Elevated pulmonary levels of Axin2 in mice exposed to herbicide 2,4-D with or without endotoxin.

2,4-Dichlorophenoxyacetic acid (2,4-D), a member of the phenoxy family of herbicides is commonly used in agriculture for controlling broadleaf weeds but its uncontrolled and incoherent use has been linked to incidences of lung toxicity. The present study aimed to understand the molecular mechanisms behind the 2,4-D alone or in combination with endotoxin (lipopolysaccharide [LPS]) induced pulmonary toxicity. Blood and lung samples were collected from Swiss albino mice (n = 48) following chronic exposure to high (37 mg/kg; 1/10th of LD50 ) and low (18.5 mg/kg; 1/20th of LD50 ) doses of 2,4-D alone or in combination with endotoxin (80 µg/animal). Transcriptome analysis revealed Wnt Canonical signaling as one of the top dysregulated pathways in mice lung following exposure to 2,4-D with and without endotoxin (LPS) co-exposure. Global view of differentially expressed genes showed increased messenger RNA expression of Axin2 by 0.26, 2.58, 3.14, 2.59, and 2.97 folds following exposure to LPS, high dose alone or in combination with LPS and low dose alone or in combination with LPS, respectively. The microarray data were validated using quantitative polymerase chain reaction and immunohistochemistry. Furthermore, the plasma concentration of Axin2 was elevated in the high dose group as revealed by Sandwich ELISA. The data taken together suggest a role of Axin2 to activate the Canonical Wnt signaling pathway in 2,4-D and or endotoxin-induced lung damage in mice.

The effect of baicalein on Wnt/ß-catenin pathway and miR-25 expression in Saos-2 osteosarcoma cell line

Background/aim: Osteosarcoma is the most common primary bone malignancy that occurs frequently in children and adolescents. Baicalein, a flavonoid that has attracted great attention in recent years with its strong antitumor activity, shows a wide range of biological and pharmaceutical effects.MicroRNAs have been found to be involved in many critical processes in cancers. This study aimed to investigate the effect of baicalein and miR-25 on Wnt/ß-catenin signaling pathway of osteosarcoma cell line Saos-2. Materials and methods: Cell viability was assessed, and qRT-PCR and Western blot were performed to study the effects of baicalein on expression of Wnt/ß-catenin signaling pathway-realted genes (ß-catenin, GSK-3ß, and Axin2) of Saos-2 cells. Results: Our results indicated that baicalein can inhibit the proliferation (IC50 value 35 µM), regulate Wnt/ß-catenin pathway and also increase miR-25 expression of Saos-2. Baicalein and also miR-25 decreased the expression of ß-catenin and Axin2, while increasing the expression of GSK-3ß. Down regulation of miR-25 decreased the expression of GSK-3ß, while ß-catenin and Axin2 expression increased. Conclusion: These findings demonstrate that baicalein may target genes related to the Wnt/ß-catenin pathway by regulating miR-25 expression and may be a potential Wnt/ß-catenin pathway inhibitor for osteosarcoma therapy.

Chinese Herbal Formulas Miao-Yi-Ai-Tang Inhibits the Proliferation and Migration of Lung Cancer Cells through Targeting ß-Catenin/AXIN and Presents Synergistic Effect with Cisplatin Suppressing Lung Cancer.

OBJECTIVE: Lung cancer is one of the major causes of cancer deaths worldwide, and the five-year survival still remains low despite the improvement of screening, prevention, and treatment methods. Chinese herbal medicines have been widely used for tumor prevention and treatment. Miao-Yi-Ai-Tang (Miao) is a novel herbal formulation and shows a potential anticancer effect. Materials and Methods. Human Small Cell Lung Cancer Cell was used for study in vitro. After treatments by Miao and Cisplatin (DDP), the invasion, migration, proliferation, and apoptosis of cells were detected by transwell, wound healing, CCK-8, and flow cytometry, respectively. The expression of ß-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of ß-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of. RESULTS: We found that Miao could inhibit invasion, migration, and proliferation and promote apoptosis of human lung cancer cells. Meanwhile, Miao and DDP presented synergy regulating the proliferation and apoptosis of lung cancer cells. The percentage of lung cancer cells in S and G2 stages was increased markedly by Miao. Besides, the expression of c-myc, AXIN, and ß-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of. CONCLUSIONS: Chinese herbal formulas Miao could suppress lung cancer through targeting the ß-catenin/AXIN signaling pathway. Therefore, our findings may provide a novel strategy for the prevention and treatment of lung cancer.ß-catenin, AXIN, and c-myc was detected by qRT-PCR and immunohistochemistry staining. Western blotting was applied for measuring the protein expression of.

