There have been many conflicting reports concerning the association of the DRD2 locus with alcohol dependence (AD). To investigate whether these findings could be reconciled by considering the genomic region of DRD2 in greater detail, we conducted two separate association studies of AD in <em>12</em>20 European-American subjects using family-based (488 subjects) and case-control (318 cases and 414 controls) designs, and 43 single nucleotide polymorphisms mapped to the gene cluster of NCAM1, TTC<em>12</em>, ANKK1 and DRD2. We used a generalized linear model and haplotype score tests for the case-control sample, and the family-based association test for the family sample. Haplotype associations centered on TTC<em>12</em> exon 3 [rs1893699-rs723077; optimal individual haplotype simulated P-value (P(oihs)) = 0.00021] in both independent samples (family and case-control). Additional AD-associated haplotypes centered around NCAM1 exon <em>12</em> in the family sample (P(oihs) = 0.0032), and at exons 2 and 5 of ANKK1 in the case-control sample (P(oihs) = 0.00058). LD contrasts between cases and controls support selection at TTC<em>12</em> exon 3 and ANKK1 exon 2. The armadillo repeat domains encoded by TTC<em>12</em> and dopamine interact in the <em>Wnt</em> pathway and may have effects on dopamine cell development in the ventral midbrain. We conclude that risk for AD is attributable in part to variants in four regions within this cluster: exon 3 of TTC<em>12</em>, exon <em>12</em>/intron13 of NCAM1 and exons 2 and 5 of ANKK1. The complexity of these relationships, many of which replicate between our independent samples, may explain prior inconsistent results.

Hip osteoarthritis (HOA) is one of the most disabling and common joint disorders with a large genetic component that is, however, still ill-defined. To date, genome-wide association studies (GWAS) in osteoarthritis (OA) and specifically in HOA have yielded only few loci, which is partly explained by heterogeneity in the OA definition. Therefore, we here focused on radiographically measured joint-space width (JSW), a proxy for cartilage thickness and an important underlying intermediate trait for HOA. In a GWAS of 6,523 individuals on hip-JSW, we identified the G allele of rs<em>12</em>982744 on chromosome 19p13.3 to be associated with a 5% larger JSW (P = 4.8 × 10(-10)). The association was replicated in 4,442 individuals from three United Kingdom cohorts with an overall meta-analysis P value of 1.1 × 10(-11). The SNP was also strongly associated with a <em>12</em>% reduced risk for HOA (P = 1 × 10(-4)). The SNP is located in the DOT1L gene, which is an evolutionarily conserved histone methyltransferase, recently identified as a potentially dedicated enzyme for <em>Wnt</em> target-gene activation in leukemia. Immunohistochemical staining of the DOT1L protein in mouse limbs supports a role for DOT1L in chondrogenic differentiation and adult articular cartilage. DOT1L is also expressed in OA articular chondrocytes. Silencing of Dot1l inhibited chondrogenesis in vitro. Dot1l knockdown reduces proteoglycan and collagen content, and mineralization during chondrogenesis. In the ATDC5 chondrogenesis model system, DOT1L interacts with TCF and <em>Wnt</em> signaling. These data are a further step to better understand the role of <em>Wnt</em>-signaling during chondrogenesis and cartilage homeostasis. DOT1L may represent a therapeutic target for OA.

