After 2 to 4 weeks of intervention, Micro CT scan and X-ray scan were used to observe the fracture healing status; After 4 weeks of intervention, the wet weight of the tibia was detected; Hematoxylin eosin (HE) staining was used to observe the pathological changes of callus tissue; The Western blot method was used to detect the expression level of callus tissue β- catenin (β-catenin), p-β-catenin, glycogen synthase kinase 3β (GSK-3 β) and Runt related transcription factor 2 (Runx2) protein.Results:After 2 and 4 weeks of intervention, the bone volume fraction (BV/TV), number of trabeculae (Tb.N), Lane Sandhu score, and callus volume in the intervention group were higher than those in the model group (all P<0.05); After 2 and 4 weeks of intervention, the BV/TV, Tb.N, Lane Sandhu score, and callus volume in the combined group were lower than those in the intervention group (all P<0.05). The wet weight of the tibia in the intervention group was higher than that in the model group at 4 weeks after intervention ( P<0.05); The wet weight of the tibia in the combined group was lower than that in the intervention group ( P<0.05). The HE staining results showed that the model group had fibrous tissue hyperplasia and more bone trabeculae, but the maturity was not high and the thickening was not significant; The intervention group formed more bony callus, with orderly arrangement of bone trabeculae, partially mature, and obvious mineralization, consistent with the direction of stress; The combined group formed more cartilaginous and fibrous callus, with more mineralization at the edge of the cartilaginous callus and the formation of bone trabeculae. Abundant capillaries can be observed in the gaps. The expression level of Runx2 and p-β-catenin/β-catenin protein in callus tissue of the intervention group was higher than that of the model group, the protein expression GSK-3 β level was lower than that of the model group (all P<0.05); The expression level of Runx2 and p-β-catenin/β-catenin protein in the callus tissue of the combined group was lower than that of the intervention group; the protein expression level of GSK-3β was higher than that of the intervention group (all P<0.05). Conclusions:Dipsacus saponins Ⅵ can effectively promote fracture repair in tibial fracture model rats; It is possible to plays a role by activating the Wnt/β-catenin signaling pathway.

Gone with the Wnt(less): a mechanistic perspective on the journey of Wnt.

Wnts are short-range signaling proteins, expressed in all metazoans from sponges to humans, critical for cell development and fate. There are 19 different Wnts in the human genome with varying expression levels and patterns, and post-translational modifications. Common to essentially all Wnts is the palmitoleation of a conserved serine by the O-acyltransferase PORCN in the endoplasmic reticulum (ER). All lipidated Wnts then bind a dedicated carrier Wntless (WLS), endowed with the task of transporting them from the ER to the plasma membrane, and ultimately facilitating their release to receptors on the Wnt-receiving cell to initiate signaling. Here, we will focus on the WLS-mediated transport step. There are currently two published structures, both obtained by single-particle cryo-electron microscopy of the Wnt/WLS complex: human Wnt8A-bound and human Wnt3A-bound WLS. We analyze the two Wnt/WLS structures - remarkably similar despite the sequence similarity between Wnt8A and Wnt3A being only ∼39% - to begin to understand the conserved nature of this binding mechanism, and ultimately how one carrier can accommodate a family of 19 different Wnts. By comparing how Wnt associates with WLS with how it binds to PORCN and FZD receptors, we can begin to speculate on mechanisms of Wnt transfer from PORCN to WLS, and from WLS to FZD, thus providing molecular-level insight into these essential steps of the Wnt signaling pathway.

Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor ß1 (TGFß) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors. ß-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFß signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in ß-catenin-positive cells. In vitro, activation of Wnt-ß-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of ß-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFß increased ß-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFß up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a ß-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFß signalling pathways in CD intestinal fibrosis.

Prediction of recurrence free survival for esophageal cancer patients using a protein signature based risk model.

