Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone (DEX) activates glycogen synthase kinase-3beta (GSK3beta) and inhibits a differentiation-related cell cycle that occurs at a commitment stage immediately after confluence. Here we show that DEX inhibition of the differentiation-related cell cycle is associated with a decrease in beta-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3beta inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase B/Akt-Ser(473) within minutes, and this inhibition was also observed after <em>12</em> h. When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by wortmannin, DEX no longer inhibited beta-catenin levels. Furthermore, DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in the presence of dominant negative forms of either PI3K or protein kinase B/Akt. These results suggest cross-talk between the PI3K/Akt and <em>Wnt</em> signaling pathways. Consistent with a role for <em>Wnt</em> signaling in the osteoblast differentiation-related cell cycle, wortmannin partially negated the DEX inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1, which is known to inhibit LEF/TCF transcriptional activity. Overexpression of HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity. In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated inhibition of the differentiation-related cell cycle was partially negated. When administered together, wortmannin and trichostatin A completely negated the inhibitory effect of DEX on the differentiation-related cell cycle. These results suggest that inhibition of a PI3K/Akt/GSK3beta/beta-catenin/LEF axis and stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on <em>Wnt</em> signaling and the osteoblast differentiation-related cell cycle.

Amplification of 8p11-<em>12</em> in human breast cancers is associated with increased proliferation and tumor grade and reduced metastasis-free patient survival. We identified Zeppo1 (zinc finger elbow-related proline domain protein 1) (FLJ14299/ZNF703) within this amplicon as a regulator of cell adhesion, migration, and proliferation in mammary epithelial cells. Overexpression of Zeppo1 reduces cell-cell adhesion and stimulates migration and proliferation. Knockdown of Zeppo1 induces adhesion and lumen formation. Zeppo1 regulates transcription, complexing with Groucho and repressing E-cadherin expression and <em>Wnt</em> and TGFβ reporter expression. Zeppo1 promotes expression of metastasis-associated p<em>12</em>0-catenin isoform 1 and alters p<em>12</em>0-catenin localization upon cell contact with the extracellular matrix. Significantly, Zeppo1 overexpression in a mouse breast cancer model increases lung metastases, while reducing Zeppo1 expression reduces both tumor size and the number of lung metastases. These results indicate that Zeppo1 is a key regulator of breast cancer progression.

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from <em>12</em> age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the <em>Wnt</em> pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

BACKGROUND

Microarray technology is widely employed for studying the molecular mechanisms underlying complex diseases. However, analyses of individual diseases or models of diseases frequently yield extensive lists of differentially expressed genes with uncertain relationships to disease pathogenesis.

OBJECTIVE

To compare gene expression changes in a heterogeneous set of lung disease models in order to identify common gene expression changes seen in diverse forms of lung pathology, as well as relatively small subsets of genes likely to be involved in specific pathophysiological processes.

METHODS

We profiled lung gene expression in <em>12</em> mouse models of infection, allergy, and lung injury. A linear model was used to estimate transcript expression changes for each model, and hierarchical clustering was used to compare expression patterns between models. Selected expression changes were verified by quantitative polymerase chain reaction.

RESULTS

A total of 24 transcripts, including many involved in inflammation and immune activation, were differentially expressed in a substantial majority (9 or more) of the models. Expression patterns distinguished three groups of models: (1) bacterial infection (n = 5), with changes in 89 transcripts, including many related to nuclear factor-kappaB signaling, cytokines, chemokines, and their receptors; (2) bleomycin-induced diseases (n = 2), with changes in 53 transcripts, including many related to matrix remodeling and Wnt signaling; and (3) T helper cell type 2 (allergic) inflammation (n = 5), with changes in 26 transcripts, including many encoding epithelial secreted molecules, ion channels, and transporters.

CONCLUSIONS

This multimodel dataset highlights novel genes likely involved in various pathophysiological processes and will be a valuable resource for the investigation of molecular mechanisms underlying lung disease pathogenesis.