Curcumin and quercetin synergistically inhibit cancer cell proliferation in multiple cancer cells and modulate Wnt/ß-catenin signaling and apoptotic pathways in A375 cells.

BACKGROUND: Traditional therapy using natural products, especially flavonoids and alkaloids have been in practice for a long time. Among flavonoids, curcumin, quercetin, berberine, and epigallocatechin have been studied in greater detail in terms of their anticancer and anti-inflammatory activities. Although many studies focused on the PI3K, MAP kinase and NF-κB pathways, a thorough investigation of modulation of players in the apoptotic and Wnt/ß-catenin signaling pathway by curcumin and quercetin has not been done. Also, only few studies have been carried out on curcumin and quercetin co-treatment studies. HYPOTHESIS/PURPOSE: We hypothesized that the combination of natural products will have synergistic effects and the antiproliferative effect will be attenuated via apoptotic as well as Wnt/ß-catenin signaling pathways. STUDY DESIGN AND METHODS: To test our hypothesis, we compared potency of natural anticancer agents in four cancer cell lines, A549, HCT116, MCF7, and A375 by MTT and colony proliferation assays and investigated mechanism of anticancer activities by analyzing players in apoptotic and Wnt/ß-catenin signaling pathways in A375 cells treated with test agents individually or in combination. RESULTS: Epicatechins, up to 100 µM concentration, did not inhibit cancer cell proliferation, while curcumin inhibited proliferation in A549 and HCT116 cancer cell lines with an IC50 of 3 to 8.5 µM. Quercetin showed stronger inhibition of cell proliferation than berberine. Combination study with two most potent agents, curcumin and quercetin, in 4 cancer cell lines, suggested synergistic effect on cell proliferation with several fold decreases in IC50. Further investigation of the mechanism of action of curcumin and quercetin in melanoma cells, A375, suggested that inhibition of cell proliferation occurred through down-regulation of Wnt/ß-catenin signaling pathway proteins, DVL2, ß-catenin, cyclin D1, Cox2, and Axin2. In addition, both curcumin and quercetin induced apoptosis by down-regulating BCL2 and inducing caspase 3/7 through PARP cleavage. CONCLUSION: These results demonstrate that curcumin and quercetin inhibit cancer cell proliferation synergistically and Wnt/ß-catenin signaling and apoptotic pathways are partly responsible for antiproliferative activities.

Wnt/ß-catenin signaling pathway is involved in regulating the migration by an effective natural compound brucine in LoVo cells.

BACKGROUND: Colorectal cancer remains the third most common malignancies and migration is one of the main factors for its high mortality rate. Brucine, a natural plant alkaloid, has been proved to possess a variety of pharmacological functions including anti-tumor activities. PURPOSE: The aim of this study was to investigate the inhibitory effect of brucine on the colorectal cancer and the underlying mechanism. METHODS: In this study, colony formation assay and transwell assay were used to investigate the effect of brucine on LoVo cells viability and migration. Immunofluorescence assay, western blot assay and Gelatin zymography assay were used to study the mechanism of brucine. Xenograft model in nude mice was induced to investigate the in vivo effect of brucine on LoVo cells. RESULTS: Brucine could significantly decrease the viability, inhibit the colony formation and induce the apoptosis of LoVo cells. Brucine could also suppress the migration of LoVo cells in a dose-dependent manner. Western blot analysis elucidated that the inhibition of migration was associated with the decreasing expression of matrix metalloproteinases including MMP2, MMP3 and MMP9. Moreover, we found that treatment of brucine could downregulate the expression of Frizzled-8, Wnt5a, APC and GSNK1A1, and increase the expression of AXIN1. Meanwhile, brucine also decreased the phosphorylation level of LRP5/6 and GSK3ß, and increased the level of p-ß-catenin. Xenografted model in nude mice study also revealed that oral administration of brucine could inhibit the growth and migration of LoVo cells by activating the expression of AXIN1 and p-ß-catenin. CONCLUSION: Brucine could suppress the migration of the colorectal cancer in vitro and in vivo and the effect was associated with the inhibition of the Wnt/ß-catenin signaling pathway.

[Does Malnutrition during Fetal Life Have a Potential to Be a Precipitating Factor for Developmental Disorders?]