BACKGROUND: Biomarkers to predict the risk of disease recurrence in Esophageal squamous cell carcinoma (ESCC) patients are urgently needed to improve treatment. We developed proteins expression-based risk model to predict recurrence free survival for ESCC patients. METHODS: Alterations in Wnt pathway components expression and subcellular localization were analyzed by immunohistochemistry in 80 ESCCs, 61 esophageal dysplastic and 47 normal tissues; correlated with clinicopathological parameters and clinical outcome over 86 months by survival analysis. Significant prognostic factors were identified by multivariable Cox regression analysis. RESULTS: Biomarker signature score based on cytoplasmic ß-catenin, nuclear c-Myc, nuclear DVL and membrane α-catenin was associated with recurrence free survival [Hazard ratio = 1.11 (95% CI = 1.05, 1.17), p < 0.001, C-index = 0.68] and added significant prognostic value over clinical parameters (p < 0.001). The inclusion of Slug further improved prognostic utility (p < 0.001, C-index = 0.71). Biomarker Signature Scoreslug improved risk classification abilities for clinical outcomes at 3 years, accurately predicting recurrence in 79% patients in 1 year and 97% in 3 years in high risk group; 73% patients within low risk group did not have recurrence in 1 year, with AUC of 0.76. CONCLUSIONS: Our comprehensive risk model predictive for recurrence allowed us to determine the robustness of our biomarker panel in stratification of ESCC patients at high or low risk of disease recurrence; high risk patients are stratified for more rigorous personalized treatment while the low risk patients may be spared from harmful side effects of toxic therapy.

Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/ß-Catenin Signaling.

Background: Kidney formation requires coordinated interactions between multiple cell types. Input from the interstitial progenitor cells is implicated in multiple aspects of kidney development. We previously reported that transcription factor 21 (Tcf21) is required for ureteric bud branching. Here, we show that Tcf21 in Foxd1+ interstitial progenitors regulates stromal formation and differentiation via interaction with ß-catenin. Methods: We utilized the Foxd1Cre;Tcf21f/f murine kidney for morphologic analysis. We used the murine clonal mesenchymal cell lines MK3/M15 to study Tcf21 interaction with Wnt/ß-catenin. Results: Absence of Tcf21 from Foxd1+ stromal progenitors caused a decrease in stromal cell proliferation, leading to marked reduction of the medullary stromal space. Lack of Tcf21 in the Foxd1+ stromal cells also led to defective differentiation of interstitial cells to smooth-muscle cells, perivascular pericytes, and mesangial cells. Foxd1Cre;Tcf21f/f kidney showed an abnormal pattern of the renal vascular tree. The stroma of Foxd1Cre;Tcf21f/f kidney demonstrated marked reduction in ß-catenin protein expression compared with wild type. Tcf21 was bound to ß-catenin both upon ß-catenin stabilization and at basal state as demonstrated by immunoprecipitation in vitro. In MK3/M15 metanephric mesenchymal cells, Tcf21 enhanced TCF/LEF promoter activity upon ß-catenin stabilization, whereas DNA-binding deficient mutated Tcf21 did not enhance TCF/LEF promoter activity. Kidney explants of Foxd1Cre;Tcf21f/f showed low mRNA expression of stromal Wnt target genes. Treatment of the explants with CHIR, a Wnt ligand mimetic, restored Wnt target gene expression. Here, we also corroborated previous evidence that normal development of the kidney stroma is required for normal development of the Six2+ nephron progenitor cells, loop of Henle, and the collecting ducts. Conclusions: These findings suggest that stromal Tcf21 facilitates medullary stroma development by enhancing Wnt/ß-catenin signaling and promotes stromal cell proliferation and differentiation. Stromal Tcf21 is also required for the development of the adjacent nephron epithelia.

WNT signaling and cancer stemness.

