This study examined resting levels of catabolic and anabolic osteokines related to <em>Wnt</em> signaling and their responses to a single bout of plyometric exercise in child and adolescent females. Fourteen premenarcheal girls [10.5 (1.8) y old] and <em>12</em> postmenarcheal adolescent girls [15.0 (1.0) y old] performed a plyometric exercise trial. One resting and 3 postexercise blood samples (5 min, 1 h, and 24 h postexercise) were analyzed for sclerostin, dickkopf-1 (DKK-1), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-β ligand (RANKL), and transforming growth factors (TGF-β1, TGF-β2, and TGF-β3). Premenarcheal girls had significantly higher resting sclerostin, TGF-β1, TGF-β2, and TGF-β3 than the postmenarcheal girls, with no significant time effect or group-by-time interaction. DKK-1 was higher in premenarcheal compared with postmenarcheal girls. There was an overall significant DKK-1 decrease from baseline to 1 h postexercise, which remained lower than baseline 24 h postexercise in both groups. There was neither a significant group effect nor group-by-time interaction in OPG, RANKL, and their ratio. RANKL decreased 5 min postexercise compared with baseline and remained significantly lower from baseline 24 h following the exercise. No changes were observed in OPG. OPG/RANKL ratio was significantly elevated compared with resting values 1 h postexercise. In young females, high-impact exercise induces an overall osteogenic effect through a transitory suppression of catabolic osteokines up to 24 h following exercise.

E-cadherin is a tumor suppressor gene in invasive lobular breast cancer. However, a proportion of high-grade ductal carcinoma shows reduced/loss of E-cadherin. In this study, we assessed the underlying mechanisms and molecular implications of E-cadherin loss in invasive ductal carcinoma. This study used large, well-characterized cohorts of early-stage breast cancer-evaluated E-cadherin expression via various platforms including immunohistochemistry, microarray analysis using Illumina HT-<em>12</em> v3, copy number analysis using Affymetrix SNP 6.0 arrays, and next-generation sequencing for differential gene expression. Our results showed 27% of high-grade invasive ductal carcinoma showed reduced/loss of E-cadherin membranous expression. CDH1 copy number loss was in 21% of invasive ductal carcinoma, which also showed low CDH1 mRNA expression (p = 0.003). CDH1 copy number was associated with copy number loss of TP53, ATM, BRCA1, and BRCA2 (p < 0.001). Seventy-nine percent of invasive ductal carcinoma with reduced CDH1 mRNA expression showed elevated expression of E-cadherin transcription suppressors TWIST2, ZEB2, NFKB1, LLGL2, CTNNB1 (p < 0.01). Reduced/loss E-cadherin expression was associated with differential expression of 2143 genes including those regulating <em>Wnt</em> (FZD2, GNG5, HLTF, WNT2, and CER1) and PIK3-AKT (FGFR2, GNF5, GNGT1, IFNA17, and IGF1) signaling pathways. Interestingly, key genes differentially expressed between invasive lobular carcinoma and invasive ductal tumors did not show association with E-cadherin loss in invasive ductal carcinoma. We conclude that E-cadherin loss in invasive ductal carcinoma is likely a consequence of genomic instability occurring during carcinogenesis. Potential novel regulators controlling E-cadherin expression in invasive ductal carcinoma warrant further investigation.

The aim of this study was to assess the expression of IGF-I and IGF-II in phyllodes tumours and fibroadenomas and to see if there is any correlation between nuclear beta-catenin expression and IGF-I and IGF-II expression in these tumours. In a previous study, it has been shown that <em>Wnt</em> signalling is important in the pathogenesis of phyllodes tumours of the breast. Epithelial <em>Wnt</em>5a overexpression and stromal <em>Wnt</em>2 overexpression were associated with abnormal, nuclear localization of beta-catenin in the stromal cells of these tumours. However, not all tumours with beta-catenin accumulation showed <em>Wnt</em> overexpression. One other possible cause of beta-catenin accumulation is overexpression of insulin-like growth factors (IGFs), as both IGF-I and IGF-II have been shown to stabilize beta-catenin. In this study, 30 fibroadenomas of the breast were assessed for beta-catenin expression using immunohistochemistry and the results were compared with previous data from 119 phyllodes tumours. In situ hybridization was used to assess IGF-I and IGF-II expression in 23 phyllodes tumours and 16 fibroadenomas. Nineteen phyllodes tumours (83%) showed widespread overexpression of IGF-II and 5/23 (22%) showed widespread overexpression of IGF-I. IGF-I expression correlated with nuclear beta-catenin staining in phyllodes tumours. Malignant phyllodes tumours showed no or little IGF-I expression. There was a degree of nuclear beta-catenin expression in the stroma (weak in 33%, moderate in 27%, and strong in 40%) in all fibroadenomas and nuclear beta-catenin staining correlated with IGF-I overexpression. Extensive IGF-II overexpression was also found in the majority of fibroadenomas (<em>12</em>/16). These results support the hypothesis that IGF-I and IGF-II overexpression may be important in the pathogenesis of fibroepithelial neoplasms of the breast and that IGF-I overexpression is likely to be contributing to the nuclear beta-catenin localization observed in the tumours.

