<em>Wnt</em> signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to <em>Wnt</em>, Norrin activates the <em>Wnt</em>/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-<em>12</em> and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus <em>Wnt</em>8-mouse Fz8CRD complex reveals Norrin mimics <em>Wnt</em> for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/<em>Wnt</em> signalling.

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical <em>wnt</em> pathway in the differentiation of mouse bone marrow-derived MSCs (mMSCs) into AT II cells. Using a modified co-culture system with murine lung epithelial-<em>12</em> (MLE-<em>12</em>) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3β and β-catenin in the canonical <em>wnt</em> pathway were up-regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co-culture system to activate <em>wnt</em>/β-catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of <em>wnt</em>/β-catenin signaling promoted mMSCs migration towards ALI mouse-derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl-2/Bax induced by H(2) O(2), which simultaneously caused reduced GSK 3β and β-catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical <em>wnt</em> pathway activation, which may be significant to their application in ALI.

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼<em>12</em>-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular <em>Wnt</em> signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

The cellular retinol-binding protein-1 (CRBP-1) plays a key role in the esterification and intercellular transfer of retinol. By in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy (CLSM), we show that, in normal liver, CRBP-1 is strongly expressed in the cytoplasm of hepatic stellate cells (HSCs) and myofibroblasts (MFs) with only low CRBP-1 levels in hepatocytes. By contrast, in 196 hepatocellular carcinoma (HCC) specimens CRBP-1 expression in MFs was down-regulated in 83%. Patients with high CRBP-1 expression in MFs had a significantly higher 2-year survival as compared with patients with low CRBP-1 expression (52% vs. 29%, respectively; P =.034). An aberrant nuclear CRBP-1 accumulation resulting from cytoplasmic invagination was found in 29% of HCCs. Nuclear CRBP-1 staining correlated positively with a favorable tumor stage (Okuda stage I; P =.01) and negatively with the Ki-67(+) proliferation fraction (PF). A Ki-67(+) PF of>> or =10% was associated with a lower 2-year survival probability as compared with patients with a Ki-67(+) PF of <10% (<em>12</em>% vs. 40%, respectively; P =.015). Prognosis did not correlate with the nuclear beta-catenin expression. There was, however, a close correlation between nuclear CRBP-1 inclusions and nuclear beta-catenin staining in HCCs (P =.008), suggesting a cross talk between CRBP-1 and the <em>Wnt</em>/wingless signal transduction pathway. In conclusion, our findings demonstrate that CRBP-1 detection may be useful for the discrimination between nonneoplastic and neoplastic liver cells and suggest that modulation of CRBP-1 expression in HCCs contributes to tumor growth and progression via retinoid-mediated signaling and disruption of cellular vitamin A homeostasis.

OBJECTIVE

Abnormal localization of beta-catenin is frequently observed in human gastric cancers. The aim of the present study was to evaluate relationships among gastrointestinal differentiation phenotypes, beta-catenin localization and mutations of Wnt signalling genes.

RESULTS

Seventy-seven regions in 39 gastric adenocarcinomas were classified according to beta-catenin localization and gastric and intestinal phenotypes. Cases with membranous beta-catenin localization showed a gradual decrease from gastric (G) (55% = 6/11) and gastric-and-intestinal-mixed (GI) (17% = 5/29) to intestinal (I) (0% = 0/21) phenotypes, while those with nuclear localization showed a concomitant increase: 18% (2/11), 41% (12/29), 95% (20/21) and 63% (10/16) for G, GI, I and null type (N), respectively (P < 0.001, membranous versus nuclear localization in G, GI through I). Mutations in exon 3 of the beta-catenin gene were found in G (50% = 1/2), GI (67% = 8/12), I (45% = 9/20) and N (0% = 0/10) regions with nuclear beta-catenin localization (GI versus N, P < 0.01; I versus N, P < 0.05). Adenomatous polyposis coli (APC) gene mutations were demonstrated only in GI, I and N types, irrespective of beta-catenin localization. Molecular analysis of these genes revealed 10 tumours to be heterogeneous out of 16 informative cases (62.5%).