OBJECTIVES: The developmental origins of health and disease paradigm (DOHaD) is a concept that fetal environmental factors affect adult phenotypes. We performed experiments to evaluate the DOHaD theory in developmental disorders using mouse models. METHODS: In vitro fertilization and embryo transfer techniques were used for mouse production. The AIN93G-control diet, which contains 20% protein (CD), 5% protein-restricted diet (PR), and PR with supplemental folic acid (FA) were provided as experimental diets to mothers. The body weights (BWs) of mothers and offspring, and the blood-clinical biochemistry results of mothers were examined. In addition, gene expression and genomic methylation in the brain of adult offspring and behavioral phenotypes of adult offspring were examined. RESULTS: Pregnant mothers that consumed the protein-restricted diets, namely, PR and FA, exhibited reduction in BW. The values of protein-related parameters determined by blood-clinical biochemistry decreased in the PR fed groups. The BWs of neonates and adult offspring did not change. The offspring exposed to maternal hyponutrition exhibited increased activity in the home cage and enhanced fear and anxiety-like behavior. The adult offspring of the PR-fed group and FA-fed groups exhibited different patterns of mRNA expression and genomic methylation in the brain. CONCLUSIONS: The maternal PR diet affected the progenies' behavioral phenotypes and epigenetic outcomes in the brain. However, the behavioral changes induced by maternal protein restriction were very slight. Hence, interactions between several genetic factors and environmental exposures such as maternal malnutrition may cause developmental and psychiatric disorders.

Major Reduction in Axillary Lymph Node Dissections After Neoadjuvant Systemic Therapy for Node-Positive Breast Cancer by combining PET/CT and the MARI Procedure.

BACKGROUND: Axillary lymph node dissection (ALND) is frequently performed for node-positive (cN+) breast cancer patients. Combining positron emission tomography/computed tomography (PET/CT) before-NST and the MARI (marking axillary lymph nodes with radioactive iodine seeds) procedure after neoadjuvant systemic therapy (NST) has the potential for avoiding unnecessary ALNDs. This report presents the results from implementation of this strategy. METHODS: All breast cancer patients treated with NST at the Netherlands Cancer Institute who underwent a PET/CT and the MARI procedure from July 2014 to July 2017 were included in the study. All the patients underwent tailored axillary treatment according to a protocol based on the combined results of PET/CT before NST and the MARI procedure after NST. With this protocol, patients showing one to three FDG-avid axillary lymph nodes (ALNs) on PET/CT (cN<4) and a tumor-negative MARI node receive no further axillary treatment. All cN (<4) patients with a tumor-positive MARI node receive locoregional radiotherapy, as well as patients with four or more FDG-avid ALNs [cN(4+)] and a tumor-negative MARI node after NST. An ALND is performed only for cN(4+) patients with a tumor-positive MARI node. RESULTS: The data of 159 patients who received a PET/CT before NST and a MARI procedure after NST were analyzed. Of these patients, 110 had one to three FDG-avid ALNs and 49 patients showed four or more FDG-avid ALNs on PET/CT before NST. For 130 patients (82%), ALND was omitted. Locoregional radiotherapy was administered to 91 patients (57%), and 39 patients (25%) received no further axillary treatment. CONCLUSION: Combining pre-NST axillary staging with PET/CT and post-NST staging with the MARI procedure resulted in an 82% reduction of ALNDs for cN + breast cancer patients.

Photoperiodic and Diurnal Regulation of WNT Signaling in the Arcuate Nucleus of the Female Djungarian Hamster, Phodopus sungorus.

The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Retinoic Acid Ameliorates Pancreatic Fibrosis and Inhibits the Activation of Pancreatic Stellate Cells in Mice with Experimental Chronic Pancreatitis via Suppressing the Wnt/ß-Catenin Signaling Pathway.