Cancer stemness, defined as the self-renewal and tumor-initiation potential of cancer stem cells (CSCs), is a cancer biology property featuring activation of CSC signaling networks. Canonical WNT signaling through Frizzled and LRP5/6 receptors is transmitted to the ß-catenin-TCF/LEF-dependent transcription machinery to up-regulate MYC, CCND1, LGR5, SNAI1, IFNG, CCL28, CD274 (PD-L1) and other target genes. Canonical WNT signaling causes expansion of rapidly cycling CSCs and modulates both immune surveillance and immune tolerance. In contrast, noncanonical WNT signaling through Frizzled or the ROR1/2 receptors is transmitted to phospholipase C, Rac1 and RhoA to control transcriptional outputs mediated by NFAT, AP-1 and YAP-TEAD, respectively. Noncanonical WNT signaling supports maintenance of slowly cycling, quiescent or dormant CSCs and promotes epithelial-mesenchymal transition via crosstalk with TGFß (transforming growth factor-ß) signaling cascades, while the TGFß signaling network induces immune evasion. The WNT signaling network orchestrates the functions of cancer-associated fibroblasts, endothelial cells and immune cells in the tumor microenvironment and fine-tunes stemness in human cancers, such as breast, colorectal, gastric and lung cancers. Here, WNT-related cancer stemness features, including proliferation/dormancy plasticity, epithelial-mesenchymal plasticity and immune-landscape plasticity, will be discussed. Porcupine inhibitors, ß-catenin protein-protein interaction inhibitors, ß-catenin proteolysis targeting chimeras, ROR1 inhibitors and ROR1-targeted biologics are investigational drugs targeting WNT signaling cascades. Mechanisms of cancer plasticity regulated by the WNT signaling network are promising targets for therapeutic intervention; however, further understanding of context-dependent reprogramming trajectories might be necessary to optimize the clinical benefits of WNT-targeted monotherapy and applied combination therapy for patients with cancer.

A WNT protein therapeutic accelerates consolidation of a bone graft substitute in a pre-clinical sinus augmentation model.

AIM: Autologous bone grafts consolidate faster than bone graft substitutes (BGSs) but resorb over time, which compromises implant support. We hypothesized that differences in consolidation rates affected the mechanical properties of grafts and implant stability, and tested whether a pro-osteogenic protein, liposomal WNT3A (L-WNT3A), could accelerate graft consolidation. MATERIALS AND METHODS: A transgenic mouse model of sinus augmentation with immunohistochemistry, enzymatic assays, and histology were used to quantitatively evaluate the osteogenic properties of autografts and BGSs. Composite and finite element modelling compared changes in the mechanical properties of grafts during healing until consolidation, and secondary implant stability following remodelling activities. BGSs were combined with L-WNT3A and tested for its osteogenic potential. RESULTS: Compared with autografts, BGSs were bioinert and lacked osteoprogenitor cells. While in autografted sinuses, new bone arose evenly from all living autograft particles, new bone around BGSs solely initiated at the sinus floor, from the internal maxillary periosteum. WNT treatment of BGSs resulted in significantly higher expression levels of pro-osteogenic proteins (Osterix, Collagen I, alkaline phosphatase) and lower levels of bone-resorbing activity (tartrate-resistant acid phosphatase activity); together, these features culminated in faster new bone formation, comparable to that of an autograft. CONCLUSIONS: WNT-treated BGSs supported faster consolidation, and because BGSs typically resist resorption, their use may be superior to autografts for sinus augmentation.

Wnt signaling pathway: A comprehensive review.

Wnt signaling is an evolutionary cell-to-cell coordination mechanism and it is highly critical for a variety of physiological processes of an organism's body, including stem cell regeneration, proliferation, division, migration, polarity of a cell, determining fate of the cell and specification of neural crest, neural symmetry and morphogenesis. Wnts are extracellular secreted glycol proteins, consisted of a family of 19 human proteins that represent the complex nature of the regulatory structure and physiological efficiency of signaling. Moreover, a Wnt/ß-catenin-dependent pathway and the ß-catenin-independent pathway that is further classified into the Planar Cell Polarity and Wnt/Ca2+ pathways have been established as key signaling nodes downstream of the frizzled (Fz/Fzd) receptor, and these nodes are extensively analyzed at biochemical and molecular levels. Genetic and epigenetic activities that ultimately characterize the pathway and its subsequent responses contribute to Wnt-ß-catenin signaling pathway hypo or hyper-activation and is associated with the variety of human disorders progression most significantly cancers. Recognizing how this mechanism operates is crucial to the advancement of cancer prevention therapies or regenerative medicine methods.

