Mechanical induction of osteoanabolic Wnt1 promotes osteoblast differentiation via Plat.

Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating ß-Catenin. Silencing Wnt1 impairs mechanically induced ß-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/ß-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.

Wnt1 gene expression in the heart left ventricle as a response to the various durations of the intensive exercise: An experimental study.

Objective. Myocardial fibrosis is a devastating condition causing millions of deaths yearly. Several factors, such as aging, cause myocardial fibrosis. The Wnt/ß-catenin pathway is one of the critical intracellular signaling for the development of cardiac fibrosis. Molecular and cellular mechanism of myocardial fibrosis induced by intensive exercise is not well-understood. The current study evaluates the effects of short- and long-term intensive exercise on the Wnt1 gene expression in a heart left ventricle in an animal model. Methods. Twenty-one male Wistar rats (mean weight 250±50 g) were divided into three groups (n=7): 1) control group (C); 2) short-term regular intensive exercise group (S-RIE, high-intensity exercise for one month six days weekly for 60 min with speed of 35 m/min), and 3) long-term regular intensive exercise group (L-RIE, high-intensity exercise for six months six days daily for 60 min with speed of 35 m/min). The heart left ventricle was isolated at the end of the experiment, and the relative gene expression of the Wnt1 gene was measured by the Real-Time PCR. Results. The L-RIE group showed a significant increase in the Wnt1 expression compared to the S-RIE and the control group. Although no difference was observed in the Wnt1 mRNA level in the S-RIE group compared to the control group, Wnt1 mRNA level increased in the L-RIE group compared to the S-RIE group. Conclusion. The exercise duration was of a great importance in the Wnt1 gene expression. Regular intensive exercise may be involved in the formation of the myocardial fibrosis by increasing the expression of the Wnt1 gene.

HuR-dependent expression of Wisp1 is necessary for TGFß-induced cardiac myofibroblast activity.

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFß-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFß-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFß treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFß. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFß, in part through post-transcriptional regulation of Wisp1.

KLF3 Transcription Activates WNT1 and Promotes the Growth and Metastasis of Gastric Cancer via Activation of the WNT/ß-Catenin Signaling Pathway.

The transcription factor Krüppel-like factor (KLF) 3 is one of the members of the KLF family, which plays an important role in tumor progression. Nevertheless, the role of KLF3 in the growth and metastasis of gastric cancer (GC) still needs to be elucidated. Bioinformatics analysis showed that KLF3 was overexpressed in patients with GC, and the high expression of KLF3 was correlated with poor survival. KLF3 was also overexpressed in GC clinical samples and cell lines. In vitro functional role of KLF3 in GC cells was explored by a gain-of-function and loss-of-function assay. Overexpressed KLF3 promoted the cell proliferation, migration, invasion, and epithelial-mesenchymal transition of GC cells, whereas suppressed KLF3 inhibited these biological behaviors. The clinical samples and bioinformatics analysis showed that WNT1 was also highly expressed in GC tumor tissues and positively correlated with KLF3 expression. The luciferase reporter assay and chromatin immunoprecipitation result confirmed that KLF3 could directly bind to the WNT1 promoter to increase the transcriptional activity of WNT1, thus regulating its expression. Overexpressed KLF3 enhanced the protein expression level of p-GSK3ß(Ser9) and ß-catenin, the key elements in the WNT/ß-catenin signaling pathway. Repression of KLF3 decreased the level of p-GSK3ß(Ser9) and ß-catenin. Immunofluorescence images showed that KLF3 promoted nuclear ß-catenin accumulation. Inhibition of WNT1 attenuated the proliferation, migration, and invasiveness of KLF3-overexpressing GC cells. Moreover, the xenograft mouse model confirmed that KLF3 promotes GC tumor growth and metastasis in vivo. Our results demonstrated that KLF3 activates the WNT/ß-catenin signaling pathway via WNT1 to promote GC tumor growth and metastasis, indicating that repression of KLF3 may act as a potential therapeutic target for patients with GC.