Current evidence supports the notion that the amyloid beta-peptide (Abeta) plays a major role in the neurotoxicity observed in the brain in Alzheimer's disease. However, the signal transduction mechanisms involved still remain unknown. In the present work, we analyzed the effect of protein kinase C (PKC) on some members of the <em>Wnt</em> signaling pathway and its implications for Abeta neurotoxicity. Activation of PKC by phorbol <em>12</em>-myristate 13-acetate protected rat hippocampal neurons from Abeta toxicity. This effect was accomplished by inhibition of glycogen synthase kinase-3beta (GSK-3beta) activity, which led to the accumulation of cytoplasmic beta-catenin and transcriptional activation via beta-catenin/T-cell factor/lymphoid enhancer factor-1 (TCF/LEF-1) of <em>Wnt</em> target genes, which in the present study were engrailed-1 (en-1) and cyclin D1 (cycD1,). In contrast, inhibition of Ca2+-dependent PKC isoforms activated GSK-3beta and offered no protection from Ab neurotoxicity. <em>Wnt</em>-3a and lithium salts, classical activators of the <em>Wnt</em> pathway, mimicked PKC activation. Our results suggest that regulation of members of the <em>Wnt</em> signaling pathway by Ca2+-dependent PKC isoforms may be important in controlling the neurotoxic process induced by Ab.

Acute liver failure (ALF) remains a disease with poor patient outcome. Improved prognosis is associated with spontaneous liver regeneration, which supports the relevance of exploring 'regenerative' therapies. Therefore, the role of the <em>Wnt</em>/beta-catenin pathway in liver regeneration following ALF was investigated. ALF was induced in mice by acetaminophen overdose, which is also a leading cause of liver failure in patients. beta-catenin distribution was also studied in liver sections from acetaminophen-induced ALF patients. A nonlethal dose of acetaminophen, which induces liver regeneration, led to stabilization and activation of beta-catenin for 1 to <em>12</em> hours. These data were also verified by increased expression of the beta-catenin surrogate target glutamine synthetase. Beta-catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3beta and an increase in levels of casein kinase 2alpha, and led to increased cyclin-D1, another known beta-catenin target. These observations were next substantiated in beta-catenin conditional-null mice (beta-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in beta-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in beta-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear beta-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of beta-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.

We have isolated a common insertion site, <em>Wnt</em>-3, for proviruses of the mouse mammary tumor virus (MMTV). Of mammary tumors induced by the GR variant of MMTV, 5% contains a provirus at <em>Wnt</em>-3, which is located on mouse chromosome 11. The gene is transcribed into a 3.8-kilobase (kb) mRNA in tumors with nearby proviral insertions but not in tumors with proviruses at other loci or in most adult tissues. Normal expression of <em>Wnt</em>-3 is detected in mouse embryos (with a peak around day <em>12</em> of gestation) and at low levels in adult brain. The transcriptional unit of the <em>Wnt</em>-3 gene spans approximately 55 kb, with a first intron of 36 kb. The deduced amino acid sequence of the <em>Wnt</em>-3 protein is 47% identical to the int-1/<em>Wnt</em>-1 gene product.

Alterations of the <em>Wnt</em>/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated <em>Wnt</em>/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between <em>12</em> and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of <em>Wnt</em> signaling with other genetic and epigenetic signals in prostate carcinogenesis.

BACKGROUND

Gastric cancer (GC) is one of the most common malignancy and primary cause of death in Chinese cancer patients. Recurrence is a major factor leading to treatment failure and low level of 5-year survival rate in GC patients following surgical resection. Therefore, identification of biomarkers with potential in predicting recurrence risk is the key problem of the prognosis in GC patients.

METHODS

A total of 74 GC patients were selected for systematic analysis, consisting of 31 patients with recurrence and 43 patients without recurrence. Firstly, miRNAs microarray and bioinformatics methods were used to characterize differential expressed miRNAs from primary tumor samples. Following, we used a ROC method to select signature with best sensitivity and specificity. Finally, we validated the signature in GC samples (frozen fresh and blood samples) using quantitative PCR.

RESULTS

We have identified <em>12</em> differential miRNAs including 7 up-regulated and 5 down-regulated miRNAs in recurrence group. Using ROC method, we further ascertained hsa-miR-335 as a signature to recognize recurrence and non-recurrence cases in the training samples. Moreover, we validated this signature using quantitative PCR method in 64 test samples with consistent result with training set. A high frequency recurrence and poor survival were observed in GC cases with high level of hsa-miR-335 (P<0.001). In addition, we evaluated that hsa-miR-335 were involved in regulating target genes in several oncogenic signal-pathways, such as p53, MAPK, TGF-β, <em>Wnt</em>, ERbB, mTOR, Toll-like receptor and focal adhesion.