Expresión génica de mediadores moleculares para potencial terapia celular en el síndrome gastrointestinal inducido por radiación / Genetic expression of molecular mediators for potential cell therapy in radiation-induced gastrointestinal syndrome

Antecedentes: Las células madres intestinales generan las distintas estirpes celulares a dicho nivel. Estas se regulan por interacciones entre el epitelio y las células del nicho celular anexo. Estas se pueden ver dañadas en tratamientos con radiación, generando el síndrome gastrointestinal inducido por radiación. Se ha visto que células madre mesenquimales (MSC) y macrófagos de médula ósea (BMM) tienen propiedades de regeneración tisular. Objetivos: Evaluar la expresión génica de IL-4, Wnt6, VEGF y bFGF, a partir de cultivos celulares primarios independientes de MSC derivadas de tejido adiposo y BMM de ratones C57BL/6, por medio de PCR en tiempo real (qRT-PCR). Diseño experimental: A partir de un análisis in silico, se confeccionaron primers para evaluar la expresión génica de las moléculas propuestas, en los cultivos primarios por medio de qRT-PCR y electroforesis. Resultados y proyecciones: IL-4 y Wnt6 no son expresadas en las muestras de BMM y MSC. VEGF y bFGF son expresadas por diferentes células, dando expresión diferenciada. A futuro, se deben evaluar las mismas estirpes celulares en un ambiente inflamatorio y su efecto en la expresión génica, en especial VEGF y bFGF. Limitaciones: El número de moléculas en estudio es limitado y la expresión se evalúo solo a nivel genético.

Background: Intestinal stem cell generates diferents cellular types in their niche. They're regulated by interactions between epithelium and niche's cells, and can be damaged by medical radiation treatments causing radiation-induced gastrointestinal syndrome. It has seen that mesenchymal stem cells (MSC) d and bone marrow-derived macrophages (BMM) have propierties of tissular regeneration. Objectives: Determinated genetic expression of IL-4, Wnt6, VEGF and bFGF, in primary cellular cultures of MSC derivated of adipose tissue and BMM of C57BL/6 mice, through real time PCR (qRT-PCR). Methods: By an in silico analysis, we created primers to evaluate the proposed molecules in the primary cellular cultives, with qRT-PCR and electrophoresis. Results and projections: IL-4 and Wnt6 were not expressed in the MSC and BMM samples. VEGF and bFGF were expressed by different cells, giving differential expression. In the future, the same samples should be analyzed in an inflammatory environment, especially VEGF and bFGF. Limitations: The number of molecules are limited and the expression of them is only in a genetic level.

Relationship between spinal lncRNA and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain / 中华麻醉学杂志