Osteoporosis related to WNT1 variants: a not infrequent cause of osteoporosis.

Nearly 10% of subjects with severe idiopathic osteoporosis present pathogenic WNT1 mutations. Clinical characteristics include a family history of osteoporosis, early adulthood onset, and fragility fractures which may evolve to pseudoarthrosis. WNT1 should be genetically screened in these patients as the phenotype is often variable and therapeutic approaches may differ. INTRODUCTION: Recent studies have shown that homozygous WNT1 gene mutations may be related to severe osteoporosis resembling osteogenesis imperfecta (OI). Conversely, heterozygous WNT1 mutations are linked to a milder phenotype of early-onset osteoporosis. Treatment with bisphosphonates is reported to be unsatisfactory. Our aim was to analyze the presence and prevalence of WNT1 mutations and the main associated clinical characteristics in subjects with primary early-onset osteoporosis. METHODS: A cohort comprising 56 subjects (aged 19-60 years) with severe, early-onset osteoporosis was screened by massive parallel sequencing with a 23-gene panel. The gene panel included 19 genes known to cause OI (including the WNT1 gene), three genes related to osteoporosis, and the gene related to hypophosphatasia (ALPL). RESULTS: We identified five patients (3 men) with heterozygous WNT1 variants. All presented severe osteoporosis with early fracture onset and a family history of fragility fractures. None presented a characteristic phenotype of OI or skeletal deformities. One patient was previously treated with bisphosphonates, presenting inadequate response to treatment and two developed pseudoarthrosis after upper arm fractures. All subjects were diagnosed in adulthood. CONCLUSIONS: Nearly 1/10 adult subjects with severe idiopathic osteoporosis may present pathogenic WNT1 mutations. Clinical characteristics commonly include a family history of osteoporosis, onset in early adulthood, marked decrease in bone mass, and prevalent fractures, particularly vertebral. WNT1 should be genetically screened in these subjects as the phenotype is often variable and the therapeutic approach may differ. The role of WNT1 mutations in the development of pseudoarthrosis should also be elucidated.

LMP2 and TAP2 impair tumor growth and metastasis by inhibiting Wnt/ß-catenin signaling pathway and EMT in cervical cancer.

BACKGROUND: The roles of low molecular mass polypeptide 2 (LMP2) and transporter-associated with antigen processing (TAP2) in tumorigenesis are controversial. Here we aimed to explore the effect of LMP2 and TAP2 on the oncogenesis and metastasis of cervical cancer cells. METHODS: The expressions of LMP2 and TAP2 in cervical cancer and normal tissues were determined by qPCR. Plate colony formation, cell counting kit-8 analysis and in vivo tumor xenograft assays were used to detect the tumor growth. Wound healing and transwell assays were used to detect the metastasis of cervical cancer. Gelatin zymography and western blotting assays were used to detect the effect of LMP2 and TAP2 on the EMT and Wnt/ß-catenin pathway in cervical cancer cells. RESULTS: In the present study, we reported that LMP2 and TAP2 levels were overexpressed in cervical cancer. Overexpression of LMP2 and TAP2 impaired the proliferation of Hela cells. In vivo studies substantiated that LMP2 and TAP2 antagonized tumor growth. Likewise, LMP2 and TAP2 overexpression decreased the migration and invasion ability of Hela cells by regulating the process of epithelial-mesenchymal transition (EMT). Mechanically, LMP2 and TAP2 subverted the protein abundance of Wnt1 and ß-catenin, thereby downregulating their downstream targets Cyclin D1 and c-Myc. In addition, Wnt1 overexpression partially rescued the observed consequences of ectopic expression of LMP2 and TAP2 in cervical cancer cells. Taken together, our study revealed that LMP2 and TAP2 suppress the oncogenesis and metastasis of cervical cancer cells by Wnt/ß-catenin pathway and altering EMT. CONCLUSION: LMP2 and TAP2 may inhibit the oncogenesis and metastasis of cervical cancer cells by inhibiting the process of EMT and the Wnt/ß-catenin signaling pathway, which may provide important insight into prospective targets for the treatment of cervical cancer.