Pancreatic fibrosis, a prominent feature of chronic pancreatitis (CP), induces persistent and permanent damage in the pancreas. Pancreatic stellate cells (PSCs) provide a major source of extracellular matrix (ECM) deposition during pancreatic injury, and persistent activation of PSCs plays a vital role in the progression of pancreatic fibrosis. Retinoic acid (RA), a retinoid, has a broad range of biological functions, including regulation of cell differentiation and proliferation, attenuating progressive fibrosis of multiple organs. In the present study, we investigated the effects of RA on fibrosis in experimental CP and cultured PSCs. CP was induced in mice by repetitive cerulein injection in vivo, and mouse PSCs were isolated and activated in vitro. Suppression of pancreatic fibrosis upon administration of RA was confirmed based on reduction of histological damage, α-smooth muscle actin (α-SMA) expression and mRNA levels of ß-catenin, platelet-derived growth factor (PDGF)-Rß transforming growth factor (TGF)-ßRII and collagen 1α1 in vivo. Wnt 2 and ß-catenin protein levels were markedly down-regulated, while Axin 2 expression level was up-regulated in the presence of RA, both in vivo and in vitro. Nuclear translation of ß-catenin was significantly decreased following RA treatment, compared with cerulein-induced CP in mice and activated PSCs. Furthermore, RA induced significant PSC apoptosis, inhibited proliferation, suppressed TCF/LEF-dependent transcriptional activity and ECM production of PSC via down-regulation of TGFßRII, PDGFRß and collagen 1α1 in vitro. These results indicate a critical role of the Wnt/ß-catenin signaling pathway in RA-induced effects on CP and PSC regulation and support the potential of RA as a suppressor of pancreatic fibrosis in mice.

Effects of a grape-supplemented diet on proliferation and Wnt signaling in the colonic mucosa are greatest for those over age 50 and with high arginine consumption.

A diet rich in fruits and vegetables, and a grape-derived compound, resveratrol, have been linked to a reduced incidence of colon cancer. In vitro and in vivo, resveratrol suppresses Wnt signaling, a pathway constitutively activated in over 85 % of colon cancers.Thirty participants were placed on a low resveratrol diet and subsequently allocated to one of three groups ingesting 1/3-to-1 lb (0.15-0.45 kg) of grapes per day for 2 weeks. Dietary information was collected via 24-h recall. Colon biopsies for biomarker analysis were obtained pre- and post-grape and evaluated for the expression of Wnt pathway target genes and for markers of proliferation by RT-PCR and immunohistochemistry.Participants lost an average of 2 · 6 lb (1.2 kg, p = 0 · 0018) during the period of grape ingestion. The expression of CyclinD1 (p < 0 · 01), AXIN2, CD133 (p = 0 · 02) and Ki67 (p = 0 · 002) were all reduced after grape ingestion. Individuals over 50 years of age and those with high dietary arginine consumption had increased basal expression of CyclinD1, AXIN2, cMYC and CD133 (p value range 0 · 04 to <0 · 001) that, following grape ingestion, were reduced to levels seen in younger participants.The reduction in Wnt signaling and mucosal proliferation seen following short-term ingestion of 1/3-1 lb (0.15-0.45 kg) of grapes per day may reduce the risk of mutational events that can facilitate colon carcinogenesis. The potential benefit is most marked for high-risk older individuals and individuals whose diet is high in arginine intake. Dietary grape supplementation may play a role in colon cancer prevention for high-risk individuals.

[Changes in Wnt pathway inhibiting factors in nitrosamine-induced esophageal precancerosis lesions and effect of gexia zhuyu decoction].

OBJECTIVE: To discuss the changes in Wnt pathway inhibiting factors in esophageal precancerosis lesions induced by methyl benzyl nitrosamine (MBNA) and the effect of Gexia Zhuyu decoction. METHOD: Wistar rats were subcutaneously injected with MBNA (3.5 mg x kg(-1) for twice per week to establish the model. Since the 1st day after the model establishment, they were orally administered with Gexia Zhuyu decoction (16, 8 mg x kg(-1)). At the 10th week, esophageal tissues were collected to observe the pathological changes of esophageal mucosa, detect SFRP1, sFRP4, Axin1, Axin2 and GSK-3ß mRNA levels.by fluorescent quantitation PCR analysis and ß-catenin protein level by Western blotting. RESULT: Being induced by MBNA, rats in the model group showed slight atypical hyperplasia in the histopathological examination. Compared with the normal group, Gexia Zhuyu decoction dose high and low groups showed no significant pathomorphological and histological changes. The model group showed lower gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and higher ß-catenin protein expression level (P < 0.01) than the normal control group. The Gexia Zhuyu decoction low dose group showed higher gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and lower ß-catenin protein expression level (P < 0.01) than the normal control group. CONCLUSION: Up-regulated ß-catenin protein level and down-regulated Wnt pathway could enhance Wnt pathway activity of MBNA-induced esophageal precancerous lesions. Gexia Zhuyu decoction could down-regulate the ß-catenin protein level and up-regulate the transcription level of Wnt pathway inhibiting factors, but could not block MBNA-induced esophageal precancerosis lesions.