To investigate the role of <em>Wnt</em>-beta-catenin signaling in bone remodeling, we analyzed the bone phenotype of female Axin2-lacZ knockout (KO) mice. We found that trabecular bone mass was significantly increased in 6- and <em>12</em>-month-old Axin2 KO mice and that bone formation rates were also significantly increased in 6-month-old Axin2 KO mice compared with wild-type (WT) littermates. In vitro studies were performed using bone marrow stromal (BMS) cells isolated from 6-month-old WT and Axin2 KO mice. Osteoblast proliferation and differentiation were significantly increased and osteoclast formation was significantly reduced in Axin2 KO mice. Nuclear beta-catenin protein levels were significantly increased in BMS cells derived from Axin2 KO mice. In vitro deletion of the beta-catenin gene under Axin2 KO background significantly reversed the increased alkaline phosphatase activity and the expression of osteoblast marker genes observed in Axin2 KO BMS cells. We also found that mRNA expression of Bmp2 and Bmp4 and phosphorylated Smad1/5 protein levels were significantly increased in BMS cells derived from Axin2 KO mice. The chemical compound BIO, an inhibitor of glycogen synthase kinase 3beta, was utilized for in vitro signaling studies in which upregulated Bmp2 and Bmp4 expression was measured in primary calvarial osteoblasts. Primary calvarial osteoblasts were isolated from Bmp2(fx/fx);Bmp4(fx/fx) mice and infected with adenovirus-expressing Cre recombinase. BIO induced Osx, Col1, Alp and Oc mRNA expression in WT cells and these effects were significantly inhibited in Bmp2/4-deleted osteoblasts, suggesting that BIO-induced Osx and marker gene expression were Bmp2/4-dependent. We further demonstrated that BIO-induced osteoblast marker gene expression was significantly inhibited by Osx siRNA. Taken together, our findings demonstrate that Axin2 is a key negative regulator in bone remodeling in adult mice and regulates osteoblast differentiation through the beta-catenin-BMP2/4-Osx signaling pathway in osteoblasts.

<em>Wnt</em> signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the <em>Wnt</em> pathway can promote stem cell self-renewal and/or direct lineage commitment. <em>Wnt</em> proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing <em>Wnt</em> signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of <em>Wnt</em> proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on <em>Wnt</em> proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize <em>Wnt</em>3a proteins on microbeads or a glass surface. This procedure requires ∼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active <em>Wnt</em> proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for <em>Wnt</em>/β-catenin-dependent transcription. Immobilization efficiency can be measured <em>12</em>-18 h after seeding the cells and takes 2-4 h. The covalent immobilization of <em>Wnt</em> proteins can also be used for single-cell analysis using <em>Wnt</em>-coated microbeads (<em>12</em>-18 h of live imaging) and to create a <em>Wnt</em> platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for <em>Wnt</em> immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed.

There has been substantial progress in the management of patients with osteoporosis and the prevention of osteoporotic fractures. Currently available strong anti-resorptive agents are bisphosphonates and an anti-receptor activator of nuclear factor-kappa B ligand (RANKL) antibody, denosumab. Although bisphosphonates and denosumab both inhibit bone resorption and prevent vertebral and non-vertebral fractures, their mechanisms of action are different. Whereas bisphosphonates' effects on bone mineral density and fracture peak around 3 to 5 years and become plateaued, those of denosumab are maintained for up to 10 years. There are differences in the modes of action of these two drugs. Bisphosphonates accumulate on the mineralized bone surface and are released by the acid environment under osteoclastic bone resorption, whereas denosumab is not accumulated on bone but directly binds RANKL and inhibits its binding to the receptor RANK. Thus, the reduction in denosumab concentration 4 to 6 months after injection may enable RANK to bind to RANKL, where it is highly expressed, such as in damaged bone regions. As anabolic agents, only teriparatide has been available for a long time, but abaloparatide, a synthetic analog of PTHrP(1-34), is currently under development. Because of the difference in the preferential binding conformations of PTH1 receptor between teriparatide and abaloparatide, the latter shows anabolic effects with fewer bone resorptive effects. Romosozumab, an anti-sclerostin antibody, inhibits the action of sclerostin, a canonical <em>Wnt</em> signal inhibitor secreted from osteocytes, and enhances canonical <em>Wnt</em> signaling. Romosozumab robustly increases vertebral and proximal femoral bone mineral density within <em>12</em> months and inhibits vertebral and clinical fractures in patients with osteoporosis by enhancing bone formation and inhibiting bone resorption. In this review, we summarize the recent advances in therapeutic agents for the treatment of osteoporosis and discuss future prospects with their use.