Medical Care Use Among Patients with Monogenic Osteoporosis Due to Rare Variants in LRP5, PLS3, or WNT1.

Pathogenic variants in the LRP5, PLS3, or WNT1 genes can significantly affect bone mineral density, causing monogenic osteoporosis. Much remains to be discovered about the phenotype and medical care needs of these patients. The purpose of this study was to examine the use of medical care among Dutch individuals identified between 2014 and 2021 with a pathogenic or suspicious rare variant in LRP5, PLS3, or WNT1. In addition, the aim was to compare their medical care utilization to both the overall Dutch population and the Dutch Osteogenesis Imperfecta (OI) population. The Amsterdam UMC Genome Database was used to match 92 patients with the Statistics Netherlands (CBS) cohort. Patients were categorized based on their harbored variants: LRP5, PLS3, or WNT1. Hospital admissions, outpatient visits, medication data, and diagnosis treatment combinations (DTCs) were compared between the variant groups and, when possible, to the total population and OI population. Compared to the total population, patients with an LRP5, PLS3, or WNT1 variant had 1.63 times more hospital admissions, 2.0 times more opened DTCs, and a greater proportion using medication. Compared to OI patients, they had 0.62 times fewer admissions. Dutch patients with an LRP5, PLS3, or WNT1 variant appear to require on average more medical care than the total population. As expected, they made higher use of care at the surgical and orthopedic departments. Additionally, they used more care at the audiological centers and the otorhinolaryngology (ENT) department, suggesting a higher risk of hearing-related problems.

R-spondin-3 is an oncogenic driver of poorly differentiated invasive breast cancer.

R-spondins (RSPOs) are influential signaling molecules that promote the Wnt/ß-catenin pathway and self-renewal of stem cells. Currently, RSPOs are emerging as clinically relevant oncogenes, being linked to cancer development in multiple organs. Although this has instigated the rapid development and testing of therapeutic antibodies targeting RSPOs, functional evidence that RSPO causally drives cancer has focused primarily on the intestinal tract. Here, we assess the oncogenic capacity of RSPO in breast cancer in a direct fashion by generating and characterizing a novel mouse model with conditional Rspo3 expression in the mammary gland. We also address the prevalence of RSPO gene alterations in breast cancer patients. We found that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number amplifications, which are associated with lack of steroid hormone receptor expression and reduced patient survival. Foremost, we demonstrate the causal oncogenic capacity of RSPO3 in the breast, as conditional Rspo3 overexpression consistently drives the development of mammary adenocarcinomas in our novel Rspo3 breast cancer model. RSPO3-driven mammary tumors typically show poor differentiation, areas of epithelial-to-mesenchymal transition, and metastatic potential. Given the reported interplay in the Wnt/ß-catenin pathway, we comparatively analyzed RSPO3-driven mouse mammary tumors versus classical WNT1-driven analogues. This revealed that RSPO3-driven tumors are distinct, as the poorly differentiated tumor morphology and metastatic potential were observed in RSPO3-driven tumorigenesis exclusively, further substantiated by differentiating gene expression profiles. Co-expression of Rspo3 and Wnt1 transduced mammary tumors with a mixed phenotype harboring morphological features characteristic of both transgenes. In summary, we report that a quarter of breast cancer patients harbor RSPO2/RSPO3 copy number gains, and these patients have a worse prognosis, whilst providing in vivo evidence that RSPO3 drives poorly differentiated invasive breast cancer in mice. Herewith, we establish RSPO3 as a driver of breast cancer with clinical relevance, proposing RSPO3 as a novel candidate target for therapy in breast cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Midbrain Preisthmus: A Poorly Known Effect of the Isthmic Organizer.