CONCLUSIONS

Our results indicate that the hsa-miR-335 has the potential to recognize the recurrence risk and relate to the prognosis of GC patients.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is a devastating disease, for which no causal therapy is available.

OBJECTIVE

To characterize WNT/β-catenin signaling in COPD in humans and elucidate its potential role as a preventive and therapeutic target in experimental emphysema in mice.

METHODS

The expression, localization, and activity of WNT/β-catenin signaling was assessed in 12 COPD and 12 transplant donor samples using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and Western blotting. The role of WNT/β-catenin signaling was assessed in elastase- and cigarette smoke-induced emphysema and therapeutic modulation thereof in elastase-induced emphysema in TOPGAL reporter and wild-type mice in vivo.

RESULTS

No differences in the mRNA expression profile of the main WNT/β-catenin signaling components were observed comparing COPD and donor lung homogenates. Immunohistochemical analysis revealed reduced numbers of nuclear β-catenin-positive alveolar epithelial cells in COPD. Similarly, WNT/β-catenin signaling was down-regulated in both experimental emphysema models. Preventive and therapeutic, WNT/β-catenin activation by lithium chloride attenuated experimental emphysema, as assessed by decreased airspace enlargement, improved lung function, reduced collagen content, and elevated expression of alveolar epithelial cell markers.

CONCLUSIONS

Decreased WNT/β-catenin signaling is involved in parenchymal tissue destruction and impaired repair capacity in emphysema. These data indicate a crucial role of WNT/β-catenin signaling in lung repair mechanisms in vivo, and highlight WNT/β-catenin activation as a future therapeutic approach for emphysema.

PTH, via the PTH/PTH-related protein receptor type 1 that couples to both protein kinase A (PKA) and protein kinase C (PKC) pathways, and the canonical <em>Wnt</em>-beta-catenin signaling pathway play important roles in bone formation. In the present study we have examined the interaction between the PTH and <em>Wnt</em> signaling pathways in mouse osteoblastic MC3T3-E1 cells. PTH dose- and time-dependently increased the concentrations of beta-catenin. The PKA activator, forskolin, and the PKC activator, phorbol <em>12</em>-myristate-13-acetate, as well as the PTH analog, [Nle(8,18),Tyr(34)]human PTH-(3-34)amide, all increased beta-catenin levels. Both H-89, a specific PKA inhibitor, and PKC inhibitors, staurosporine and calphostin C, antagonized PTH stimulation of beta-catenin levels. TGF-beta as well as transfection of the TGF-beta-signaling molecule, Smad3, enhanced beta-catenin levels, and this was antagonized by transfection of a dominant-negative Smad3. The transcriptional activity of transfected dominant-active beta-catenin was enhanced by PTH, an effect that was antagonized by cotransfection of a dominant-negative Smad3. PTH as well as LiCl(2), which mimics the effects of the <em>Wnt</em>-beta-catenin pathway, rescued the dexamethasone- and etoposide-induced apoptosis of osteoblastic cells. In conclusion, the data demonstrate that PTH stimulates osteoblast beta-catenin levels via Smad3, and that both PKA and PKC pathways are involved. The canonical <em>Wnt</em>-beta-catenin pathway is likely to be involved in the antiapoptotic actions of PTH by acting through Smad3 in osteoblasts.