Changes in Wnt pathway inhibiting factors in nitrosamine-induced esophageal precancerosis lesions and effect of gexia zhuyu decoction / 中国中药杂志

<p><b>OBJECTIVE</b>To discuss the changes in Wnt pathway inhibiting factors in esophageal precancerosis lesions induced by methyl benzyl nitrosamine (MBNA) and the effect of Gexia Zhuyu decoction.</p><p><b>METHOD</b>Wistar rats were subcutaneously injected with MBNA (3.5 mg x kg(-1) for twice per week to establish the model. Since the 1st day after the model establishment, they were orally administered with Gexia Zhuyu decoction (16, 8 mg x kg(-1)). At the 10th week, esophageal tissues were collected to observe the pathological changes of esophageal mucosa, detect SFRP1, sFRP4, Axin1, Axin2 and GSK-3β mRNA levels.by fluorescent quantitation PCR analysis and β-catenin protein level by Western blotting.</p><p><b>RESULT</b>Being induced by MBNA, rats in the model group showed slight atypical hyperplasia in the histopathological examination. Compared with the normal group, Gexia Zhuyu decoction dose high and low groups showed no significant pathomorphological and histological changes. The model group showed lower gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and higher β-catenin protein expression level (P < 0.01) than the normal control group. The Gexia Zhuyu decoction low dose group showed higher gene transcription levels of esophageal tissues sFRP1, sFRP4, Axin1 and Axin2 (P < 0.05 or P < 0.01) and lower β-catenin protein expression level (P < 0.01) than the normal control group.</p><p><b>CONCLUSION</b>Up-regulated β-catenin protein level and down-regulated Wnt pathway could enhance Wnt pathway activity of MBNA-induced esophageal precancerous lesions. Gexia Zhuyu decoction could down-regulate the β-catenin protein level and up-regulate the transcription level of Wnt pathway inhibiting factors, but could not block MBNA-induced esophageal precancerosis lesions.</p>

A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3ß protein-dependent activation of the Wnt/ß-catenin signaling pathway.

Estrogen promotes growth in many tissues by activating Wnt/ß-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/ß-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced ß-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of ß-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of ß-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3ß. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/ß-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3ß-dependent activation of ß-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.

Differential activity of Wnt/beta-catenin signaling in the embryonic mouse thalamus.

In neural development, several Wnt genes are expressed in the vertebrate diencephalon, including the thalamus. However, roles of Wnt signaling in the thalamus during neurogenesis are not well understood. We examined Wnt/beta-catenin activity in embryonic mouse thalamus and found that a Wnt target gene Axin2 and reporter activity of BAT-gal transgenic mice show similar, differential patterns within the thalamic ventricular zone, where ventral and rostral regions had lower activity than other regions. Expression of Wnt ligands and signaling components also showed complex, differential patterns. Finally, based on partially reciprocal patterns of Wnt and Shh signals in the thalamic ventricular zone, we tested if Shh signal is sufficient or necessary for the differential Axin2 expression. Analysis of mice with enhanced or reduced Shh signal showed that Axin2 expression is similar to controls. These results suggest that differential Wnt signaling may play a role in patterning the thalamus independent of Shh signaling.

Influence of an LRP5 cytoplasmic SNP on Wnt signaling and osteoblastic differentiation.

The low density lipoprotein receptor-related protein 5 (LRP5) is a key determinant of bone mass, via the Wnt signaling pathway control of osteoblast function. This study examined human LRP5 signaling and the effects of an intracellular domain single nucleotide polymorphism (SNP: p.V1525A) on osteoblast differentiation and mineralization. Constitutively active LRP5 was constructed by deletion of the extracellular domain of LRP5 (LRP5DeltaN). Expression of LRP5DeltaN-V, which carries the allele p.1525V, induced higher beta-catenin/TCF-LEF activity compared to LRP5DeltaN-A, which carries the allele p.1525A. In a yeast two-hybrid assay, LRP5DeltaN-V also demonstrated a stronger interaction with AXIN than LRP5DeltaN-A. Expression of either of the alleles did not change cell proliferation. However, cells expressing LRP5DeltaN-V showed increased alkaline phosphatase activity and bone nodule formation compared to cells transfected with empty vector or LRP5DeltaN-A after osteogenic supplement (OS: beta-glycerophosphate and l-ascorbic acid) treatment. Cells expressing LRP5DeltaN-V revealed significantly increased bone sialoprotein (BSP) expression after 7 days of OS treatment and maintained elevated expression until day 21. Osteocalcin (OCN) mRNA levels were increased after 14-21 days of OS treatment in LRP5DeltaN-V expressing cells. LRP5DeltaN-V expressing cells demonstrated positive interaction with BMP-2 signaling of transcription at the SBE-luc promoter. LRP5 signaling is affected by the cytoplasmic SNP, p.V1525A. mRNA levels of Runx2 and Osterix were not affected by this SNP.