This essay reexamines molecular evidence supporting the existence of the 'preisthmus', a caudal midbrain domain present in vertebrates (studied here in the mouse). It is thought to derive from the embryonic m2 mesomere and appears intercalated between the isthmus (caudally) and the inferior colliculus (rostrally). Among a substantial list of gene expression mappings examined from the Allen Developing and Adult Brain Atlases, a number of quite consistent selective positive markers, plus some neatly negative markers, were followed across embryonic stages E11.5, E13.5, E15.5, E18.5, and several postnatal stages up to the adult brain. Both alar and basal subdomains of this transverse territory were explored and illustrated. It is argued that the peculiar molecular and structural profile of the preisthmus is due to its position as rostrally adjacent to the isthmic organizer, where high levels of both FGF8 and WNT1 morphogens must exist at early embryonic stages. Isthmic patterning of the midbrain is discussed in this context. Studies of the effects of the isthmic morphogens usually do not attend to the largely unknown preisthmic complex. The adult alar derivatives of the preisthmus were confirmed to comprise a specific preisthmic sector of the periaqueductal gray, an intermediate stratum represented by the classic cuneiform nucleus, and a superficial stratum containing the subbrachial nucleus. The basal derivatives, occupying a narrow retrorubral domain intercalated between the oculomotor and trochlear motor nuclei, include dopaminergic and serotonergic neurons, as well as a variety of peptidergic neuron types.

Association of Membranous WNT-1 and Nuclear mTOR with Endometrial Cancer Grade.

Endometrial cancer remains a common cancer affecting the female reproductive system. There is still a need for more efficient ways of determining the degree of malignancy and optimizing treatment. WNT and mTOR are components of signaling pathways within tumor cells, and dysfunction of either protein is associated with the pathogenesis of neoplasms. Therefore, the aim of our study was to assess the impact of subcellular WNT-1 and mTOR levels on the clinical course of endometrial cancer. WNT-1 and mTOR levels in the plasma membrane, nucleus, and cytoplasm were evaluated using immunohistochemical staining in a group of 64 patients with endometrial cancer of grades 1-3 and FIGO stages I-IV. We discovered that the levels of WNT-1 and mTOR expression in the cellular compartments were associated with tumor grade and staging. Membranous WNT-1 was negatively associated, whereas cytoplasmic WNT-1 and nuclear mTOR were positively associated with higher grading of endometrial cancer. Furthermore, nuclear mTOR was positively associated with FIGO stages IB-IV. To conclude, we found that the assessment of WNT-1 in the cell membrane may be useful for exclusion of grade 3 neoplasms, whereas cytoplasmic WNT-1 and nuclear mTOR may be used as indicators for confirmation of grade 3 neoplasms.

MiRNA-21a, miRNA-145, and miRNA-221 Expression and Their Correlations with WNT Proteins in Patients with Obstructive and Non-Obstructive Coronary Artery Disease.