Twist, a transcription factor of the basic helix-loop-helix class, has been suggested to have oncogenic properties. We reported Twist expression was regulated by <em>Wnt</em>/beta-catenin signaling and that both <em>Wnt</em>-1 and Twist could contribute to mammary tumorigenesis. The aim of this study was to demonstrate the expression of Twist, <em>Wnt</em>-1 and <em>Wnt</em>-2 in human breast cancer tissue. We examined the expression in patients with breast cancer by RT-PCR and immunohistochemistry. RT-PCR of twenty-three pairs of cancer and normal breast tissue revealed that Twist was up-regulated in 69.6% (16/23) of the cancer lesions and 21.7% (5/23) of the normal breast tissues. <em>Wnt</em>-2 was up-regulated in all of the cancer lesions and 13.0% (3/23) of the normal breast tissues, whereas <em>Wnt</em>-1 was expressed in both the cancer and normal breast tissues of the five cases examined. Immunohistochemical analyses revealed that Twist was positively expressed in 52.2% (<em>12</em>/23) of the cancer lesions and 34.8% (8/23) of the normal breast tissues. Twist and <em>Wnt</em>-2 are highly expressed in breast cancer tissue, suggesting that both molecules could play important roles in mammary carcinogenesis.

We characterized the temporal changes in chondrogenic genes and developed a staging scheme for in vitro chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in three-dimensional (3D) alginate gels. A time-dependent accumulation of glycosaminoglycans, aggrecan, and type II collagen was observed in chondrogenic but not in basal constructs over 24 days. qRT-PCR demonstrated a largely characteristic temporal pattern of chondrogenic markers and provided a basis for staging the cellular phenotype into four stages. Stage I (days 0-6) was defined by collagen types I and VI, Sox 4, and BMP-2 showing peak expression levels. In stage II (days 6-<em>12</em>), gene expression for cartilage oligomeric matrix protein, HAPLN1, collagen type XI, and Sox 9 reached peak levels, while gene expression of matrilin 3, Ihh, Homeobox 7, chondroadherin, and <em>WNT</em> 11 peaked at stage III (days <em>12</em>-18). Finally, cells in stage IV (days 18-24) attained peak levels of aggrecan; collagen IX, II, and X; osteocalcin; fibromodulin; PTHrP; and alkaline phosphatase. Gene profiles at stages III and IV were analogous to those in juvenile articular and adult nucleus pulposus chondrocytes. Gene ontology analyses also demonstrated a specific expression pattern of several putative novel marker genes. These data provide comprehensive insights on chondrogenesis of hMSCs in 3D gels. The derivation of this staging scheme may aid in defining maximally responsive time points for mechanobiological modulation of constructs to produce optimally engineered tissues.

The heritable component to attempted and completed suicide is partly related to psychiatric disorders and also partly independent of them. Although attempted suicide linkage regions have been identified on 2p11-<em>12</em> and 6q25-26, there are likely many more such loci, the discovery of which will require a much higher resolution approach, such as the genome-wide association study (GWAS). With this in mind, we conducted an attempted suicide GWAS that compared the single-nucleotide polymorphism (SNP) genotypes of <em>12</em>01 bipolar (BP) subjects with a history of suicide attempts to the genotypes of 1497 BP subjects without a history of suicide attempts. In all, 2507 SNPs with evidence for association at P<0.001 were identified. These associated SNPs were subsequently tested for association in a large and independent BP sample set. None of these SNPs were significantly associated in the replication sample after correcting for multiple testing, but the combined analysis of the two sample sets produced an association signal on 2p25 (rs300774) at the threshold of genome-wide significance (P=5.07 × 10(-8)). The associated SNPs on 2p25 fall in a large linkage disequilibrium block containing the ACP1 (acid phosphatase 1) gene, a gene whose expression is significantly elevated in BP subjects who have completed suicide. Furthermore, the ACP1 protein is a tyrosine phosphatase that influences <em>Wnt</em> signaling, a pathway regulated by lithium, making ACP1 a functional candidate for involvement in the phenotype. Larger GWAS sample sets will be required to confirm the signal on 2p25 and to identify additional genetic risk factors increasing susceptibility for attempted suicide.