<AbstractText>Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective.</AbstractText><AbstractText>Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day <em>12</em>.</AbstractText><AbstractText>We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-β and <em>Wnt</em> signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-β/<em>Wnt</em> signaling activity.</AbstractText><AbstractText>Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.</AbstractText>

<AbstractText>A chemical approach to examine the role of <em>Wnt</em> signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs).</AbstractText><AbstractText>LSCs were isolated from human donor eyes and cultured as single cells for <em>12</em> to 14 days with the following small molecules: IIIC3, an antagonist of the <em>Wnt</em> signaling inhibitor Dickkopf (DKK), and IC15, a <em>Wnt</em> signaling inhibitor. Proliferation of LSCs in the presence of IIIC3 and IC15 was determined by the number of cells and colonies established. Maintenance of stemness was determined by p63α, cytokeratin (K)<em>12</em>, and K14 expression.</AbstractText><AbstractText>Activation of <em>Wnt</em>, through IIIC3-mediated DKK inhibition, resulted in similar colony forming efficiency (CFE) as in the untreated LSCs, but significantly increased the number of cultivated cells 7.21% with 5 μM. Inhibition of <em>Wnt</em> with IC15 significantly reduced the CFE (P ≤ 0.01) and the number of cultivated cells by 16% to 29%. Percentage of cells expressing high levels of p63α (p63αbright) and quantity of small cells (≤<em>12</em> μm), which contain the LSCs, increased 4.71% and 11.26% (both P < 0.05), respectively, with 5 μM IIIC3. All concentrations of IIIC3 and IC15 retained the K14 undifferentiated marker (97%), while differentiation, as detected by expression of K<em>12</em>, was found in up to 2% of cells in 1 μM IIIC3, 1 μM IC15, or 5 μM IIIC3.</AbstractText><AbstractText><em>Wnt</em> signaling is required in LSC proliferation and maintenance of an undifferentiated state. The current study is a proof of concept that the <em>Wnt</em> pathway could be modulated in LSCs to enhance or decrease the efficiency of human LSC expansion.</AbstractText>

Lung cancer causes more deaths worldwide than any other cancer. In addition to cigarette smoking, dietary factors may contribute to lung carcinogenesis. Epidemiologic studies, including the environment and genetics in lung cancer etiology (EAGLE), have reported increased consumption of red/processed meats to be associated with higher risk of lung cancer. Heme-iron toxicity may link meat intake with cancer. We investigated this hypothesis in meat-related lung carcinogenesis using whole genome expression. We measured genome-wide expression (HG-U133A) in 49 tumor and 42 non-involved fresh frozen lung tissues of 64 adenocarcinoma EAGLE patients. We studied gene expression profiles by high-versus-low meat consumption, with and without adjustment by sex, age, and smoking. Threshold for significance was a false discovery rate (FDR) ≤ 0.15. We studied whether the identified genes played a role in heme-iron related processes by means of manually curated literature search and gene ontology-based pathway analysis. We found that gene expression of 232 annotated genes in tumor tissue significantly distinguished lung adenocarcinoma cases who consumed above/below the median intake of fresh red meats (FDR = 0.<em>12</em>). Sixty-three (∼ 28%) of the 232 identified genes (<em>12</em> expected by chance, P-value < 0.001) were involved in heme binding, absorption, transport, and <em>Wnt</em> signaling pathway (e.g., CYPs, TPO, HPX, HFE, SLCs, and <em>WNTs</em>). We also identified several genes involved in lipid metabolism (e.g., NCR1, TNF, and UCP3) and oxidative stress (e.g., TPO, SGK2, and MTHFR) that may be indirectly related to heme-toxicity. The study's results provide preliminary evidence that heme-iron toxicity might be one underlying mechanism linking fresh red meat intake and lung cancer.