Objective:To investigate the relationship between spinal long chain noncoding RNA (lncRNA) and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain (NP).Methods:The experiment was performed in two parts.Experiment Ⅰ Sprague-Dawley rats of both sexes, aged 7 days, weighing 15-20 g, were selected.Rats were sacrificed, the dorsal horn of spinal cord was removed, and the primary astrocytes were extracted and cultured.Lipopolysaccharide 1 μg/ml was added to induce the activation of astrocytes for 24 h. The lncRNA binding to kindlin-1 was identified using PCR immunoprecipitation method.The localization of lncRNA FOXF1-AS1 in astrocytes was observed by fluorescence in situ hybridization, and the binding between lncRNA FOXF1-AS1 and kindlin-1 was detected by biotin-labeled magnetic bead method.Experiment Ⅱ Thirty clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 5 groups ( n=6 each) using a random number table method: sham operation control group (group C), NP group, lncRNA FOXF1-AS1 overexpression group (group F), lncRNA FOXF1-AS1 overexpression plus kindlin-1 shRNA group (group FK) and lncRNA FOXF1-AS1 overexpression + Wnt inhibitor group (group FW). NP was induced by chronic constrictive injury in anesthetized animals.In group F, lncRNA FOXF1-AS1 overexpression lentivirus 10 μl was intrathecally injected at 28 days before operation, and vector virus 10 μl was intrathecally injected in the other groups.In FK group, kindlin-1 interfering shRNA interference adenovirus 10 μl, and vector virus 10 μl was intrathecally injected in the other groups.In group FW, Wnt inhibitor IWP-2 10 μl was intrathecally injected at 1-3 days after operation, artificial cerebrospinal fluid 10 μl was intrathecally injected at the same time point in the other groups.Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 1 day before operation, at 4 days and 7 days after operation.The animals were sacrificed at the end of measurement of pain threshold at 7 days after operation, and the spinal cord tissues were taken for determination of the expression of kindlin-1, Wnt3a and glial fibrillary acidic protein (GFAP) (by Western blot) and the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay). Results:ExperimentⅠ lncRNA FOXF1-AS1, which was expressed in the cytoplasm of astrocytes, combined with kindlin-1.Experiment Ⅱ Compared with C group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in group NP ( P<0.05). Compared with NP group, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, the expression of kindlin-1, Wnt3a and GFAP in spinal cord was up-regulated, and the contents of TNF-α and IL-1β were increased in F group, MWT was increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and the expression of kindlin-1 was up-regulated, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Compared with group F, MWT was significantly increased, TWL was prolonged at 4 and 7 days after operation, and the contents of TNF-α and IL-1β were decreased in group FK and group FW, the expression of spinal kindlin-1, Wnt3a and GFAP was down-regulated in group FK, and expression of Wnt3a and GFAP was down-regulated in group FW ( P<0.05). Conclusion:lncRNA FOXF1-AS1 can up-regulate kindlin-1 expression, activate Wnt3a signaling pathway, promote astrocyte activation, and then regulate inflammatory responses and is involved in the process of neuropathic pain in rats.

Effect of ulinastatin on hyperoxia-induced acute lung injury in infantile rats: relationship with Wnt/β-catenin signaling pathway / 中华麻醉学杂志

Objective:To evaluate the effect of ulinastatin on hyperoxia-induced acute lung injury (ALI) and its relationship with Wnt/β-catenin signaling pathway in infantile rats.Methods:A total of 36 clean-grade Sprague-Dawley rats, aged 14 days, weighing 40-50 g, were divided into 3 groups ( n=12 each): control group (C group), hyperoxia-induced ALI group (ALI group) and ulinastatin group (UTI group). Hyperoxia-induced ALI was induced by inhaling oxygen at concentration greater than 90% for 72 h. At 1 day after the model was established successfully, ulinastatin 50 000 U/kg was injected intraperitoneally daily at the same time for 3 consecutive days in group UTI, while the equal volume of normal saline was injected intraperitoneally at the same time point in C and ALI groups.The animals were sacrificed at 4 days after the model was established successfully, the lung tissues were taken for determination of the wet/dry weight ratio (W/D ratio), for microscopic examination of the pathological changes which were scored, for measurement of interleukin-6 (IL-6) IL-1β and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay) and for detection of the expression of phosphorylated glycogen synthase kinase (p-GSK-3β), Wnt3a and β-catenin by Western blot, and ultrastructure was examined with with an electron microscope. Results:Compared with C group, W/D ratio and lung injury score were significantly increased, the contents of IL-6, IL-1β and TNF-α were increased, and the expression of p-GSK-3β, Wnt3a and β-catenin were up-regulated in lung tissues in group ALI ( P<0.05). Compared with group ALI, W/D ratio and lung injury score were significantly decreased, the contents of IL-6, IL-1β and TNF-α were decreased, and the expression of p-GSK-3β, Wnt3a and β-catenin were down-regulated in lung tissues in group UTI ( P<0.05). The ultrastructure injury in group UTI was reduced as compared with group ALI. Conclusion:The mechanism by which ulinastatin can alleviate hyperoxia-induced ALI is related to inhibiting the activation of Wnt/β-catenin signaling pathway and decreasing inflammatory response in infantile rats.

Relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain / 中华麻醉学杂志

Objective:To evaluate the relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain (NP).Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (SH group), NP group, kindlin-1 shRNA group (K group) and Wnt3a inhibition group (W group). NP was induced by chronic constrictive injury in anesthetized animals.At 21 days before operation, kindlin-1 shRNA adenovirus vector 10 μl was intrathecally injected in group K, and empty viral vector 10 μl was intrathecally injected in SH, NP and W groups.Wnt inhibitor IWP-2 10 μl was intrathecally injected in group W, and artificial cerebrospinal fluid 10 μl was intrathecally injected in SH, NP and K groups at 1-3 days after operation.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 4 and 7 days after operation, respectively.At the end of pain threshold measurement at 7 days after operation, the animals were sacrificed and the lumbar segments (L 4-6) of the spinal cord were obtained for determination of the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay) and the expression of kindlin-1 and Wnt3a (by Western blot). Results:Compared with group SH, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, and the contents of TNF-α and IL-1β in spinal cord were increased in NP, K, and W groups, the expression of kindlin-1 and Wnt3a was up-regulated in NP and W groups, and expression of Wnt3a was up-regulated in group K ( P<0.05). Compared with group NP, MWT was significantly increased and TWL was prolonged at 4 and 7 days after operation in K and W groups, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of kindlin-1 and Wnt3a was down-regulated in group K, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of Wnt3a was down-regulated in group W ( P<0.05), and no significant change was found in kindlin-1 expression ( P>0.05). Conclusion:Spinal kindlin-1 regulates the inflammatory response by up-regulating the expression of Wnt3a, and it is involved in the maintenance of NP in rats.

Research progress on the role of the Wnt signaling pathway in the development of intrauterine adhesions and its significance in treatment / 中华老年医学杂志

The Wnt signaling pathway also plays a role in fibrotic diseases of various tissues and organs, such as pulmonary fibrosis, liver fibrosis, renal fibrosis and skin fibrosis.Activation of the Wnt signaling pathway is related to the fibrosis of the endometrium and ectopic endometrial tissue, and it is a key component of the potential mechanisms for the development intrauterine adhesions.It could also be a target for treatment via promoting endometrial proliferation and angiogenesis and enhancing colony formation and self-renewal ability of endometrial stem cells.Wnt signaling pathways may regulate the complicated cross-talking networks and balance the dynamic processes of fibrogenesis and repair in intrauterine adhesions, thus potentially providing a new route for studying the pathological mechanisms of intrauterine adhesions and finding therapeutic targets.

Functions and regulation of the serine/threonine protein kinase CK1 family: moving beyond promiscuity.

Regarded as constitutively active enzymes, known to participate in many, diverse biological processes, the intracellular regulation bestowed on the CK1 family of serine/threonine protein kinases is critically important, yet poorly understood. Here, we provide an overview of the known CK1-dependent cellular functions and review the emerging roles of CK1-regulating proteins in these processes. We go on to discuss the advances, limitations and pitfalls that CK1 researchers encounter when attempting to define relationships between CK1 isoforms and their substrates, and the challenges associated with ascertaining the correct physiological CK1 isoform for the substrate of interest. With increasing interest in CK1 isoforms as therapeutic targets, methods of selectively inhibiting CK1 isoform-specific processes is warranted, yet challenging to achieve given their participation in such a vast plethora of signalling pathways. Here, we discuss how one might shut down CK1-specific processes, without impacting other aspects of CK1 biology.

WNT10A Mutation Causes Ectodermal Dysplasia in a Patient Mosaic for Turner Syndrome.

WNT10A plays a role in the proper proliferation and differentiation of ectodermal structures. Mutations in this gene can be responsible for a highly phenotypically variable range of disorders termed ectodermal dysplasias. Here, we describe the case of a five-year-old male patient who is mosaic for Turner syndrome (45,X [90%]/46,X isodicentric Y [10%]) and who presented to dermatology with anhidrosis, conical-shaped teeth, and a slowed rate of hair growth with genetic testing subsequently revealing a likely pathogenic heterozygous variant in WNT10A (c.682T>A; p.Phe228Ile). Future investigation into the WNT10A pathway, which is regulated downstream by ß-catenin, might allow topical therapeutics to be developed that promote normal ectodermal growth and differentiation. Current management for this patient includes precautions taken to prevent overheating and heat stroke and close dermatological and dental monitoring.

The structural biology of canonical Wnt signalling.