Among the multiple cellular effects mediated by lysophosphatidic acid (LPA), the effect on cell proliferation has extensively been investigated. A recent study showed that LPA-mediated proliferation of colon cancer cells requires activation of beta-catenin. However, the majority of colon cancer cells have deregulation of the <em>Wnt</em>/beta-catenin pathway. This prompted us to hypothesize the presence of additional pathway(s) activated by LPA resulting in an increase in the proliferation of colon cancer cells. Krüppel-like factor 5 (KLF5) is a transcriptional factor highly expressed in the crypt compartment of the intestinal epithelium. In this work, we investigated a role of KLF5 in LPA-mediated proliferation. We show that LPA stimulated the expression levels of KLF5 mRNA and protein in colon cancer cells and this stimulation was mediated by LPA(2) and LPA(3). Silencing of KLF5 expression by small interfering RNA significantly attenuated LPA-mediated proliferation of SW480 and HCT116 cells. LPA-mediated KLF5 induction was partially blocked by inhibition of the mitogen-activated protein kinase kinase and protein kinase C-delta. Moreover, we observed that LPA regulates KLF5 expression via eukaryotic elongation factor 2 kinase (eEF2k). Inhibition of calmodulin or silencing of eEF2k blocked the stimulation in KLF5 expression. Knockdown of eEF2k specifically inhibited KLF5 induction by LPA but not by fetal bovine serum or phorbol <em>12</em>-myristate 13-acetate. These results identify KLF5 as a target of LPA-mediated signaling and suggest a role of KLF5 in promoting proliferation of intestinal epithelia in response to LPA.

BACKGROUND

Activation of the Wnt signaling pathway is frequently observed in hepatocellular carcinoma (HCC), though mutation of three of its components, CTNNB1, AXIN1, and AXIN2, is observed substantially less often.

METHODS

We examined the relationship between Wnt signaling and epigenetic alteration of secreted frizzled-related protein (SFRP) genes in HCC.

RESULTS

We frequently detected the active form of beta-catenin and accumulation of nuclear beta-catenin in liver cancer cell lines. We detected methylation of SFRP family genes in liver cancer cell lines (SFRP1, 9/12, 75%; SFRP2, 7/12, 58%; SFRP4, 3/12, 25%; SFRP5, 7/12, 58%) and primary HCCs (SFRP1, 9/19, 47%; SFRP2, 12/19, 63%; SFRP5, 8/19, 42%), though methylation of SFRP4 was not found in primary HCCs. SFRP methylation also was detected in hepatitis B or C virus-associated chronic hepatitis (SFRP1, 6/37, 16%; SFRP2, 14/37, 38%; SFRP5, 5/37, 14%) and liver cirrhosis (SFRP1, 10/28, 36%; SFRP2, 9/28, 32%; SFRP5, 3/28, 11%), suggesting that methylation of these genes is an early event in liver carcinogenesis. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcriptional activity in liver cancer cells, while overexpression of a beta-catenin mutant and depletion of SFRP1 using siRNA synergistically upregulated TCF/LEF transcriptional activity.

CONCLUSIONS

Our results confirm the frequent methylation and silencing of Wnt antagonist genes in HCC, and suggest that their loss of function contributes to activation of Wnt signaling during hepatocarcinogenesis.

The <em>Wnt</em> genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least <em>12</em> subfamilies of <em>Wnt</em> genes [2]. <em>Wnt</em>3 and <em>Wnt</em>8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific <em>Wnt</em> ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of <em>Wnt</em>8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of <em>Wnt</em>8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, <em>Wnt</em>8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that <em>Wnt</em>8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.