MicroRNAs and the WNT signaling cascade regulate the pathogenetic mechanisms of atherosclerotic coronary artery disease (CAD) development. OBJECTIVE: To evaluate the expression of microRNAs (miR-21a, miR-145, and miR-221) and the role of the WNT signaling cascade (WNT1, WNT3a, WNT4, and WNT5a) in obstructive CAD and ischemia with no obstructive coronary arteries (INOCA). METHOD: The cross-sectional observational study comprised 94 subjects. The expression of miR-21a, miR-145, miR-221 (RT-PCR) and the protein levels of WNT1, WNT3a, WNT4, WNT5a, LRP6, and SIRT1 (ELISA) were estimated in the plasma of 20 patients with INOCA (66.5 [62.8; 71.2] years; 25% men), 44 patients with obstructive CAD (64.0 [56.5; 71,0] years; 63.6% men), and 30 healthy volunteers without risk factors for cardiovascular diseases (CVD). RESULTS: Higher levels of WNT1 (0.189 [0.184; 0.193] ng/mL vs. 0.15 [0.15-0.16] ng/mL, p < 0.001) and WNT3a (0.227 [0.181; 0.252] vs. 0.115 [0.07; 0.16] p < 0.001) were found in plasma samples from patients with obstructive CAD, whereas the INOCA group was characterized by higher concentrations of WNT4 (0.345 [0.278; 0.492] ng/mL vs. 0.203 [0.112; 0.378] ng/mL, p = 0.025) and WNT5a (0.17 [0.16; 0.17] ng/mL vs. 0.01 [0.007; 0.018] ng/mL, p < 0.001). MiR-221 expression level was higher in all CAD groups compared to the control group (p < 0.001), whereas miR-21a was more highly expressed in the control group than in the obstructive (p = 0.012) and INOCA (p = 0.003) groups. Correlation analysis revealed associations of miR-21a expression with WNT1 (r = -0.32; p = 0.028) and SIRT1 (r = 0.399; p = 0.005) protein levels in all CAD groups. A positive correlation between miR-145 expression and the WNT4 protein level was observed in patients with obstructive CAD (r = 0.436; p = 0.016). Based on multivariate regression analysis, a mathematical model was constructed that predicts the type of coronary lesion. WNT3a and LRP6 were the independent predictors of INOCA (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Activation of the canonical cascade of WNT-ß-catenin prevailed in patients with obstructive CAD, whereas in the INOCA and control groups, the activity of the non-canonical pathway was higher. It can be assumed that miR-21a has a negative effect on the formation of atherosclerotic CAD. Alternatively, miR-145 could be involved in the development of coronary artery obstruction, presumably through the regulation of the WNT4 protein. A mathematical model with WNT3a and LRP6 as predictors allows for the prediction of the type of coronary artery lesion.

Hepatoprotective effect of protocatechuic acid against type 2 diabetes-induced liver injury.

CONTEXT: Protocatechuic acid (PCA) has a protective effect on alcoholic liver injury, but the role of PCA in type 2 diabetes-induced liver injury is not well known. OBJECTIVES: This study explores the therapeutic effect and potential mechanism of PCA on type 2 diabetes-induced liver injury. MATERIALS AND METHODS: An insulin resistance/type 2 diabetic (IR/D) model was established by high-fat diet for 4 weeks + streptozotocin (35 mg/kg; i.p) in male Wistar rats pretreated with or without PCA (15 or 30 mg/kg for 6 d). RESULTS: PCA at 15 and 30 mg/kg significantly upregulated the levels of body weight (BW; 230.2, 257.8 g), high density lipids (22.68, 34.78 mg/dL), glutathione (10.24, 16.21 nmol/mg), superoxide dismutase (21.62, 29.34 U/mg), glucagon-like peptide-1, glucose transporter-4, Wnt1, and ß-catenin, while downregulating those of liver weight (LW; 9.4, 6.7 g), BW/LW (4.1, 2.6%), serum glucose (165, 120 mg/dL), serum insulin (13.46, 8.67 µIU/mL), homeostatic model assessment of insulin resistance (5.48, 2.57), total cholesterol (68.52, 54.31 mg/dL), triglycerides (72.15, 59.64 mg/dL), low density lipids (42.18, 30.71), aspartate aminotransferase (54.34 and 38.68 U/L), alanine aminotransferase (42.87, 29.98 U/L), alkaline phosphatase (210.16, 126.47 U/L), malondialdehyde (16.52, 10.35), pro-inflammatory markers (tumor necrosis factor α (TNF-α (149.67, 120.33 pg/mg)) , IL-6 (89.79, 73.69 pg/mg) and IL-1ß (49.67, 38.73 pg/mg)), nuclear factor kappa B (NF-κB), and interleukin-1ß, and ameliorated the abnormal pathological changes in IR/D rats. DISCUSSION AND CONCLUSION: PCA mitigates the IR, lipid accumulation, oxidative stress, and inflammation in liver tissues of IR/D rats by modulating the NF-κB and Wnt1/ß-catenin pathways.