CONCLUSIONS

Intestinal phenotypic expression is accompanied by a shift from membranous to cytoplasmic/nuclear accumulation of beta-catenin. In contrast, N-type regions may progress along a different pathway.

OBJECTIVE

The therapeutic effects of pulsed electromagnetic fields (PEMFs) on osteoporosis have been documented. However, the precise mechanisms by which PEMFs elicit these favorable biological responses are still not fully understood. This study aimed to systematically investigate the effects of PEMFs on bone mass and Wnt/β-catenin signaling pathway in ovariectomized rats.

METHODS

Thirty 3-month-old female Sprague Dawley rats were randomly assigned to one of three groups: sham-operated control (sham), ovariectomy (OVX), and ovariectomy with PEMFs treatment (PEMFs). One week following ovariectomy surgery, rats in the PEMFs group were exposed to PEMFs for 40 min/day, 5 days/week, for 12 weeks.

RESULTS

After 12-week interventions, serum 17β-estradiol and bone-specific alkaline phosphatase levels increased in the PEMFs group. Bone mineral density of the femur and the fifth lumbar vertebral body also increased in the PEMFs group. Histomorphometrical studies showed that PEMFs improved trabecular area, trabecular width, and trabecular number by 77.50%, 17.38% and 51.06%, respectively, and reduced trabecular separation by 44.28% compared with the OVX group. Biomechanical studies showed that PEMFs increased maximum load and energy to failure in the fifth lumbar vertebral body. Quantitative real-time RT-PCR analysis showed that PEMFs increased the mRNA expressions of <em>Wnt</em>3a, low-density lipoprotein receptor-related protein 5(LRP5), β-catenin, c-myc and runt-related gene 2 (Runx2), and reduced dickkopf1 (DKK1) in ovariectomized rats. However, mRNA expression of Axin2 was not affected by PEMFs.

CONCLUSIONS

PEMFs can prevent ovariectomy-induced bone loss and deterioration of bone microarchitecture and strength, at least partly, through activation of Wnt/β-catenin signaling pathway.

AKI with incomplete epithelial repair is a major contributor to CKD characterized by tubulointerstitial fibrosis. Injury-induced epithelial secretion of profibrotic factors is hypothesized to underlie this link, but the identity of these factors and whether epithelial injury is required remain undefined. We previously showed that activation of the canonical Wnt signaling pathway in interstitial pericytes cell autonomously drives myofibroblast activation in vivo. Here, we show that inhibition of canonical Wnt signaling also substantially prevented TGFβ-dependent myofibroblast activation in vitro. To investigate whether Wnt ligand derived from proximal tubule is sufficient for renal fibrogenesis, we generated a novel mouse strain with inducible proximal tubule Wnt or 24 weeks postinjection. Compared with vehicle-treated controls, kidneys with tamoxifen-induced WntWnt target gene expression compared with controls. Notably, fibrotic kidneys had no evidence of inflammatory cytokine expression, leukocyte infiltration, or epithelial injury, despite the close histologic correlation of each with CKD. These results provide the first example of noninflammatory renal fibrosis. The fact that epithelial-derived Wnt ligand is sufficient to drive interstitial fibrosis provides strong support for the maladaptive repair hypothesis in the AKI to CKD transition.

Appropriate and controlled chondrogenesis and endochondral ossification play fundamental roles in the fracture healing cascade, a regenerative process involved in highly coordinated biological events, including the <em>Wnt</em>/β-catenin signaling pathway. To examine the role and importance of this pathway in chondrocytes, we studied bone repair of closed tibias fractures in Col2a1-ICAT transgenic mice, in which the <em>Wnt</em>/β-catenin signaling pathway is specially inhibited in chondrocytes. Radiological, histological, and histomorphometric analyses at 7, 9, <em>12</em>, 14, 21, and 28 days after fracture demonstrated the bone repairs were retarded in Col2a1-ICAT transgenic mice, due to reduced and delayed cartilage formation, chondrocyte hypertrophy, and bone generation. In addition, at 5 weeks, Col2a1-ICAT transgenic mice exhibited a weak mechanical tolerance to four-point bending. Furthermore, quantitative-PCR analysis revealed that the expression of genes associated specifically with cartilage extracellular matrix formation (collagen II, collagen X, and mmp13), bone remodeling (alp, collagen I, and osteocalcin), and vascular extravagation (vegf), and transcriptional activators involved in cartilage generation and ossification (sox9 and runx2) was decreased and delayed in the fracture sites of Col2a1-ICAT transgenic mice during healing. Collectively, these results suggest that <em>Wnt</em>/β-catenin signaling is critical for fracture healing, especially with respect to chondrogenesis and endochondral ossification. Thus, our study provides insight into the possible mechanisms of and therapeutic targets for improving normal facture repair and the healing of non-union fractures.