Cenani-Lenz syndrome (CLS) is an autosomal-recessive congenital disorder affecting distal limb development. It is characterized mainly by syndactyly and/or oligodactyly and is now shown to be commonly associated with kidney anomalies. We used a homozygosity-mapping approach to map the CLS1 locus to chromosome 11p11.2-q13.1. By sequencing candidate genes, we identified recessive LRP4 mutations in <em>12</em> families with CLS. LRP4 belongs to the low-density lipoprotein (LDL) receptor-related proteins (LRPs), which are essential for various developmental processes. LRP4 is known to antagonize LRP6-mediated activation of canonical <em>Wnt</em> signaling, a function that is lost by the identified mutations. Our findings increase the spectrum of congenital anomalies associated with abnormal lipoprotein receptor-dependent signaling.

We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts associated with abnormal Fgf23 production and mineralization in Hyp mice. We found evidence that elevation of Fgf23 expression in osteocytes is associated with increments in Fgf1, Fgf7, and Egr2 and decrements in Sost, an inhibitor in the <em>Wnt</em>-signaling pathway, were observed in Hyp bone. beta-Catenin levels were increased in Hyp cortical bone, and TOPflash luciferase reporter assay showed increased transcriptional activity in Hyp-derived osteoblasts, consistent with <em>Wnt</em> activation. Moreover, activation of Fgf and <em>Wnt</em>-signaling stimulated Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the extracellular matrix protein Dmp1. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced posttranslational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regard to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH-altering factors, carbonic anhydrase <em>12</em> (Car<em>12</em>) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone.

OBJECTIVE

To investigate the expression and role of the Wnt signaling pathway in human limbal stem cells (LSCs).

METHODS

Total RNA was isolated from the human limbus and central cornea. Limbal or cornea-specific transcripts were identified through quantitative real-time PCR. Protein expression of Wnt molecules was confirmed by immunohistochemistry on human ocular tissue. Activation of Wnt signaling using lithium chloride was achieved in vitro and its effects on LSC differentiation and proliferation were evaluated.

RESULTS

Expression of WntWntWntWntWnt inhibitors were specific to the limbal region, whereas WntWntWntWntWnt/β-catenin signaling increased the proliferation and colony-forming efficiency of primary human LSCs. The stem cell phenotype was maintained, as shown by higher expression levels of putative corneal epithelial stem cell markers, ATP-binding cassette family G2 and ΔNp63α, and low expression levels of mature cornea epithelial cell marker, cytokeratin 12.

CONCLUSIONS

These findings demonstrate for the first time that Wnt signaling is present in the ocular surface epithelium and plays an important role in the regulation of LSC proliferation. Modulation of Wnt signaling could be of clinical application to increase the efficiency of ex vivo expansion of corneal epithelial stem/progenitor cells for transplantation.

BACKGROUND

Obesity is frequently complicated by comorbid conditions, yet how excess adipose contributes is poorly understood. Although adipocytes in obese individuals induce systemic inflammation via secreted cytokines, another potential mediator has recently been identified (i.e., adipocyte-derived exosomes). We hypothesized that adipocyte-derived exosomes contain mediators capable of activating end-organ inflammatory and fibrotic signaling pathways.

METHODS

We developed techniques to quantify and characterize exosomes shed by adipocytes from seven obese (age: <em>12</em>-17.5 y, BMI: 33-50 kg/m(2)) and five lean (age: 11-19 y, BMI: 22-25 kg/m(2)) subjects.

RESULTS

Abundant exosomal miRNAs, but no mRNAs, were detected. Comparison of obese vs. lean visceral adipose donors detected 55 differentially expressed miRNAs (P < 0.05; fold change ≥|1.2|). qRT-PCR confirmed downregulation of miR-148b (ratio = 0.2 (95% confidence interval = 0.1, 0.6)) and miR-4269 (0.3 (0.1, 0.8)), and upregulation of miR-23b (6.2 (2.2, 17.8)) and miR-4429 (3.8 (1.1-13.4)). Pathways analysis identified TGF-β signaling and Wnt/β-catenin signaling among the top canonical pathways expected to be altered with visceral adiposity based on projected mRNA targets for the 55 differentially expressed miRNAs. A select mRNA target was validated in vitro.