The Wnt1-Cre2 transgene is active in the male germline.

The Wnt1-Cre transgenic mouse line is widely used to express the CRE recombinase in neural crest lineages, but it overexpresses WNT1 itself, which can cause undesired phenotypes. To address this, we and others previously developed a Wnt1-Cre2 line based on the same regulatory elements as Wnt1-Cre but without ectopic Wnt1 expression. However, while Wnt1-Cre2 exhibits normal activity when transmitted from female mice, it exhibits unexpected activity in the male germline. The Wnt1-Cre2 transgene was previously mapped to the E2f1 locus. Several genes in this genomic region exhibit significant expression in spermatogonia or spermatocytes, suggesting that local regulatory elements may be driving ectopic transgene expression. The Wnt1-Cre2 line can therefore be used both as a neural crest specific and a general deleter, and care should be taken when setting up genetic crosses.

Diverse cellular origins of adult blood vascular endothelial cells.

During embryonic stages, vascular endothelial cells (ECs) originate from the mesoderm, at specific extraembryonic and embryonic regions, through a process called vasculogenesis. In the adult, EC renewal/replacement mostly depend on local resident ECs or endothelial progenitor cells (EPCs). Nevertheless, contribution from circulating ECs/EPCs was also reported. In addition, cells lacking from EC/EPC markers with in vitro extended plasticity were shown to originate endothelial-like cells (ELCs). Most of these cells consist of mesenchymal stromal progenitors, which would eventually get mobilized from the bone marrow after injury. Based on that, current knowledge on different mouse and human bone marrow stromal cell (BM-SC) subpopulations, able to contribute with mesenchymal stromal/stem cells (MSCs), is herein reviewed. Such analyses underline an unexpected heterogeneity among sinusoidal LepR+ stromal/CAR cells. For instance, in a recent report a subgroup of LepR+ stromal/CAR progenitors, which express GLAST and is traced in Wnt1Cre;R26RTom mice, was found to contribute with ELCs in vivo. These GLAST â€‹+ â€‹Wnt1+ BM-SCs were shown to get mobilized to the peripheral blood and to contribute with liver regeneration. Other sources of ELCs, such as adipose, neural and dental pulp tissues, were also published. Finally, mechanisms likely involved in the enhanced cellular plasticity properties of bone marrow/adipose tissue stromal cells, able to originate ELCs, are assessed. In the future, strategies to analyze the in vivo expression profile of stromal cells, with MSC properties, in combination with screening of active genomic regions at the single cell-level, during early postnatal development and/or after injury, will likely help understanding properties of these ELC sources.

The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

M2-like tumor-associated macrophages-secreted Wnt1 and Wnt3a promotes dedifferentiation and metastasis via activating ß-catenin pathway in thyroid cancer.

BACKGROUND: Thyroid carcinoma (TC) has been a global issue for its rapid increasing incidence worldwide. Although most TC was not so aggressive with a good prognosis, treatment against anaplastic TC was relatively limited and the mechanisms are not well elucidated yet. METHODS: TC cell lines (IHH4 and TPC-1) were used. Flow cytometry was used to identify the surface marker of M2-like tumor-associated macrophages (TAMs) from cell culture. Quantitative real-time polymerase chain reaction, western blot analysis, immunostaining, and immunohistochemistry were used to detect the expression of Wnt1, Wnt3a, components of Wnt/ß-catenin pathway, and proliferation/epithelial-mesenchymal transition (EMT)-related proteins. Alkaline phosphatase activity assay, colony formation assay, and transwell assay were used to examine the roles of Wnt1, Wnt3a, and ß-catenin pathway in cell dedifferentiation, proliferation, migration, and invasion of TC cells, respectively. Subcutaneous tumor growth was monitored in nude mice. RESULTS: Coculture with M2-like TAMs facilitated dedifferentiation, proliferation, migration, and invasion in TC cells. EMT and proliferation-related proteins were also promoted in cocultured TC cells. The level of Wnt1 and Wnt3a was increased in the coculture system. Block of Wnt1 or Wnt3a suppressed malignant behaviors in cocultured tumor cells. Furthermore, Wnt1 or Wnt3a knockdown inhibited Wnt/ß-catenin signaling pathway, and suppressed EMT and proliferation-related signals in cocultured tumor cells. Knockdown of Wnt1 or Wnt3a inhibited tumor growth in xenograft model. CONCLUSION: M2-like TAMs promoted dedifferentiation, proliferation, and metastasis of TC by Wnt1 and Wnt3a secretion and ensuing ß-catenin activation.