BACKGROUND

Hypoxic tumor microenvironment and maintenance of stemness contribute to drug resistance in breast cancer. However, whether Hypoxia-inducible factor-2α (HIF-2α) in hypoxic tumor microenvironment mediates conversion to a stem cell phenotype and chemoresistance of breast tumors has not been elucidated.

METHODS

The mRNA and protein expressions of HIF-1α, HIF-2α, Wnt and Notch pathway were determined using qRT-PCR and western blot. Cell viability and renew ability were assessed by MTT, Flow cytometric analysis and soft agar colony formation.

RESULTS

In our study, acute hypoxia (6-12 h) briefly increased HIF-1α expression, while chronic hypoxia (48 h) continuously enhanced HIF-2α expression and induced the resistance of breast cancer cells to Paclitaxel (PTX). Furthermore, HIF-2α overexpression induced a stem cell phenotype, the resistance to PTX and enhanced protein expression of stem cell markers, c-Myc, OCT4 and Nanog. Most importantly, Wnt and Notch signaling, but not including Shh, pathways were both activated by HIF-2α overexpression. Dickkopf-1 (DKK-1), a Wnt pathway inhibitor, and L685,458, an inhibitor of the Notch pathway, reversed the resistance to PTX and stem phenotype conversion induced by HIF-2α overexpression. In addition, HIF-2α overexpression enhanced tumorigenicity and resistance of xenograft tumors to PTX, increased activation of the Wnt and Notch pathways and induced a stem cell phenotype in vivo.

CONCLUSIONS

In conclusion, HIF-2α promoted stem phenotype conversion and induced resistance to PTX by activating Wnt and Notch pathways.

BACKGROUND

Macrophages are a heterogeneous cell population which in response to the cytokine milieu polarize in either classically activated macrophages (M1) or alternatively activated macrophages (M2). This plasticity makes macrophages essential in regulating inflammation, immune response and tissue remodeling and a novel therapeutic target in inflammatory diseases such as atherosclerosis. The aim of the study was to describe the transcriptomic profiles of differently polarized human macrophages to generate new hypotheses on the biological function of the different macrophage subtypes.

RESULTS

Polarization of circulating monocytes/macrophages of blood donors was induced in vitro by IFN-γ and LPS (M1), by IL-4 (M2a), and by IL-10 (M2c). Unstimulated cells (RM) served as time controls. Gene expression profile of M1, M2a, M2c and RM was assessed at 6, <em>12</em> and 24h after polarization with Whole Human Genome Agilent Microarray technique. When compared to RM, M1 significantly upregulated pathways involved in immunity and inflammation, whereas M2a did the opposite. Conversely, decreased and increased expression of mitochondrial metabolism, consistent with insulin resistant and insulin sensitive patterns, was seen in M1 and M2a, respectively. The time sequence in the expression of some pathways appeared to have some specific bearing on M1 function. Finally, canonical and non-canonical <em>Wnt</em> genes and gene groups, promoting inflammation and tissue remodeling, were upregulated in M2a compared to RM.

CONCLUSIONS

Our data in in vitro polarized human macrophages: 1. confirm and extend known inflammatory and anti-inflammatory gene expression patterns; 2. demonstrate changes in mitochondrial metabolism associated to insulin resistance and insulin sensitivity in M1 and M2a, respectively; 3. highlight the potential relevance of gene expression timing in M1 function; 4. unveil enhanced expression of Wnt pathways in M2a suggesting a potential dual (pro-inflammatory and anti-inflammatory) role of M2a in inflammatory diseases.

Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand <em>12</em> (CXCL<em>12</em>). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Cell-lineage analysis using Cxcl<em>12</em>-creER mice reveals that quiescent Cxcl<em>12</em>-creER⁺ perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical <em>Wnt</em> signaling components along the single-cell trajectory. β-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration. Therefore, quiescent Cxcl<em>12</em>-creER⁺ BMSCs transform into osteoblast precursor cells in a manner mediated by canonical <em>Wnt</em> signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration.

Most of colorectal adenocarcinomas are believed to arise from adenomas, which are premalignant lesions. Sequencing the whole exome of the adenoma will help identifying molecular biomarkers that can predict the occurrence of adenocarcinoma more precisely and help understanding the molecular pathways underlying the initial stage of colorectal tumorigenesis. We performed the exome capture sequencing of the normal mucosa, adenoma and adenocarcinoma tissues from the same patient and sequenced the identified mutations in additional 73 adenomas and 288 adenocarcinomas. Somatic single nucleotide variations (SNVs) were identified in both the adenoma and adenocarcinoma by comparing with the normal control from the same patient. We identified <em>12</em> nonsynonymous somatic SNVs in the adenoma and 42 nonsynonymous somatic SNVs in the adenocarcinoma. Most of these mutations including OR6X1, SLC15A3, KRTHB4, RBFOX1, LAMA3, CDH20, BIRC6, NMBR, GLCCI1, EFR3A, and FTHL17 were newly reported in colorectal adenomas. Functional annotation of these mutated genes showed that multiple cellular pathways including <em>Wnt</em>, cell adhesion and ubiquitin mediated proteolysis pathways were altered genetically in the adenoma and that the genetic alterations in the same pathways persist in the adenocarcinoma. CDH20 and LAMA3 were mutated in the adenoma while NRXN3 and COL4A6 were mutated in the adenocarcinoma from the same patient, suggesting for the first time that genetic alterations in the cell adhesion pathway occur as early as in the adenoma. Thus, the comparison of genomic mutations between adenoma and adenocarcinoma provides us a new insight into the molecular events governing the early step of colorectal tumorigenesis.

Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland^1,2. FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours^3-8. However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive^9-11 and oncogenic^{<em>12</em>-14} roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and-through TLE3 inactivation-promote metastasis driven by the <em>WNT</em> pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element-herein denoted FOXA1 mastermind (FOXMIND)-to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies.

OBJECTIVE

This review examines recent advances in the analysis of genetic determinants of bone mass. It addresses both human and animal linkage studies as well as genetic manipulations in animals, inbred mouse models, and candidate gene analyses.

RESULTS

Recent studies have implicated novel regulatory pathways in bone biology including both the neuroendocrine system and metabolic pathways linked to lipid metabolism. Variations in the lipoprotein receptor-related protein 5 (LRP5), part of the <em>Wnt</em>-frizzled pathway, were independently identified by linkage in high and low bone mass families. Subsequently, other high bone mass syndromes have been shown to have mutations in this gene. Neural studies have shown the skeletal regulatory activity of leptin and neuropeptide Y receptors via the hypothalamus. Subsequently, the beta-adrenergic pathway has been implicated, with important changes in bone mass. The lipoxygenase <em>12</em>/15 pathway, identified through inbred mouse models and through pharmacologic studies with specific inhibitors, has also been shown to have important effects on bone mass. These studies exemplify the value of genetic models both to identify and then confirm pathways by mutational study and pharmacologic interventions. Continuing candidate gene studies often performed with multiple loci complement such discoveries. However, these studies have not focused on the clinical endpoint of fracture and few have included large enough groups to engender confidence in the associations reported, as such studies may require thousands of individuals. Interestingly, results often differ by ethnicity, age, or gender. A small proportion have examined whether relevant genes influence response to treatment.

CONCLUSIONS

The combinations of human and animal genetic linkage studies have advanced understanding of the regulation of bone mass. Studies ranging from linkage to pharmacology provide optimism for new targets and treatments for osteoporosis.