Polycystic ovary syndrome (PCOS) is an endocrine disorder in women of reproductive age. In addition to anovulation, endometrial dysfunction can reduce fertility in PCOS. The cyclical changes of endometrium are controlled by estrogen and progesterone via modulating <em>Wnt</em>/B-catenin pathway. Clomiphene citrate (CC) and letrozole are used to induce ovulation; unlike letrozole, there is a discrepancy between ovulation and pregnancy rates in CC-treated cycles. Because of the anti-estrogenic effects of CC on endometrium, we compared the expression of the key molecules of <em>Wnt</em>/B-catenin pathway in the endometrium of women taking CC and letrozole. This study included PCOS and healthy women divided into the groups stimulated with letrozole (5 mg) or CC (100 mg) as well as NO-treatment groups. The endometrial thickness and hormonal profile were measured on day <em>12</em> of the menses.Using real-time polymerase chain reaction and western blot, we evaluated mRNA and protein expression of B-catenin, glycogen synthase kinase 3 beta (GSK3B), dickkopf <em>Wnt</em> signaling pathway inhibitor 1 (DKK1), and estrogen receptor 1 (ESR1) in the endometrial samples. Significantly, the mean serum estrogen and progesterone were lower and higher, respectively, in letrozole than CC groups. The endometrial thickness was significantly reduced in CC. The proteins expression of active B-catenin, inactive GSK3B, and ESR1 were significantly decreased in CC-treated groups. The mRNA and protein assessment of DKK1 showed significantly higher expression in CC. Our results indicate that letrozole can provide an acceptable activation of <em>Wnt</em>/B-catenin pathway, resulting in adequate proliferation of endometrium in the women receiving letrozole compared to CC.

UNASSIGNED

Urothelial bladder cancer (UBC) is one of the most lethal urological malignancies in the world. Patients with UBC are routinely given chemotherapy which results in a median survival of <em>12</em>-15 months. Nuclear-enriched abundant transcript 1 (NEAT1) functions as an oncogene and could be used as a therapeutic target for human UBC. However, the involvement of NEAT1 in doxorubicin (DOX) resistance of UBC has been poorly demonstrated.

UNASSIGNED

Quantitative Real-time PCR (qRT-PCR) was used to detect the expression levels of NEAT1 and miR-214-3p in UBC tissues and cells. Bioinformatics prediction, RNA pull-down and qRT-PCR were used to assay the regulation manner of NEAT1 and miR-214-3p. Loss/gain function of NEAT1 and miR-214-3p together with western blot, drug resistance assay and flow cytometry were used to explore the influence of NEAT1 in DOX resistance was correlative with miR-214-3p. Finally, luciferase assay system was applied to determine the Wnt/β-catenin signal activity.

UNASSIGNED

NEAT1 was upregulated and miR-214-3p was downregulated in DOX-resistant UBC tissues and cells. NEAT1 knockdown inhibited J82 and T24 cells to DOX chemosensitivity by negatively regulating miR-214-3p expression. NEAT1/miR-214-3p contributed to DOX resistance of UBC preliminary through the Wnt/β-catenin pathway.

UNASSIGNED

NEAT1 contributed to DOX resistance of UBC through the Wnt/β-catenin pathway partly by negatively regulating miR-214-3p expression. Our findings will provide a promising ncRNA targeted therapeutic strategy for UBC with DOX resistance.

Metallothionein 1H (MT1H) expression level is downregulated in several kinds of tumors, including hepatocellular cancer (HCC). However, its biological functions and underlying mechanisms in HCC is largely unknown. The current study aimed to demonstrate the expression status, biological roles and potential mechanisms of MT1H in HCC.

We investigated the expression level of MT1H in the Cancer Genome Atlas (TCGA) dataset and a panel of <em>12</em> paired tumor/non-tumor tissues. In vitro, gain-of-function experiments were performed to examine the role of MT1H on HCC cell proliferation, invasion, and migration. Using bioinformatics assay, reporter assays, quantitative real-time PCR, and western blotting, we explored the possible mechanisms underlying the role of MT1H in HCC cells. In vivo nude mice experiments were performed to assess the anti-proliferative role of MT1H in HCC.

Downregulation of MT1H was observed in TCGA dataset and a panel of <em>12</em> paired tumor/non-tumor tissues. Ectopic overexpression of MT1H in HepG2 and Hep3B cells inhibited cell proliferation, invasion, and migration. Gene Set Enrichment Analysis (GSEA) showed that MT1H might involve in regulation of Wnt/β-catenin pathway. Top/Fop reporter assay confirmed that MT1H had an effect on Wnt/β-catenin signaling. Real-time PCR showed MT1H expression decreased the expression of Wnt/β-catenin target genes. Western blotting assay showed that overexpression of MT1H inhibited the nuclear translocation of β-catenin and that the Akt/GSK-3β axis mediated the modulatory role of MT1H on Wnt/β-catenin signaling in HCC. In vivo nude mice experiments demonstrated that MT1H suppressed the proliferation of HCC cells. Taken together, MT1H suppressed the proliferation, invasion and migration of HCC cells via regulating Wnt/β-catenin signaling pathway.

This study demonstrated that through inhibiting Wnt/β-catenin pathway, MT1H suppresses the proliferation and invasion of HCC cells. MT1H may be a potential target for HCC therapy.