The <em>wnt</em> gene family encodes a set of secreted glycoproteins involved in key developmental processes, including cell fate specification and regulation of posterior growth (Cadigan KM, Nusse R. 1997. <em>Wnt</em> signaling: a common theme in animal development. Genes Dev. 11:3286-3305.; Martin BL, Kimelman D. 2009. <em>Wnt</em> signaling and the evolution of embryonic posterior development. Curr Biol. 19:R215-R219.). As for many other gene families, evidence for expansion and/or contraction of the <em>wnt</em> family is available from deuterostomes (e.g., echinoderms and vertebrates [Nusse R, Varmus HE. 1992. <em>Wnt</em> genes. Cell. 69:1073-1087.; Schubert M, Holland LZ, Holland ND, Jacobs DK. 2000. A phylogenetic tree of the <em>Wnt</em> genes based on all available full-length sequences, including five from the cephalochordate amphioxus. Mol Biol Evol. 17:1896-1903.; Croce JC, Wu SY, Byrum C, Xu R, Duloquin L, Wikramanayake AH, Gache C, McClay DR. 2006. A genome-wide survey of the evolutionarily conserved <em>Wnt</em> pathways in the sea urchin Strongylocentrotus purpuratus. Dev Biol. 300:<em>12</em>1-131.]) and ecdysozoans (e.g., arthropods and nematodes [Eisenmann DM. 2005. <em>Wnt</em> signaling. WormBook. 1-17.; Bolognesi R, Farzana L, Fischer TD, Brown SJ. 2008. Multiple <em>Wnt</em> genes are required for segmentation in the short-germ embryo of Tribolium castaneum. Curr Biol. 18:1624-1629.]), but little is known from the third major bilaterian group, the lophotrochozoans (e.g., mollusks and annelids [Prud'homme B, Lartillot N, Balavoine G, Adoutte A, Vervoort M. 2002. Phylogenetic analysis of the <em>Wnt</em> gene family. Insights from lophotrochozoan members. Curr Biol. <em>12</em>:1395.]). To obtain a more comprehensive scenario of the evolutionary dynamics of this gene family, we exhaustively mined <em>wnt</em> gene sequences from the whole genome assemblies of a mollusk (Lottia gigantea) and two annelids (Capitella teleta and Helobdella robusta) and examined them by phylogenetic, genetic linkage, intron-exon structure, and embryonic expression analyses. The 36 <em>wnt</em> genes obtained represent 11, <em>12</em>, and 9 distinct <em>wnt</em> subfamilies in Lottia, Capitella, and Helobdella, respectively. Thus, two of the three analyzed lophotrochozoan genomes retained an almost complete ancestral complement of <em>wnt</em> genes emphasizing the importance and complexity of this gene family across metazoans. The genome of the leech Helobdella reflects significantly more dynamism than those of Lottia and Capitella, as judged by gene duplications and losses, branch length, and changes in genetic linkage. Finally, we performed a detailed expression analysis for all the Helobdella <em>wnt</em> genes during embryonic development. We find that, although the patterns show substantial overlap during early cleavage stages, each <em>wnt</em> gene has a unique expression pattern in the germinal plate and during tissue morphogenesis. Comparisons of the embryonic expression patterns of the duplicated <em>wnt</em> genes in Helobdella with their orthologs in Capitella reveal extensive regulatory diversification of the duplicated leech <em>wnt</em> genes.

Beta-catenin is critical for intercellular adhesion and also plays a role as a transcription activating protein in the <em>Wnt</em> signalling pathway. Increased protein levels and mutation of the beta-catenin gene have been demonstrated in various cancers; however, the role of beta-catenin in gastric cancer remains largely unknown. Using gastric cancer tissues and normal adjacent gastric mucosa obtained from 20 patients with gastric cancer (eight diffuse-type, <em>12</em> intestinal-type) undergoing gastric resection or endoscopy, we assessed the expression of beta-catenin by immunohistochemistry and quantitative PCR analysis. Furthermore, the tumour suppressor gene APC, which down-regulates the beta-catenin levels was analysed for mutations. Overall mRNA levels of beta-catenin were significantly increased in the tumour samples compared with the matched normal gastric mucosa (P < 0.05). Increased beta-catenin mRNA levels were significantly more frequent in intestinal-type gastric cancers as compared to diffuse-type gastric cancers (P < 0.01). Six out of 20 tumours exhibited >6-fold increased beta-catenin mRNA levels as compared with normal mucosa. APC gene mutations were found in four cases. A beta-catenin gene mutation was identified only in one intestinal-type gastric cancer exhibiting a massive overexpression of beta-catenin mRNA in the tumour. In intestinal-type gastric cancers beta-catenin mRNA levels are greatly enhanced. APC and beta-catenin gene mutations are also present primarily in intestinal-type gastric cancers. These findings support the hypothesis that in intestinal-type gastric cancers the accumulation of beta-catenin protein may result from impaired degradation of the beta-catenin protein due to alterations of the beta-catenin and APC genes, as well as from enhanced beta-catenin transcription which is present in the great majority of intestinal-type gastric cancers.