Downregulation of tumor-derived exosomal miR-34c induces cancer-associated fibroblast activation to promote cholangiocarcinoma progress.

BACKGROUND: This study aimed to investigate the exact regulatory mechanisms of exosomal miR-34c in mediating communication between cholangiocarcinoma cells and fibroblasts. METHODS: Exosomes were isolated from HuCCT-1 and HIBEC cells using differential ultracentrifugation and identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) method. Real-time quantitative PCR (qRT-PCR) and western blotting analyses were performed to assess the levels of pro-inflammatory factors, and fibroblast-related proteins and Wnt-linked signaling pathway proteins, respectively. Exosome-tracking was performed with confocal microscopy. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Transwell assays were used to measure cell proliferation and migration, respectively. Further, the oncogenicity of cholangiocarcinoma cells was analyzed in nude mice transplanted tumor model. RESULTS: The analysis suggested that the expression of miR-34c was decreased in exosomes from HuCCT-1 cells. Moreover, miR-34c in exosomes mediated fibroblast activation by directly targeting WNT1. Additionally, cancer-associated fibroblasts (CAFs) activated by downregulation of exosomal miR-34c promoted cholangiocarcinoma progression. CONCLUSIONS: Thus, miR-34c in exosomes was found to be a key player in regulating intercellular communication between tumor cells and fibroblasts.

ODAM promotes junctional epithelium-related gene expression via activation of WNT1 signaling pathway in an ameloblast-like cell line ALC.

OBJECTIVE: In this study, we investigated the potential and mechanism of odontogenic ameloblast-associated protein (ODAM) in the promoting junctional epithelium-related gene expression in an ameloblast-like cell line ALC. BACKGROUND: ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM-ARHGEF5-RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear. METHODS: A murine ameloblast-like cell line, ALC, was used to investigate the effects of ODAM on the JE-like changes of ALC cells in an epithelium-induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real-time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays. RESULTS: ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM-overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells. CONCLUSION: In conclusion, the research work showed that ODAM promotes junctional epithelium-related gene expression in ALC via the ODAM-WNT1 axis, which may provide new insight into the function of ODAM and JE formation.

Exendin-4 Attenuates Remodeling in the Remote Myocardium of Rats After an Acute Myocardial Infarction by Activating ß-Arrestin-2, Protein Phosphatase 2A, and Glycogen Synthase Kinase-3 and Inhibiting ß-Catenin.