CONCLUSIONS

These data show that visceral adipocytes shed exosomal-mediators predicted to regulate key end-organ inflammatory and fibrotic signaling pathways.

BACKGROUND

The low-density lipoprotein receptor-related protein 5 (LRP5) gene, essential for glucose and cholesterol metabolism, may have a role in the aetiology of obesity, an important risk factor for diabetes.

METHODS

To investigate the association between LRP5 polymorphisms and obesity, 27 single-nucleotide polymorphisms (SNPs), spacing about 5 kb apart on average and covering the full transcript length of the LRP5 gene, were genotyped in 1873 Caucasian people from 405 nuclear families. Obesity (defined as body mass index (BMI) >30 kg/m(2)) and three obesity-related phenotypes (BMI, fat mass and percentage of fat mass (PFM)) were investigated.

RESULTS

Single markers (<em>12</em> tagging SNPs and 4 untaggable SNPs) and haplotypes (5 blocks) were tested for associations, using family-based designs. SNP4 (rs4988300) and SNP6 (rs634008) located in block 2 (intron 1) showed significant associations with obesity and BMI after Bonferroni correction (SNP4: p<0.001 and p = 0.001, respectively; SNP6: p = 0.002 and 0.003, respectively). The common allele A for SNP4 and minor allele G for SNP6 were associated with an increased risk of obesity. Significant associations were also observed between common haplotype A-G-G-G of block 2 with obesity, BMI, fat mass and PFM with global empirical values p<0.001, p<0.001, p = 0.003 and p = 0.074, respectively. Subsequent sex-stratified analyses showed that the association in the total sample between block 2 and obesity may be mainly driven by female subjects.

CONCLUSIONS

Intronic variants of the LRP5 gene are markedly associated with obesity. We hypothesise that such an association may be due to the role of LRP5 in the WNT signalling pathway or lipid metabolism. Further functional studies are needed to elucidate the exact molecular mechanism underlying our finding.

BACKGROUND

Androgen-deprivation therapy (ADT) is standard treatment for locally advanced or metastatic prostate cancer (PCa). Many patients develop castration resistance (castration-resistant PCa [CRPC]) after approximately 2-3 yr, with a poor prognosis. The molecular mechanisms underlying CRPC progression are unclear.

OBJECTIVE

To undertake quantitative tumour transcriptome profiling prior to and following ADT to identify functionally important androgen-regulated pathways or genes that may be reactivated in CRPC.

METHODS

RNA sequencing (RNA-seq) was performed on tumour-rich, targeted prostatic biopsies from seven patients with locally advanced or metastatic PCa before and approximately 22 wk after ADT initiation. Differentially regulated genes were identified in treatment pairs and further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on cell lines and immunohistochemistry on a separate CRPC patient cohort. Functional assays were used to determine the effect of pathway modulation on cell phenotypes.

METHODS

We searched for gene expression changes affecting key cell signalling pathways that may be targeted as proof of principle in a CRPC in vitro cell line model.

CONCLUSIONS

We identified ADT-regulated signalling pathways, including the <em>Wnt</em>/β-catenin signalling pathway, and observed overexpression of β-catenin in a subset of CRPC by immunohistochemistry. We validated 6 of <em>12</em> (50%) pathway members by qRT-PCR on LNCaP/LNCaP-AI cell RNAs, of which 4 (67%) demonstrated expression changes consistent with RNA-seq data. We show that the tankyrase inhibitor XAV939 (which promotes β-catenin degradation) reduced androgen-independent LNCaP-AI cell line growth compared with androgen-responsive LNCaP cells via an accumulation of cell proportions in the G0/G1 phase and reduction in the S and G2/M phases. Our biopsy protocol did not account for tumour heterogeneity, and pathway inhibition was limited to pharmacologic approaches.