<em>Wnt</em> ligands and Frizzled (Fz) G protein-coupled receptors impact cell fate, including embryonic development of the ovary. Because the role of these regulatory molecules during follicular development in the adult is not known, an RT-PCR survey was done. <em>Wnt</em>-4, Fz-4, and Fz-1 were among the transcripts detected, and each exhibited a specific pattern of expression. Fz-1 mRNA was low in preovulatory follicles of PMSG-treated mice but was increased within 4-<em>12</em> h after an ovulatory surge of human CG. By in situ analysis, Fz-1 transcripts increased first in the theca cells and then in the granulosa cells of ovulating follicles but were low in corpora lutea. In contrast, <em>Wnt</em>-4, a critical factor in early ovarian development, was expressed in small preantral follicles. In addition, <em>Wnt</em>-4 was detected in preovulatory follicles and exhibited high levels in corpora lutea. A potential receptor for <em>Wnt</em>-4 in corpora lutea is Fz-4 that was also elevated in this tissue. Although <em>Wnt</em>-4 has been shown to function downstream of the PR in other tissues, <em>Wnt</em>-4 was not altered in follicles of PR-null mice that fail to ovulate. Rather expression of Fz-1 was lower in ovaries of PR knockout mice, compared with normal littermates. Thus, specific <em>Wnt</em>/Fz are expressed at distinct stages of follicular development, suggesting multiple functions for this signaling pathway in the ovary.

Transcriptional activation of <em>Wnt</em>/Wg-responsive genes requires the stabilization and nuclear accumulation of beta-catenin, a dedicated coactivator of LEF/TCF enhancer-binding proteins. Here we report that recombinant beta-catenin strongly enhances binding and transactivation by LEF-1 on chromatin templates in vitro. Interestingly, different LEF-1 isoforms vary in their ability to bind nucleosomal templates in the absence of beta-catenin, owing to N-terminal residues that repress binding to chromatin, but not nonchromatin, templates. Transcriptional activation in vitro requires both the armadillo (ARM) repeats and the C terminus of beta-catenin, whereas the phosphorylated N terminus is inhibitory to transcription. A fragment spanning the C terminus (CT) and ARM repeats 11 and <em>12</em> (CT-ARM), but not the CT alone, functions as a dominant negative inhibitor of LEF-1-beta-cat activity in vitro and can block ATP-dependent binding of the complex to chromatin. LEF-1-beta-cat transactivation in vitro was also repressed by inhibitor of beta-catenin and Tcf-4 (ICAT), a physiological inhibitor of <em>Wnt</em>/Wg signaling that interacts with ARM repeats 11 and <em>12</em>, and by the nonsteroidal anti-inflammatory compound, sulindac. None of these transcription inhibitors (CT-ARM, ICAT, or sulindac) could disrupt the LEF-1-beta-cat complex after it was stably bound to chromatin. We conclude that the CT-ARM region of beta-catenin functions as a chromatin-specific activation domain, and that several inhibitors of the <em>Wnt</em>/Wg pathway directly modulate LEF-1-beta-cat activity on chromatin.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of <em>12</em> induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including <em>WNT</em> pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of <em>WNT</em> signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

Selenium is an essential micronutrient. Its recommended daily allowance is not attained by a significant proportion of the population in many countries and its intake has been suggested to affect colorectal carcinogenesis. Therefore, microarrays were used to determine how both selenoprotein and global gene expression patterns in the mouse colon were affected by marginal selenium deficiency comparable to variations in human dietary intakes. Two groups of <em>12</em> mice each were fed a selenium-deficient (0.086 mg Se/kg) or a selenium-adequate (0.15 mg Se/kg) diet. After 6 wk, plasma selenium level, liver, and colon glutathione peroxidase (GPx) activity in the deficient group was <em>12</em>, 34, and 50%, respectively, of that of the adequate group. Differential gene expression was analysed with mouse 44K whole genome microarrays. Pathway analysis by GenMAPP identified the protein biosynthesis pathway as most significantly affected, followed by inflammation, Delta-Notch and <em>Wnt</em> pathways. Selected gene expression changes were confirmed by quantitative real-time PCR. GPx1 and the selenoproteins W, H, and M, responded significantly to selenium intake making them candidates as biomarkers for selenium status. Thus, feeding a marginal selenium-deficient diet resulted in distinct changes in global gene expression in the mouse colon. Modulation of cancer-related pathways may contribute to the higher susceptibility to colon carcinogenesis in low selenium status.