Bone fractures in patients with diabetes mellitus heal poorly and require innovative therapies to support bone regeneration. Here, we assessed whether sulfated hyaluronan included in collagen-based scaffold coatings can improve fracture healing in diabetic rats. Macroporous thermopolymerized lactide-based scaffolds were coated with collagen including non-sulfated or sulfated hyaluronan (HA/sHA3) and inserted into 3 mm femoral defects of non-diabetic and diabetic ZDF rats. After <em>12</em> weeks, scaffolds coated with collagen/HA or collagen/sHA3 accelerated bone defect regeneration in diabetic, but not in non-diabetic rats as compared to their non-coated controls. At the tissue level, collagen/sHA3 promoted bone mineralization and decreased the amount of non-mineralized bone matrix. Moreover, collagen/sHA3-coated scaffolds from diabetic rats bound more sclerostin in vivo than the respective controls. Binding assays confirmed a high binding affinity of sHA3 to sclerostin. In vitro, sHA3 induced BMP-2 and lowered the RANKL/OPG expression ratio, regardless of the glucose concentration in osteoblastic cells. Both sHA3 and high glucose concentrations decreased the differentiation of osteoclastic cells. In summary, scaffolds coated with collagen/sHA3 represent a potentially suitable biomaterial to improve bone defect regeneration in diabetic conditions. The underlying mechanism involves improved osteoblast function and binding sclerostin, a potent inhibitor of <em>Wnt</em> signaling and osteoblast function.

UNASSIGNED

Osteoporosis and Alzheimer's disease are common diseases of aging that would seem to be unrelated, but may be linked through the influence of bone-derived signals on brain function. The aim of the current study is to investigate the relationship between circulating levels of bone-related biomarkers and cognition.

UNASSIGNED

The population included 103 community-dwelling older individuals with memory concerns but without cognitive impairment. A global cognition summary measure was collected at baseline and 6, <em>12</em>, and 18 months post-enrollment by converting raw scores from 19 cognitive function tests to z-scores and averaging. Baseline plasma concentrations of bone-related biomarkers, including undercarboxylated, carboxylated, and total osteocalcin, parathyroid hormone, C-terminal telopeptide of collagen 1 (CTX-1), procollagen type 1 amino-terminal propeptide, osteoprotegrin, osteopontin, Dickkopf <em>WNT</em> signaling pathway inhibitor 1 (Dkk1), sclerostin, and amyloid β peptides (Aβ40 and Aβ42), were measured.

UNASSIGNED

Using sex, age, and education-adjusted mixed-effects models, we found that baseline levels of TNF-related apoptosis-inducing ligand (TRAIL; p < .001), Dkk1 (p = .014), and CTX-1 (p = .046) were related to the annual rate of change of global cognition over the 18 month follow-up. In cognitive domain-specific analysis, baseline TRAIL was found to be positively related to the annual rate of change in episodic (p < .001) and working memory (p = .016), and baseline Dkk1 was positively related to semantic memory (p = .027) and negatively related to working memory (p = .016).

UNASSIGNED

These results further confirm the link between bone and brain health and suggest that circulating levels of bone-related biomarkers may have diagnostic potential to predict worsening cognition.

The alveolar epithelium consists of (ATI) and type II (ATII) cells. ATI cells cover the majority of the alveolar surface due to their thin, elongated shape and are largely responsible for barrier function and gas exchange. During lung injury, ATI cells are susceptible to injury, including cell death. Under some circumstances, ATII cells also die. To regenerate lost epithelial cells, ATII cells serve as progenitor cells. They proliferate to create new ATII cells and then differentiate into ATI cells [¹,²,³]. Regeneration of ATI cells is critical to restore normal barrier and gas exchange function. Although the signaling pathways by which ATII cells proliferate have been explored [⁴,⁵,⁶,⁷,⁸,⁹,¹⁰,¹¹,^<em>12</em>], the mechanisms of ATII-to-ATI cell differentiation have not been well studied until recently. New studies have uncovered signaling pathways that mediate ATII-to-ATI differentiation. Bone morphogenetic protein (BMP) signaling inhibits ATII proliferation and promotes differentiation. <em>Wnt</em>/β-catenin and ETS variant transcription factor 5 (Etv5) signaling promote proliferation and inhibit differentiation. Delta-like 1 homolog (Dlk1) leads to a precisely timed inhibition of Notch signaling in later stages of alveolar repair, activating differentiation. Yes-associated protein/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) signaling appears to promote both proliferation and differentiation. We recently identified a novel transitional cell state through which ATII cells pass as they differentiate into ATI cells, and this has been validated by others in various models of lung injury. This intermediate cell state is characterized by the activation of Transforming growth factor beta (TGFβ) and other pathways, and some evidence suggests that TGFβ signaling induces and maintains this state. While the abovementioned signaling pathways have all been shown to be involved in ATII-to-ATI cell differentiation during lung regeneration, there is much that remains to be understood. The up- and down-stream signaling events by which these pathways are activated and by which they induce ATI cell differentiation are unknown. In addition, it is still unknown how the various mechanistic steps from each pathway interact with one another to control differentiation. Based on these recent studies that identified major signaling pathways driving ATII-to-ATI differentiation during alveolar regeneration, additional studies can be devised to understand the interaction between these pathways as they work in a coordinated manner to regulate differentiation. Moreover, the knowledge from these studies may eventually be used to develop new clinical treatments that accelerate epithelial cell regeneration in individuals with excessive lung damage, such as patients with the Acute Respiratory Distress Syndrome (ARDS), pulmonary fibrosis, and emphysema.