Maternal methyl supplements increase offspring DNA methylation at Axin Fused.

Transient environmental exposures during mammalian development can permanently alter gene expression and metabolism by influencing the establishment of epigenetic gene regulatory mechanisms. The genomic characteristics that confer such epigenetic plasticity upon specific loci, however, have not been characterized. Methyl donor supplementation of female mice before and during pregnancy permanently increases DNA methylation at the viable yellow agouti (A(vy)) metastable epiallele in the offspring. The current study tested whether another murine metastable epiallele, axin fused (Axin(Fu)), similarly exhibits epigenetic plasticity to maternal diet. We found that methyl donor supplementation of female mice before and during pregnancy increased DNA methylation at Axin(Fu) and thereby reduced by half the incidence of tail kinking in Axin(Fu)/+ offspring. The hypermethylation was tail-specific, suggesting a mid-gestation effect. Our results indicate that stochastic establishment of epigenotype at metastable epialleles is, in general, labile to methyl donor nutrition, and such influences are not limited to early embryonic development.

Inhibition of Wnt/Axin/beta-catenin pathway activity promotes ventral CNS midline tissue to adopt hypothalamic rather than floorplate identity.

Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, we have explored the role of the Wnt/Axin/beta-catenin pathway in this process. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.

Transgenerational inheritance of epigenetic states at the murine Axin(Fu) allele occurs after maternal and paternal transmission.

Phenotypic variation that cannot be explained by genetic or environmental heterogeneity has intrigued geneticists for decades. The molecular basis of this phenomenon, however, is largely a mystery. Axin-fused (Axin(Fu)), first identified in 1937, is a classic example of a mammalian allele displaying extremely variable expression states. Here we demonstrate that the presence or absence of its characteristic phenotype, a kinked tail, correlates with differential DNA methylation at a retrotransposon within Axin(Fu) and identify mutant transcripts arising adjacent to the retrotransposon LTR that are likely to be causative of the phenotype. Furthermore, the epigenetic state at Axin(Fu) can be inherited transgenerationally after both maternal and paternal transmission. This is in contrast to epigenetic inheritance at the murine agouti-viable yellow (A(vy)) allele, which occurs through the female only. Unlike the egg, the sperm contributes very little (if any) cytoplasm to the zygote, and therefore paternal inheritance at Axin(Fu) argues against the possibility that the effects are due to cytoplasmic or metabolic influences. Consistent with the idea of transgenerational inheritance of epigenetic marks, we find that the methylation state of Axin(Fu) in mature sperm reflects the methylation state of the allele in the somatic tissue of the animal, suggesting that it does not undergo epigenetic reprogramming during gametogenesis. Finally, we show that epigenetic inheritance is influenced by strain background. These findings enable us to propose a model for transgenerational epigenetic inheritance in mammals.

Activation of AXIN2 expression by beta-catenin-T cell factor. A feedback repressor pathway regulating Wnt signaling.

The Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in the pathway play a key role in many cancers. Although Wnts act to stabilize beta-catenin levels in the cytosol and nucleus, a multiprotein complex containing adenomatous polyposis coli, glycogen synthase kinase 3beta, and Axin1 or its homolog Axin2/Axil/conductin promotes beta-catenin phosphorylation and subsequent proteasomal degradation. We found that the rat Axil gene was strongly induced upon neoplastic transformation of RK3E cells by mutant beta-catenin or gamma-catenin or after ligand-induced activation of a beta-catenin-estrogen receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells activated the conductin gene, and human cancers with defective beta-catenin regulation had elevated AXIN2 gene and protein expression. Expression of AXIN2/Axil was strongly repressed in cancer cells by restoration of wild type adenomatous polyposis coli function or expression of a dominant negative form of T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role in the ability of beta-catenin to activate AXIN2 transcription. In contrast to AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by beta-catenin-TCF. Because Axin2 can inhibit beta-catenin abundance and function, the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling. Additionally, although Axin1 and Axin2 have been thought to have comparable functions, the observation that Wnt pathway activation elevates AXIN2 but not AXIN1 expression suggests that there may be potentially significant functional differences between the two proteins.