The proteasomal degradation of beta-catenin mediated by the glycogen synthase kinase 3beta (GSK3beta) and destruction complex is the central step in the canonical <em>Wnt</em> signaling pathway. However, that there are branches of <em>Wnt</em> signaling pathways that do not depend on beta-catenin/Tcf-mediated transcription activation has long been understood. In this study, we hypothesized that there are many more GSK3 and destruction complex-dependent proteolytic target proteins that mediate <em>Wnt</em> signaling in the cell. To test this hypothesis, we have developed and carried out a screen for such candidate proteins using an in vitro expression cloning technique and biochemical reconstitution of <em>Wnt</em> signaling in Xenopus egg cytoplasmic extracts. Forty-two proteins have been identified as potential candidates for GSK3-regulated phosphorylation, proteasomal degradation, or both, of which <em>12</em> are strong candidates for <em>Wnt</em>-pathway-regulated degradation. Some of them have been reported to interact with beta-catenin and implicated in the canonical <em>Wnt</em> signaling pathway, and other targets identified include proteins with various cellular functions such as RNA processing, cytoskeletal dynamics, and cell metabolism. Thus, we propose that <em>Wnt</em>/GSK3/destruction complex signaling regulates multiple target proteins to control a broad range of cellular activities in addition to beta-catenin-mediated transcription activation.

<em>Wnt</em> signalling pathways have extremely diverse functions in animals, including induction of cell fates or tumours, guidance of cell movements during gastrulation, and the induction of cell polarity. <em>Wnt</em> can induce polar changes in cellular morphology by a remodelling of the cytoskeleton. However, how activation of the Frizzled receptor induces cytoskeleton rearrangement is not well understood. We show, by an in depth 4-D microscopy analysis, that the Caenorhabditis elegans <em>Wnt</em> pathway signals to CED-10/Rac via two separate branches to regulate modulation of the cytoskeleton in different cellular situations. Apoptotic cell clearance and migration of the distal tip cell require the MOM-5/Fz receptor, GSK-3 kinase, and APC/APR-1, which activate the CED-2/5/<em>12</em> branch of the engulfment machinery. MOM-5 (Frizzled) thus can function as an engulfment receptor in C. elegans. Our epistatic analyses also suggest that the two partially redundant signalling pathways defined earlier for engulfment may act in a single pathway in early embryos. By contrast, rearrangement of mitotic spindles requires the MOM-5/Fz receptor, GSK-3 kinase, and beta-catenins, but not the downstream factors LIT-1/NLK or POP-1/Tcf. Taken together, our results indicate that in multiple developmental processes, CED-10/Rac can link polar signals mediated by the <em>Wnt</em> pathway to rearrangements of the cytoskeleton.

Although attention has focused on the chemopreventive action of retinoic acid (RA) in hepatocarcinogenesis, the functional role of RA in the liver has yet to be clarified. To explore the role of RA in the liver, we developed transgenic mice expressing RA receptor (RAR) alpha- dominant negative form in hepatocytes using albumin promoter and enhancer. At 4 months of age, the RAR alpha- dominant negative form transgenic mice developed microvesicular steatosis and spotty focal necrosis. Mitochondrial beta-oxidation activity of fatty acids and expression of its related enzymes, including VLCAD, LCAD, and HCD, were down-regulated; on the other hand, peroxisomal beta-oxidation and its related enzymes, including AOX and BFE, were up-regulated. Expression of cytochrome p4504a10, cytochrome p4504a<em>12</em>, and cytochrome p4504a14 was increased, suggesting that omega-oxidation of fatty acids in microsomes was accelerated. In addition, formation of H2O2 and 8-hydroxy-2'-deoxyguanosine was increased. After <em>12</em> months of age, these mice developed hepatocellular carcinoma and adenoma of the liver. The incidence of tumor formation increased with age. Expression of beta-catenin and cyclin D1 was enhanced and the TCF-4/beta-catenin complex was increased, whereas the RAR alpha/ beta-catenin complex was decreased. Feeding on a high-RA diet reversed histological and biochemical abnormalities and inhibited the occurrence of liver tumors. These results suggest that hepatic loss of RA function leads to the development of steatohepatitis and liver tumors. In conclusion, RA plays an important role in preventing hepatocarcinogenesis in association with fatty acid metabolism and <em>Wnt</em> signaling.