The shortage of organs for kidney transplantation has created the need to develop new strategies to restore renal structure and function. Given our recent finding that the lymph node (LN) can serve as an in vivo factory to generate or sustain complex structures like liver, pancreas, and thymus, we investigated whether it could also support kidney organogenesis from mouse renal embryonic tissue (metanephroi). Here we provide the first evidence that metanephroi acquired a mature phenotype upon injection into LN, and host cells likely contributed to this process. Urine-like fluid-containing cysts were observed in several grafts <em>12</em> weeks post-transplantation, indicating metanephroi transplants' ability to excrete products filtered from the blood. Importantly, the kidney graft adapted to a loss of host renal mass, speeding its development. Thus, the LN might provide a unique tool for studying the mechanisms of renal maturation, cell proliferation, and fluid secretion during cyst development. Moreover, we provide evidence that inside the LN, short-term cultured embryonic kidney cells stimulated with the <em>Wnt</em> agonist R-Spondin 2 gave rise to a monomorphic neuron-like cell population expressing the neuronal 200-kDa neurofilament heavy marker. This finding indicates that the LN might be used to validate the differentiation potential of candidate stem cells in regenerative nephrology.

Increased complement component 1q (C1q) secretion with aging leads to muscle fibrosis and atrophy whereas resistance training attenuates circulating C1q levels. This study aimed to clarify whether resistance exercise-induced reduction of C1q secretion contributes to the inhibition of fibrosis and atrophy in aged muscles. Young (13-wk-old) and aged (38-wk-old) senescence-accelerated mouse prone 1 mice were randomly assigned to one of 4 groups: a young or aged sedentary control group, or a young or aged resistance training (climbing a ladder 3 d/wk for <em>12</em> wk) group. We found that resistance training ameliorated muscle fibrosis and atrophy in aged mice, concomitant with decreased circulating and muscle C1q levels and attenuated activation of muscle <em>Wnt</em> signaling (glycogen synthase kinase β/β-catenin), including β-catenin in satellite (Pax7+/DAPI+) and fibroblast (vimentin+/DAPI+) cells. Furthermore, during muscle regeneration after mice were injured by cardiotoxin injection, we observed a reduction in circulating C1q levels, the inhibition of muscle fibrosis and repair, and decreased in the activation of muscle cytoplasmic and nuclear β-catenin in aged mice from the resistance training group, but these effects were cancelled by a single preadministration of exogenous recombinant C1q. In addition, resistance training attenuated aging-related muscle loss concomitant with decreased expression of both muscle ring-finger protein 1 and muscle atrophy F-box in the muscle. Thus, resistance training-induced changes in circulating C1q levels may contribute to the prevention of muscle fibrosis and atrophy via muscle <em>Wnt</em> signaling in senescent mice.-Horii, N., Uchida, M., Hasegawa, N., Fujie, S., Oyanagi, E., Yano, H., Hashimoto, T., Iemitsu, M. Resistance training prevents muscle fibrosis and atrophy via down-regulation of C1q-induced <em>Wnt</em> signaling in senescent mice.

Identification of specific biomarkers for ischemic stroke is necessary due to their abilities to improve treatment outcomes. Many studies have demonstrated the involvement of microRNAs (miRNAs) in the pathogenesis and complications of ischemic stroke and patient outcomes. We found that the expression of miR-874-3p was downregulated in clinical samples of ischemic stroke. Thus, the present study explored the potential role of miR-874-3p in ischemic stroke and related mechanisms. A mouse model of ischemic stroke was constructed by middle cerebral artery occlusion. The relationship among miR-874-3p, C-X-C motif chemokine ligand <em>12</em> (CXCL<em>12</em>) and the <em>Wnt</em>/β-catenin pathway was explored by dual luciferase reporter assay and Western blot analysis. Angiogenesis and brain tissue apoptosis were evaluated by immunofluorescence staining and TUNEL staining, respectively. ELISA was introduced to measure levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8 and IL-10 in brain tissues. Primary hippocampal neuronal cells were isolated from the mouse model of ischemic stroke and incubated with HUVECs for HUVEC tube formation. High expression of CXCL<em>12</em> and low expression of miR-874-3p were confirmed in ischemic stroke. And miR-874-3p was found to target and downregulate CXCL<em>12</em>, thus reducing TNF-α, IL-1, IL-6 and IL-8 levels, but enhancing IL-10 level. Collectively, upregulating miR-874-3p inhibits CXCL<em>12</em> expression to promote angiogenesis and inhibit inflammation in ischemic stroke mice by activating the <em>Wnt</em>/β-catenin pathway, which may provide a new direction of ischemic stroke treatment.