CONCLUSIONS

RNA-seq of paired PCa samples revealed ADT-regulated signalling pathways. Proof-of-principle inhibition of the Wnt/β-catenin signalling pathway specifically delays androgen-independent PCa cell cycle progression and proliferation and warrants further investigation as a potential target for therapy for CRPC.

BACKGROUND

Abnormal activation of the Wnt/beta-catenin signaling pathway is common and critical in the pathogenesis of digestive cancers. In this study, the authors investigated the promoter methylation of the dickkopf homolog 3 gene Dkk-3 in these cancers and its prognostic significance in gastric cancer.

METHODS

Dkk-3 methylation was assessed in 173 patients with gastric cancers (including 104 patients who were followed for up to 4090 days) and in 128 patients with colorectal cancer. Cell growth was evaluated by using a colony-formation assay. For survival analyses, the authors used Kaplan-Meier plots, the log-rank test, and Cox proportional regression.

RESULTS

Dkk-3 was silenced or down-regulated in 12 of 17 gastric cancer cell lines (70.6%) and in 3 of 9 colon cancer cell lines (33.3%). The loss of gene expression was associated with promoter methylation, which could be restored by demethylating agents. Ectopic expression of Dkk-3 suppressed colony formation. Moreover, methylation of Dkk-3 was detected in 117 of 173 primary gastric tumors (67.6%) and in 67 of 128 colorectal tumors (52.3%). The clinical significance and the prognostic value of Dkk-3 methylation also were examined in 104 gastric cancers and in 84 colorectal cancers. Multivariate analysis indicated that Dkk-3 methylation was associated significantly and independently with poor disease survival (relative risk, 2.534; 95% confidence interval, 1.54-4.17; P=.002) in gastric cancer, but not in colorectal cancer. Kaplan-Meier survival curves revealed that patients who had Dkk-3 methylated gastric cancers had a significantly shorter survival (median, 0.76 years) compared with patients who did not have Dkk-3 methylation (median, 2.68 years; P<.0001; log-rank test).

CONCLUSIONS

Epigenetic silencing of the Dkk-3 gene by promoter methylation was a common event in gastric cancer and was associated with a poor outcome in such patients.

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex <em>12</em> (MED<em>12</em>) have been detected in a majority of fibroids but no information is available whether or not they co-occur with cytogenetic subtypes as, e.g., rearrangements of the genes encoding high mobility group AT-hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED<em>12</em> mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting <em>12</em>q14~15 or by mutations affecting exon 2 of MED<em>12</em>. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED<em>12</em> mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED<em>12</em> mutations expressed significantly higher levels of the gene encoding wingless-type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED<em>12</em> cooperate in activating the <em>Wnt</em> pathway which in turn activates β-catenin known to cause leiomyoma-like lesions in a mouse model. The occurrence of a "fibroid-type mutation" in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

The <em>Wnt</em>-beta-catenin pathway plays key roles in embryogenesis. <em>Wnt</em>-4 is known to be expressed in the mesonephric duct in embryonic development. It is tempting to speculate that the <em>Wnt</em>-4-beta-catenin pathway contributes to the recovery from acute renal failure (ARF). This study used an in vivo model of ARF rats to clarify the significance of the <em>Wnt</em>-4-beta-catenin pathway in ARF. ARF was induced by clamping the rat left renal artery for 1 h. At 3, 6, <em>12</em>, 24, 48, and 72 h after reperfusion, whole kidney homogenate and total RNA were extracted for examination by Western blot analysis and real-time RT-PCR. <em>Wnt</em>-4 mRNA and protein expression were strongly increased at 3 to <em>12</em> h and 6 to 24 h after ischemia, respectively. In immunohistologic examination, <em>Wnt</em>-4 was expressed in the proximal tubules and co-expressed with aquaporin-1, GM130, and PCNA. Cyclin D1 and cyclin A were expressed at 24 to 48 h after reperfusion. In addition, the overexpression of <em>Wnt</em>-4 and beta-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Taken together, these data suggest that the <em>Wnt</em>-4-beta-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The <em>Wnt</em>-4-beta-catenin pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