BACKGROUND

Patients with familial adenomatous polyposis (FAP) are at increased risk for the development of colorectal cancer. Surgery and chemoprevention are the most effective means to prevent cancer development. Thymoquinone (TQ) is considered the main compound of the volatile Nigella sativa seed oil and has been reported to possess anticarcinogenic properties. In this study we evaluated the chemopreventive properties of TQ in a mouse model of FAP.

METHODS

APCMin mice were fed with chow containing 37.5 mg/kg or 375 mg/kg TQ for <em>12</em> weeks. H&E stained intestine tissue sections were assessed for tumor number, localization, size, and grade. Immunohistochemistry for β-catenin, c-myc, Ki-67 and TUNEL-staining was performed to investigate TQ's effect on major colorectal cancer pathways. TQ's impact on GSK-3β and β-catenin were studied in RKO cells.

RESULTS

375 mg/kg but not 37.5 mg/kg TQ decreased the number of large polyps in the small intestine of APCMin mice. TQ induced apoptosis in the neoplastic tissue but not in the normal mucosa. Furthermore, upon TQ treatment, β-catenin was retained at the membrane and c-myc decreased in the nucleus, which was associated with a reduced cell proliferation in the villi. In vitro, TQ activated GSK-3β, which induced membranous localization of β-catenin and reduced nuclear c-myc expression.

CONCLUSIONS

In summary, TQ interferes with polyp progression in ApcMin mice through induction of tumor-cell specific apoptosis and by modulating Wnt signaling through activation of GSK-3β. Nigella sativa oil (or TQ) might be useful as nutritional supplement to complement surgery and chemoprevention in FAP.

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/<em>Wnt</em> pathway is required for many developmental decisions in all animals. One transducer of the Wingless/<em>Wnt</em> signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/<em>Wnt</em> signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified <em>12</em> regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.

BACKGROUND

The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics.

METHODS

In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation.

RESULTS

Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation.

CONCLUSIONS

CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

The <em>Wnt</em> signaling pathway plays an essential role in carcinogenesis, and the amount of fat intake and composition of dietary fatty acids are crucial factors for colon carcinogenesis. We investigated whether various dietary fats affected the <em>Wnt</em> signaling pathway of colon tumorigenesis in azoxymethane (AOM)-treated rats. Male Sprague-Dawley rats were given intraperitoneal injections of AOM and supplemented with 10% corn, olive, beef, and fish oil for 44 wk. Aberrant crypt foci (ACF) and tumors were examined at <em>12</em> and 44 wk. Normal appearing colon mucosal proliferation and apoptosis were evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and percentages of fragmented DNA, respectively. Expressions of beta-catenin, cyclin D(1), <em>Wnt</em>2, <em>Wnt</em>3, and <em>Wnt</em>5a of normal appearing colon mucosa were analyzed by Western blot analysis. Long-term dietary corn oil and beef tallow increased ACF, tumor incidence, and tumor numbers in AOM-treated rats. In contrast, both olive and fish oil inhibited them. Dietary corn oil and beef tallow increased BrdU incorporation and the expression of cytosolic beta-catenin and cyclin D(1) and decreased apoptosis in the colon mucosa. Expressions of <em>Wnt</em>2 and <em>Wnt</em>3 in rats fed with beef tallow and <em>Wnt</em>5a in rats fed with corn oil increased with or without AOM-treatment. BrdU-incorporated cells were often observed at the tops of crypts in rats fed with beef tallow, whereas this was not observed in rats fed with the other diet. Long-term high intake of corn oil and beef tallow enhanced cell proliferation through <em>Wnt</em> signaling and modulated the distribution of proliferating cells, which might contribute to promoting effects in colon tumorigenesis.