<strong class="sub-title"> Keywords: </strong> Angiogenesis; C-X-C motif chemokine ligand <em>12</em>; Inflammatory factor release; Ischemic stroke; microRNA-874-3p.

We have studied the evolution of <em>Wnt</em> genes in cnidarians and the expression pattern of all <em>Wnt</em> ligands in the hydrozoan Hydractinia echinata. Current views favor a scenario in which <em>12</em> <em>Wnt</em> sub-families were jointly inherited by cnidarians and bilaterians from their last common ancestor. Our phylogenetic analyses clustered all medusozoan genes in distinct, well-supported clades, but many orthologous relationships between medusozoan <em>Wnts</em> and anthozoan and bilaterian <em>Wnt</em> genes were poorly supported. Only seven anthozoan genes, <em>Wnt</em>2, <em>Wnt</em>4, <em>Wnt</em>5, <em>Wnt</em>6, <em>Wnt</em> 10, <em>Wnt</em>11, and <em>Wnt</em>16 were recovered with strong support with bilaterian genes and of those, only the <em>Wnt</em>2, <em>Wnt</em>5, <em>Wnt</em>11, and <em>Wnt</em>16 clades also included medusozoan genes. Although medusozoan <em>Wnt</em>8 genes clustered with anthozoan and bilaterian genes, this was not well supported. In situ hybridization studies revealed poor conservation of expression patterns of putative <em>Wnt</em> orthologs within Cnidaria. In polyps, only <em>Wnt</em>1, <em>Wnt</em>3, and <em>Wnt</em>7 were expressed at the same position in the studied cnidarian models Hydra, Hydractinia, and Nematostella. Different expression patterns are consistent with divergent functions. Our data do not fully support previous assertions regarding <em>Wnt</em> gene homology, and suggest a more complex history of <em>Wnt</em> family genes than previously suggested. This includes high rates of sequence divergence and lineage-specific duplications of <em>Wnt</em> genes within medusozoans, followed by functional divergence over evolutionary time scales.

The present study aimed to investigate the relationship between <em>wnt</em>/β-catenin signaling pathway and kidney impairment in diabetic nephropathy (DN) mice as well as the renoprotective effect of rhein (RH). Mice were randomly divided into four groups (n = 6): db/db mice treated with RH (DN + RH), db/db mice (DN), db/m mice treated with RH (NC + RH) and db/m mice (NC). RH-treated groups were administered orally at a daily dose <em>12</em>0 mg/kg. Mice were sacrificed after <em>12</em> weeks of treatments. In our study, increased albuminuria, together with weight gain and hyperglycemia was observed in the beginning of the study and continued to increase throughout the length of the study (<em>12</em> weeks). Histopathologic changes were observed in the DN group. Expectedly, mice receiving the treatment with RH were protected from this injury. Meanwhile, the expression of nephrin, a podocyte-specific marker, was significantly reduced while <em>wnt</em>1, p-GSK-3β/tGSK-3β, p-β-catenin/tβ-catenin were higher in the DN group mice when analyzed by immunofluorescence and Western blotting. RH reversed these above changes. <em>wnt</em>/β-catenin signaling pathway participates in RH ameliorating kidney injury in DN mice. The manipulation of RH might act as a promising therapeutic intervention for DN.

Glucocorticoids (GC) are used for the treatment of inflammatory diseases, including various forms of arthritis. However, their use is limited, amongst others, by adverse effects on bone. The <em>Wnt</em> and bone formation inhibitor sclerostin was recently implicated in the pathogenesis of GC-induced osteoporosis. However, data are ambiguous. The aim of this study was to assess the regulation of sclerostin by GC using several mouse models with high GC levels and two independent cohorts of patients treated with GC. Male 24-week-old C57BL/6 and 18-week-old DBA/1 mice exposed to GC and <em>12</em>-week-old mice with endogenous hypercortisolism displayed reduced bone formation as indicated by reduced levels of P1NP and increased serum sclerostin levels. The expression of sclerostin in femoral bone tissue and GC-treated bone marrow stromal cells, however, was not consistently altered. In contrast, GC dose- and time-dependently suppressed sclerostin at mRNA and protein level in human mesenchymal stromal cells, and this effect was GC receptor-dependent. In line with the human cell culture data, patients with rheumatoid arthritis (RA, n=101) and polymyalgia rheumatica (PMR, n=21) who were exposed to GC had lower serum levels of sclerostin than healthy age- and sex-matched controls (-40%, p<0.01 and -26.5%, p<0.001, respectively). In summary, sclerostin appears to be differentially regulated by GC in mice and humans as it is suppressed by GCs in humans but is not consistently altered in mice. Further studies are required to delineate the differences between GC regulation of sclerostin in mice and humans and assess whether sclerostin mediates GC-induced osteoporosis in humans.