The Dishevelled protein mediates several diverse biological processes. Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls cell fate via canonical <em>Wnt</em> signaling, it also controls cell polarity via the vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9]. The relationship between subcellular localization of Dishevelled and its signaling activities remains unclear; conflicting results have been reported depending upon the organism and cell types examined [8, 9, 10, 11, <em>12</em>, 13, 14, 15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new reagents to sequester wild-type Dishevelled protein either at the cell membrane or away from the cell membrane. Removal of Dishevelled from the cell membrane disrupts convergent extension by preventing Rho/Rac activation and mediolateral cell polarization. By manipulating the subcellular localization of K->>M (dsh1), we show that this mutation inhibits Dishevelled activation of Rac, regardless of its subcellular localization. These data demonstrate that membrane localization of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both membrane-targeted and cytoplasm-targeted Dishevelled can potently activate canonical <em>Wnt</em> signaling, suggesting that local concentration of Dishevelled protein, but not its spatial localization, is central to canonical <em>Wnt</em> signaling. These results suggest that in vertebrate embryos, subcellular localization is insufficient to account for the pathway specificity of Dishevelled in the canonical <em>Wnt</em> versus PCP signaling cascades.

Galpha(<em>12</em>/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(<em>12</em>) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(<em>12</em>/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical <em>Wnt</em> signaling. Furthermore, Galpha(<em>12</em>/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical <em>Wnt</em> signaling. These findings provide the first evidence that Galpha(<em>12</em>) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.

We identified a novel gene, which encodes a 265-amino-acid sequence with a thrombospondin (TSP) type 1 motif. Unlike the other secretory proteins of the TSP family, this gene encodes no apparent secretion cleavage site, but has a putative nuclear localization signal. Northern blot analysis showed transient expression in the central nervous system (CNS) during development. In situ hybridization showed its expression in the dorsal part of the neural tube on 10 and <em>12</em> dpc, especially in the boundary region between roof plate and neuroepithelium. This expression was enhanced in the rostral part. The signals were observed in other tissues such as truncal region neighboring forelimbs and mesenchymal tissues around the nasal cavity. We named this gene R-spondin (roof plate-specific spondin). Transfection of an epitope-tagged R-spondin into COS7 and 293 cells showed its localization in nuclei and medium, suggesting that R-spondin may become secretory or nuclear protein by some processing, while most of other proteins with TSP type 1 domain are secretory proteins. The expression of R-spondin was reduced in <em>Wnt</em>-1/3a double knockout mouse. R-spondin might be a novel marker of the boundary between the roof plate and neuroepithelium and may contribute to the development of dorsal neural tube under the regulation of <em>Wnts</em>.

The evolutionary conserved <em>Wnt</em> signaling pathway regulates cardiogenesis. However, members of the <em>Wnt</em> pathway are also expressed in the adult heart. Although <em>Wnt</em>-signaling is quiescent under normal conditions, we noticed activation on pathological stress of the heart, such as chronic afterload increase. To examine the role of <em>Wnt</em> signaling on the postnatal heart, we modified the expression and function of the <em>Wnt</em> regulator dishevelled 1 (Dvl-1) both in transgenic mice with cardiac-specific overexpression of Dvl-1 (Dvl-1-Tg) and in cultured cardiac myocytes. Dvl-1-Tg mice (3 months) had severe cardiac hypertrophy (heart weight:body weight ratio: 5.2+/-0.3 mg/g wild-type [WT] versus 6.4+/-0.7 mg/g Dvl-1-Tg; P<0.01), an increase in cardiomyocyte size (86% increase in Dvl-1-Tg compared with WT; P<0.01) and marked raise of atrial natriuretic factor expression (<em>12</em>-fold increase versus WT; P<0.01). Hypertrophy was associated with left ventricular dilatation in Dvl-1-Tg and a reduction of ejection fraction (4.4+/-0.1 mm versus 5.5+/-0.2 mm, 80+/-2% and 43+/-4% in WT versus Dvl-1-Tg, respectively; P<0.01). Transgenic animals died prematurely before 6 months of age. Both canonical as well as noncanonical <em>Wnt</em> signaling branches were activated in the Dvl-1-Tg animals. Small interfering RNA-mediated depletion of Dvl-1 was used to further characterize the role of Dvl-1 in cardiac myocytes. Whereas baseline parameters were unaltered, beta-adrenergic hypertrophic response was abrogated in Dvl-1 knockdown cardiac myocytes, indicating a mandatory role in beta-adrenergic stimulation. Therefore, activation of <em>Wnt</em> signaling is sufficient and critical for the induction of myocardial hypertrophy and cardiomyopathy.