The Wnt signalling pathways are of great importance in embryonic development and oncogenesis. Canonical and non-canonical Wnt signalling pathways are known, with the canonical (or ß-catenin dependent) pathway being perhaps the best studied of these. While structural knowledge of proteins and interactions involved in canonical Wnt signalling has accumulated over the past 20 years, the pace of discovery has increased in recent years, with the structures of several key proteins and assemblies in the pathway being released. In this review, we provide a brief overview of canonical Wnt signalling, followed by a comprehensive overview of currently available X-ray, NMR and cryoEM data elaborating the structures of proteins and interactions involved in canonical Wnt signalling. While the volume of structures available is considerable, numerous gaps in knowledge remain, particularly a comprehensive understanding of the assembly of large multiprotein complexes mediating key aspects of pathway, as well as understanding the structure and activation of membrane receptors in the pathway. Nonetheless, the presently available data affords considerable opportunities for structure-based drug design efforts targeting canonical Wnt signalling.

Optimizing autologous bone contribution to implant osseointegration.

BACKGROUND: Autologous bone can be harvested from the flutes of a conventional drill or from a bone scraper; here we compared whether autologous bone chips generated by a new slow-speed instrument were more osteogenic than the bone chips generated by conventional drills or bone scrapers. Additionally, we tested whether the osteogenic potential of bone chips could be further improved by exposure to a Wnt signaling (WNT) therapeutic. METHODS: Osteotomies were prepared in fresh rat maxillary first molar extraction sockets using a conventional drill or a new osseo-shaping instrument; titanium alloy implants were placed immediately thereafter. Using molecular/cellular and histologic analyses, the fates of the resulting bone chips were analyzed. To test whether increasing WNT signaling improved osteogenesis in an immediate post-extraction implant environment, a WNT therapeutic was introduced at the time of implant placement. RESULTS: Bone collected from a conventional drill exhibited extensive apoptosis; in contrast, bone generated by the new instrument remained in situ, which preserved their viability. Also preserved was the viability of the osteoprogenitor cells attached to the bone chips. Exogenous treatment with a WNT therapeutic increased the rate of osteogenesis around immediate post-extraction implants. CONCLUSIONS: Compared with conventional drills or bone scrapers, a new cutting instrument enabled concomitant site preparation with autologous bone chip collection. Histology/histomorphometric analyses revealed that the bone chips generated by this new tool were more osteogenic and could be further enhanced by exposure to a WNT therapeutic. Even though gaps still existed in placebo controls and liposomal WNT3A (L-WNT3A) cases, the area of peri-implant bone was significantly greater in L-WNT3A treated sites.

MiR-128 promotes osteogenic differentiation of bone marrow mesenchymal stem cells in rat by targeting DKK2.

Bone loss caused by inflammatory disease, such as peri-implantitis, poses a great challenge to clinicians for restoration. Emerging evidence indicates that microRNAs (miRNAs) are indispensable regulators of bone growth, development, and formation. In the present study, we found that microRNA-128 (miR-128) was differentially up-regulated during the osteogenic differentiation of rat bone marrow stem cells (rBMSCs). Overexpression of miR-128 promoted osteogenic differentiation of rBMSCs by up-regulating alkaline phosphatase (ALP), matrix mineralization, mRNA, and protein levels of osteogenic makers (e.g. RUNX2, BMP-2, and COLIA1), whereas inhibition of miR-128 suppressed osteoblastic differentiation in vitro. Mechanistically, miR-128 directly and functionally targeted Dickkopf2 (DKK2), which is a Wnt signaling pathway antagonist, and enhanced Wnt/ß-catenin signaling activity. Furthermore, the positive effect of miR-128 on osteogenic differentiation was apparently abrogated by DKK2 overexpression. Collectively, these results indicate that miR-128 promotes osteogenic differentiation of rBMSCs by targeting DKK2, which may provide a promising approach to the treatment of peri-implantitis.