PURPOSE: This study tested if the protective anti-remodeling effect of GLP-1 agonist Exendin-4 after an acute myocardial infarction (MI) in rats involves inhibition of the Wnt1/ß-catenin signaling pathway. METHODS: Rats were divided into sham, sham + Exendin-4 (10 µg/day, i.p), MI, and MI + Exendin-4. MI was introduced to rats by permanent left anterior descending coronary artery (LAD) ligation. RESULTS: On day 7 post-infraction, MI rats showed LV dysfunction with higher serum levels of cardiac markers. Their remote myocardia showed increased mRNA and protein levels of collagen I/III with higher levels of reactive oxygen species (ROS) and inflammatory cytokines, as well as protein levels of Wnt1, phospho-Akt, transforming growth factor (TGF-ß1), Smad, phospho-Smad3, α-SMA, caspase-3, and Bax. They also showed higher protein levels of phospho-glycogen synthase kinase-3ß (p-GSK3ß), as well as total, phosphorylated, and nuclear ß-catenin with a concomitant decrease in the levels of cyclic adenosine monophosphate (cAMP), mRNA of manganese superoxide dismutase (MnSOD), and protein levels of Bcl-2, ß-arrestin-2, and protein phosphatase-2 (PP2A). Administration of Exendin-4 to MI rats reduced the infarct size and reversed the aforementioned signaling molecules without altering protein levels of TGF-1ß and Wnt1 or Akt activation. Interestingly, Exendin-4 increased mRNA levels of MnSOD, protein levels of ß-arrestin-2 and PP2A, and ß-catenin phosphorylation but reduced the phosphorylation of GSK3ß and Smad3, and total ß-catenin levels in the LV of control rats. CONCLUSION: Exendin-4 inhibits the remodeling in the remote myocardium of rats following acute MI by attenuating ß-catenin activation and activating ß-arrestin-2, PP2A, and GSK3ß. Graphical Abstract A graphical abstract that illustrates the mechanisms by which Exendin-4 inhibits cardiac remodeling in remote myocardium of left ventricle MI-induced rats. Mechanisms are assumed to occur in the cardiomyocytes and/or other resident cells such as fibroblast. Β-catenin activation and nuclear translocation are associated with increased synthesis of inflammatory cytokines and transforming growth factor ß-1 (TGF-ß1). GSK3ß is inhibited by phosphorylation at Ser9. Under normal conditions, ß-catenin is degraded in the cytoplasm by the active GSK3ß-dependent degradation complex (un-phosphorylated) which usually phosphorylates ß-catenin at Ser33/37/Thr41. After MI, TGF-ß1, and Wnt 1 levels are significantly increased, the overproduction of Wnt1 induces ß-catenin stabilization and nuclear translocation through increasing the phosphorylation of disheveled (DVL) protein which in turn phosphorylates and inhibits GSK3ß. TGF-ß1 stimulates the phosphorylation of Smad-3 and subsequent nuclear translocation to activate the transcription of collage 1/III and α-smooth muscle actin (α-SMA). Besides, TGF-ß1 stabilizes cytoplasmic ß-catenin levels indirectly by phosphorylation of Akt at Thr308-induced inhibition of GSK3ß by increasing phosphorylation of Ser9. Exendin-4, and possibly through G protein-coupled receptors (GPCRs), increases levels of cAMP and upregulates ß-arrestin-2 levels. Both can result in a positive inotropic effect. Besides, ß-arrestin-2 can stimulate PP2A to dephosphorylation Smad3 (inhibition) and GSK3ß (activation), thus reduces fibrosis and prevents the activation of ß-catenin and collagen deposition.

Frizzled 1 and Wnt1 as new potential therapeutic targets in the traumatically injured spinal cord.

Despite the experimental evidence pointing to a significant role of the Wnt family of proteins in physiological and pathological rodent spinal cord functioning, its potential relevance in the healthy and traumatically injured human spinal cord as well as its therapeutic potential in spinal cord injury (SCI) are still poorly understood. To get further insight into these interesting issues, we first demonstrated by quantitative Real-Time PCR and simple immunohistochemistry that detectable mRNA expression of most Wnt components, as well as protein expression of all known Wnt receptors, can be found in the healthy human spinal cord, supporting its potential involvement in human spinal cord physiology. Moreover, evaluation of Frizzled (Fz) 1 expression by double immunohistochemistry showed that its spatio-temporal and cellular expression pattern in the traumatically injured human spinal cord is equivalent to that observed in a clinically relevant model of rat SCI and suggests its potential involvement in SCI progression/outcome. Accordingly, we found that long-term lentiviral-mediated overexpression of the Fz1 ligand Wnt1 after rat SCI improves motor functional recovery, increases myelin preservation and neuronal survival, and reduces early astroglial reactivity and NG2+ cell accumulation, highlighting the therapeutic potential of Wnt1 in this neuropathological situation.