Preclinical and clinical studies suggest that diminished folate status increases the risk of colorectal carcinogenesis. However, many biochemical functions of folate are dependent on the adequate availability of other 1-carbon nutrients, including riboflavin, vitamin B-6, and vitamin B-<em>12</em>. Aberrations in the <em>Wnt</em> pathway are thought to play an important role in human colorectal cancers. This study therefore investigated if mild depletion of folate combined with depletion of riboflavin, vitamin B-6, and vitamin B-<em>12</em> could induce alterations in the <em>Wnt</em> pathway in the colonic mucosa. Ninety-six mice were pair-fed diets with different combinations of B vitamin depletion for 10 wk. Genomic DNA methylation and uracil misincorporation were measured by LC/MS and GC/MS. Gene-specific methylation, strand breaks, and expressions were measured by real-time PCR and immunoblotting. Proliferation and apoptosis were determined by immunohistochemistry. DNA strand breaks within the Apc mutation cluster region were induced by folate depletion combined with inadequacies of riboflavin, vitamin B-6, and vitamin B-<em>12</em> (P < 0.05), but such effects were not induced by folate depletion alone. Similarly, minor changes in the expression of Apc, beta-catenin, and cyclin D1 produced by mild folate depletion were significantly magnified by multiple vitamin depletion. Apoptosis, which can be suppressed by increased <em>Wnt</em>-signaling, was attenuated by the combined deficiency state (P < 0.05) but not by singlet or doublet deficiencies. These findings indicate that a mild depletion of folate that is of insufficient magnitude by itself to induce alterations in components of the <em>Wnt</em> pathway may produce such effects when present in conjunction with mild inadequacies of other 1-carbon nutrients.

OBJECTIVE

Bone marrow lesions (BMLs) are well described in osteoarthritis (OA) using MRI and are associated with pain, but little is known about their pathological characteristics and gene expression. We evaluated BMLs using novel tissue analysis tools to gain a deeper understanding of their cellular and molecular expression.

METHODS

We recruited 98 participants, 72 with advanced OA requiring total knee replacement (TKR), <em>12</em> with mild OA and 14 non-OA controls. Participants were assessed for pain (using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)) and with a knee MRI (using MOAKS). Tissue was then harvested at TKR for BML analysis using histology and tissue microarray.

RESULTS

The mean (SD) WOMAC pain scores were significantly increased in advanced OA 59.4 (21.3) and mild OA 30.9 (20.3) compared with controls 0.5 (1.28) (p<0.0001). MOAKS showed all TKR tissue analysed had BMLs, and within these lesions, bone marrow volume was starkly reduced being replaced by dense fibrous connective tissue, new blood vessels, hyaline cartilage and fibrocartilage. Microarray comparing OA BML and normal bone found a significant difference in expression of 218 genes (p<0.05). The most upregulated genes included stathmin 2, thrombospondin 4, matrix metalloproteinase 13 and Wnt/Notch/catenin/chemokine signalling molecules that are known to constitute neuronal, osteogenic and chondrogenic pathways.

CONCLUSIONS

Our study is the first to employ detailed histological analysis and microarray techniques to investigate knee OA BMLs. BMLs demonstrated areas of high metabolic activity expressing pain sensitisation, neuronal, extracellular matrix and proinflammatory signalling genes that may explain their strong association with pain.

The canonical <em>Wnt</em> pathway regulates several biological processes including development, cell growth and proliferation via consecutive gene regulation. A high number of target genes of the <em>Wnt</em> pathway has been identified, but the chronological order of target gene expression is still elusive. This order is supposed to be crucial for the controlled course of events downstream of the activated <em>Wnt</em> pathway. Here we present the expression chronologies of the target genes Ccnd1 (encoding for cyclin D1), Myc (c-Myc), Cdkn1a (p21CIP1/WAF1), Tfrc (Transferrin receptor 1), Plf1 (Proliferin-1) and Ramp3 (Receptor activity-modifying protein 3) in C57MG cells after stimulation with <em>Wnt</em>-3a. We discriminated between immediate (below 1 h), early (between 1 and 6 h), intermediate (between 6 and <em>12</em> h) and late (after <em>12</em> h) targets. According to this classification Myc and Tfrc belong to the immediate target genes; Ccnd1, Plf1 and Ramp3 are early target genes and Cdkn1a is an intermediate target gene.