The <em>Wnt</em> signaling pathway is essential for a wide array of developmental and physiological processes. <em>Wnts</em> are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing beta-catenin nuclear translocation and activation. Although beta-catenin has been shown to be critical in liver biology, the expression of the 19 <em>Wnt</em> and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of <em>Wnt</em> and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 <em>Wnt</em>, 10 frizzled receptors genes, and secreted Frizzled-related protein-1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 <em>Wnts</em> and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 <em>Wnt</em> and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 <em>Wnt</em> genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although <em>12</em> <em>Wnt</em> and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 <em>Wnt</em> and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 <em>Wnt</em>, seven Fz, and Fzb genes were expressed in SECs.

CONCLUSIONS

These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways.

BACKGROUND

We investigated Nestin expression in triple-negative breast cancer and examined how the modulation of Nestin expression affects cell cycle progression, survival, invasion and regulatory signaling in breast cancer stem cells (CSC) in vitro.

METHODS

Nestin expression in 150 triple-negative breast cancer specimens were examined by immunohistochemistry. The role of Nestin expression in tumorigenesis was examined by assaying naturally occurring Nestinhigh/Nestinlow CSC from <em>12</em> breast cancer tissues, as well as CSC from 26 clinical specimens, where Nestin overexpression and silencing was achieved by genetic manipulation, for their ability to form mammospheres and induce solid tumors. Cell cycle progression, spontaneous apoptosis and invasiveness of Nestin-silenced breast CSC were investigated by flow cytometry and transwell assays. The relative levels of expression of epithelial-mesenchymal transition (EMT) and <em>Wnt</em>/β-catenin pathway-related molecules were determined by western blotting.

RESULTS

Nestin expression was significantly associated with poor survival in patients with triple-negative breast cancer (P = 0.01). Nestinhigh breast CSC rapidly formed typical mammospheres in vitro. Nestinhigh, but not Nestinlow CSC, efficiently formed solid tumors in vivo. Nestin silencing induced cell cycle arrest at G2/M (52.03% versus 19.99% in controls) and promoted apoptosis (36.45% versus 8.29% in controls). Nestin silencing also inhibited breast CSC invasiveness, and was associated with significantly upregulated E-cadherin, while N-cadherin, vimentin, a-smooth muscle actin (a-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) expression was downregulated (P <0.05 for all). Nestin silencing also upregulated Axin, glycogen synthase kinase-3 beta (GSK-3β), adenomatous polyposis coli (APC), and peroxisome proliferator-activated receptor alpha (PPARa), and downregulated β-catenin, c-Myc, cyclin D and MMP-7 expression in CSC. Inhibition of the Wnt/β-catenin pathway mitigated mammosphere formation in Nestinhigh CSC, while inhibition of GSK-3β promoted the mammosphere formation in Nestinlow CSC (P <0.05 for all).

CONCLUSIONS

Our data indicates that Nestin positively regulates the proliferation, survival and invasiveness of breast CSC by enhancing Wnt/β-catenin activation.