Avaliação da expressão gênica de agonistas e antagonistas da via Wnt em células ex-vivo de fibroblastos do tipo sinoviócitos humanos / Evaluation of gene expression of agonists and antagonists of the Wnt pathway in ex-vivo cells of human synoviocyte-like fibroblasts

INTRODUÇÃO: a via Wnt regula vários fenômenos e processos fisiológicos ao longo da vida humana incluindo o desenvolvimento ósseo. O metabolismo ósseo consiste em uma estabilidade entre a formação e a absorção do tecido. Quando esse equilíbrio é perdido as pessoas afetadas podem sofrer erosão ou osteoproliferação óssea. Atualmente para os pacientes com absorção óssea há vários medicamentos enquanto que para os pacientes com osteoproliferação óssea não há muitas opções eficazes. OBJETIVOS: analisar a expressão genética de agonistas e antagonistas da via Wnt de pacientes com artrite reumatoide- AR e espondiloartrites- EpAs (espondilite anquilosante e artrite psoriásica). METODOLOGIA: foram cultivados fibroblastos sinoviais de sete pacientes sendo quatro com artrite reumatoide e três com espondiloatrites (um com espondilite anquilosante e dois com artrite psoriásica) em meio de cultura DMEM com soro fetal bovino. Depois, dos fibroblastos foi extraído o mRNA, convertido em cDNA. Com o cDNA foi feito o RT-qPCR pelo método TAQMAN. RESULTADOS: Em todos os genes houve uma diferença de expressão entre os fibroblastos estimulados e os não estimulados, mas o resultado estatisticamente significativo foi o do gene WNT-9A. Com esse gene houve uma redução de expressão nos fibroblastos estimulados com IL-17, IL-22 e TNF-alfa nos pacientes com artrite reumatoide e com espondiloartrites. A maioria dos resultados desse estudo foram não estatisticamente significativas comprovando que a hipótese de que os estímulos alterariam a expressão gênica dos fibroblastos se tornou nula. CONCLUSÕES: Alguns fatores podem ter contribuído para esses resultados como baixo N amostral, estímulo com as citocinas, estímulo não completo em condições in vitro pois foram desconsideradas as interações entre os genes e entre os antagonistas e agonistas. Dessa forma a mais estudos se tornam necessários para que se possa confirmar ou não que os estímulos com as citocinas realmente não afetam a expressão dos genes antagonistas e agonistas que compõe a via Wnt. PALAVRAS CHAVE: proteínas Wnt, doenças reumáticas, doenças ósseas, expressão gênica.

INTRODUCTION: the Wnt pathway regulates various physiological phenomena and processes throughout human life including bone development. Bone metabolism consists of stability between tissue formation and absorption. When this balance is lost, affected people may experience erosion or bone proliferation. Currently, for patients with bone absorption have several medications available while for patients with bone proliferation there are not many effective drug options. OBJECTIVES: to analyze the gene expression of the Wnt pathway agonists and antagonists in patients with rheumatoid arthritis - RA and spondyloarthritis - SpAs (ankylosing spondylitis and psoriatic arthritis). METHODOLOGY: Synovial fibroblasts were grown from seven patients, four with rheumatoid arthritis and three with spondyloarthritis (one with ankylosing spondylitis and two with psoriatic arthritis) in DMEM culture medium with fetal bovine serum. Then, from the fibroblasts, the mRNA was extracted, converted into cDNA. RT-qPCR was performed using the cDNA using the TAQMAN method. RESULTS: In all genes, there was a difference in expression between stimulated and unstimulated fibroblasts, but the statistically significant result occurred with the WNT-9A gene. With this gene, there was a reduction in expression in fibroblasts stimulated with IL-17, IL-22 and TNF-alpha in patients with rheumatoid arthritis and spondyloarthritis. Most of the results of this study were not statistically significant, proving that the hypothesis that stimuli would alter the gene expression of fibroblasts has become null. CONCLUSIONS: Some factors may have contributed to these results, such as low N sample, stimulus with cytokines, non-complete stimulus in in vitro conditions because interactions between genes and between antagonists and agonists were disregarded. Thus, further studies are necessary to confirm or not that the stimuli with cytokines do not really affect the expression of the antagonist and agonist genes that make up the WNT pathway. KEY WORDS: Wnt proteins, rheumatic diseases, bone diseases, gene expression.