Blastoporal expression of the T-box transcription factor gene brachyury is conserved in most metazoans [1, 2]. Its role in mesoderm formation has been intensively studied in vertebrates [3-6]. However, its fundamental function near the blastopore is poorly understood in other phyla. Cnidarians are basal metazoans that are important for understanding evolution of metazoan body plans [7, 8]. Because they lack mesoderm, they have been used to investigate the evolutionary origins of mesoderm [1, 9-11]. Here, we focus on corals, a primitive clade of cnidarians that diverged from sea anemones ∼500 mya [<em>12</em>]. We developed a microinjection method for coral eggs to examine Brachyury functions during embryogenesis of the scleractinian coral, Acropora digitifera. Because Acropora embryos undergo pharynx formation after the blastopore closes completely [13-15], they are useful to understand Brachyury functions in gastrulation movement and pharynx formation. We show that blastoporal expression of brachyury is directly activated by <em>Wnt</em>/β-catenin signaling in the ectoderm of coral embryos, indicating that the regulatory axis from <em>Wnt</em>/β-catenin signaling to brachyury is highly conserved among eumetazoans. Loss-of-function analysis demonstrated that Brachyury is required for pharynx formation but not for gastrulation movement. Genome-wide transcriptome analysis demonstrated that genes positively regulated by Brachyury are expressed in the ectoderm of Acropora gastrulae, while negatively regulated genes are in endoderm. Therefore, germ layer demarcation around the blastopore appears to be the evolutionarily conserved role of Brachyury during gastrulation. Compared with Brachyury functions in vertebrate mesoderm-ectoderm and mesoderm-endoderm demarcation [4-6], our results suggest that the vertebrate-type mesoderm may have originated from brachyury-expressing ectoderm adjacent to endoderm.

BACKGROUND

It has been shown that the expression of potassium channel tetramerization domain containing <em>12</em> (KCTD<em>12</em>) as a regulator of GABAB receptor signaling is reversely associated with gastrointestinal stromal tumors. In present study we examined the probable role of KCTD<em>12</em> in regulation of several signaling pathways and chromatin remodelers in esophageal squamous cell carcinoma (ESCC).

METHODS

KCTD<em>12</em> ectopic expression was done in KYSE30 cell line. Comparative quantitative real time PCR was used to assess the expression of stem cell factors and several factors belonging to the WNT/NOTCH and chromatin remodeling in transfected cells in comparison with non-transfected cells.

RESULTS

We observed that the KCTD<em>12</em> significantly down regulated expression of NANOG, SOX2, SALL4, KLF4, MAML1, PYGO2, BMI1, BRG1, MSI1, MEIS1, EGFR, DIDO1, ABCC4, ABCG2, and CRIPTO1 in transfected cells in comparison with non-transfected cells. Migration assay showed a significant decrease in cell movement in ectopic expressed cells in comparison with non-transfected cells (p = 0.02). Moreover, KCTD<em>12</em> significantly decreased the 5FU resistance in transfected cells (p = 0.01).

CONCLUSIONS

KCTD<em>12</em> may exert its inhibitory role in ESCC through the suppression of WNT /NOTCH, stem cell factors, and chromatin remodelers and can be introduced as an efficient therapeutic marker.

BACKGROUND

MicroRNAs (miRNAs) play momentous roles in various biological processes including cell differentiation. However, little is known about the role of miRNAs in human dental pulp cells (hDPCs) during odontogenic differentiation. The aims of this study were to investigate the expression of miRNAs in the primary culture of hDPCs when incubated in odontogenic medium.

METHODS

The potential characteristics of hDPCs were investigated by miRNA microarray and real-time reverse transcriptase polymerase chain reaction. Bioinformatics (ie, target prediction, Gene Ontology analysis, and Kyoto Encyclopedia of Genes and Genomes mapping tools) were applied for predicting the complementary target genes of miRNAs and their biological functions.

RESULTS

A total of 22 miRNAs were differentially expressed in which <em>12</em> miRNAs up-regulated and 10 miRNAs down-regulated in differentiated hDPCs compared with the control. The target genes of differential miRNAs were predicted to associate with several biological functions and signaling pathways including the mitogen-activated protein kinase (MAPK) and the <em>Wnt</em> signaling pathway.

CONCLUSIONS

The differential expression miRNAs may be involved in governing hDPC odontogenic differentiation, thus contributing to the future investigations of regulatory mechanisms in reparative dentin formation and dental pulp regeneration.