There is an unmet need for therapies that can restore bone strength and reduce fracture risk among patients at high risk of osteoporotic fracture. To address this need, bone-forming therapies that increase osteoblast activity are required to help restore bone structure and strength. Sclerostin is now recognized as a target for osteoporosis therapy. Sclerostin is predominantly secreted by the osteocyte and acts as an extracellular inhibitor of canonical <em>Wnt</em> signaling by binding to the receptors lipoprotein receptor-related protein-4, 5 and 6. Monoclonal antibodies to sclerostin (Scl-Ab) have been used in both clinical and in preclinical studies of osteoporosis with beneficial outcomes for bone density, structure, strength and fracture risk reduction. In this review paper, we summarize the current literature describing the effects of Scl-Ab in animal models of osteoporosis. In addition, we report new pharmacologic data from three animal studies of Scl-Ab: 1) a <em>12</em>-month study evaluating bone quality in ovariectomized (OVX) rats; 2) a 6-month study evaluating bone structure and strength in adolescent cynomolgus monkeys; and 3) the effects of transition from Scl-Ab to vehicle or the RANKL inhibitor osteoprotegerin-Fc in OVX rats. Together, these results demonstrate that inhibition of sclerostin by Scl-Ab increased bone formation, and decreased bone resorption, leading to improved bone structure, bone mass and bone strength while maintaining bone quality in multiple animal models of osteoporosis. Further, gains in bone mass induced by Scl-Ab treatment were preserved by antiresorptive agents such as a RANKL inhibitor as a follow-on therapy. The bone-forming effects of Scl-Ab were unaffected by pre- or co-treatment with a bisphosphonate, and were restored following a treatment-free period after initial dosing. These data support the clinical development of Scl-Ab for treatment of conditions with low bone mass such as postmenopausal and male osteoporosis.

The benefits coming from long-term treatment of postmenopausal osteoporosis with bisphophonates are limited by a coupled decrease in bone formation. The objective of this study is to determine whether this decrease in bone formation is associated with changes in serum levels of the <em>WNT</em> signaling antagonist sclerostin or Dickkopf-1 (DKK1). This is an ancillary observation from patients participating in a <em>12</em> months, phase 2, randomized clinical trial. We analyzed 107 patients given either monthly intramuscular neridronate (<em>12</em>.5, 25 or 50 mg) or placebo. Serum C-terminal telopeptide of type I collagen (sCTX, a bone-resorption marker) decreased by 61%, 75% and 73% in the <em>12</em>.5, 25 and 50 mg dose groups, respectively. Mean changes in bone alkaline phosphatase (bAP) at <em>12</em> months were -47%, -60.0% and -52.6% in the groups receiving <em>12</em>.5, 25 or 50 mg neridronate, respectively. Serum DKK1 remained unchanged at all time points in the 3 groups. Serum sclerostin increased versus placebo group gradually and significantly only in patients treated with 25 or 50 mg neridronate monthly, reaching 138-148% of baseline values (P<0.001). Changes in serum sclerostin at <em>12</em> months were negatively correlated with changes in bAP (P<0.001) even when data were adjusted for sCTX changes and only treated patients were included. In conclusions, decreased bone formation after several months of bisphosphonate therapy is associated with increased serum levels of sclerostin. This might suggest that <em>Wnt</em> signaling may play a role in the coupling between resorption and formation.

The pathogenesis of androgenetic alopecia (AGA, male-pattern baldness) is driven by androgens, and genetic predisposition is the major prerequisite. Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms (SNPs) at eight different genomic loci are associated with AGA development. However, a significant fraction of the overall heritable risk still awaits identification. Furthermore, the understanding of the pathophysiology of AGA is incomplete, and each newly associated locus may provide novel insights into contributing biological pathways. The aim of this study was to identify unknown AGA risk loci by replicating SNPs at the <em>12</em> genomic loci that showed suggestive association (5 × 10(-8)<P<10(-5)) with AGA in a recent meta-analysis. We analyzed a replication set comprising 2,759 cases and 2,661 controls of European descent to confirm the association with AGA at these loci. Combined analysis of the replication and the meta-analysis data identified four genome-wide significant risk loci for AGA on chromosomes 2q35, 3q25.1, 5q33.3, and <em>12</em>p<em>12</em>.1. The strongest association signal was obtained for rs7349332 (P=3.55 × 10(-15)) on chr2q35, which is located intronically in <em>WNT</em>10A. Expression studies in human hair follicle tissue suggest that <em>WNT</em>10A has a functional role in AGA etiology. Thus, our study provides genetic evidence supporting an involvement of <em>WNT</em> signaling in AGA development.