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. <em>Wnt</em> proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of <em>Wnts</em> on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6-10 days after co-culture, cardiac differentiation was determined by alpha-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with <em>Wnt</em>11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7+/-0.3-fold), whereas <em>Wnt</em>3A-conditioned medium showed no effect. Cell fusion was not affected by <em>Wnt</em>11-conditioned medium. Because <em>Wnts</em> inhibit glycogen synthase kinase-3beta, we further determined whether the glycogen synthase kinase-3beta inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mM) did not affect CPC differentiation. In contrast, <em>Wnt</em>11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol <em>12</em>-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with <em>Wnt</em>11-conditioned medium. Our data demonstrate that <em>Wnt</em>11, but not <em>Wnt</em>3A, augments cardiomyogenic differentiation of human CPCs. <em>Wnt</em>11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.

The Polycomb group protein enhancer of zeste homolog 2 (EZH2), which has roles during development of numerous tissues, is a critical regulator of cell type identity. Overexpression of EZH2 has been detected in invasive breast carcinoma tissue samples and is observed in human breast tissue samples of morphologically normal lobules up to <em>12</em> years before the development of breast cancer. The function of EZH2 during preneoplastic progression in the mammary gland is unknown. To investigate the role of EZH2 in the mammary gland, we targeted the expression of EZH2 to mammary epithelial cells using the mouse mammary tumor virus long terminal repeat. EZH2 overexpression resulted in aberrant terminal end bud architecture. By the age of 4 months, 100% of female mouse mammary tumor virus-EZH2 virgin mice developed intraductal epithelial hyperplasia resembling the human counterpart accompanied by premature differentiation of ductal epithelial cells and up-regulation of the luminal marker GATA-3. In addition, remodeling of the mammary gland after parturition was impaired and EZH2 overexpression caused delayed involution. Mechanistically, we found that EZH2 physically interacts with beta-catenin, inducing beta-catenin nuclear accumulation in mammary epithelial cells and activating <em>Wnt</em>/beta-catenin signaling. The biological significance of these data to human hyperplasias is demonstrated by EZH2 up-regulation and colocalization with beta-catenin in human intraductal epithelial hyperplasia, the earliest histologically identifiable precursor of breast carcinoma.

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to <em>12</em> months. Genetic modulation of NOTCH and/or <em>WNT</em>/β-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs.

OBJECTIVE

Lgr5 is a member of the G protein receptor super-family and was shown recently to be a stem cell marker for cells with intestinal differentiation. Its over-expression has been demonstrated in hepatocellular, basal cell carcinoma, and ovarian cancers but the underlying mechanisms are poorly understood. The aim of this study was to investigate if Lgr5 over-expression was correlated with human colorectal carcinogenesis and its potential correlation with beta-catenin.

METHODS

The study was carried out on a tissue microarray that consisted of 102 colorectal carcinomas (CRC; M:F = 55:47), 18 colon adenoma, and <em>12</em> colon normal mucosa cases. Immunostains were performed with the standard EnVision method with primary antibodies against Lgr5, beta-catenin, and p53 antigens. Immunoreactivity of neoplastic cells to each antibody was double-blindly semi-quantified by two pathologists and the data were analyzed with the Chi-square and Spearman rank correlation tests. Subsequently, expression of Lgr5 in tissue sections of tumor centre and invasive margins of 21 cases of CRC certified to be immunoreactive of Lgr5 in TMA were evaluated and possible differences of Lgr5 expression between them were analyzed.

RESULTS

Lgr5 immunoreactivity was observed only in single cells in the base of normal colon mucosal crypts but high in 28% (five out of 18) adenomas, and significantly higher in 54% (55/102, p = 0.016) CRC cases. In normal mucosa, adenoma, and CRC, beta-catenin expression was seen in 25% (three out of <em>12</em>), 27% (five out of 18), and 81% (83/102) cases, respectively, in contrast to 0, 0, and 40% (41/102) for p53 expression, respectively. In CRC, Lgr5 expression was more intense in women than men (p < 0.0001), and positively correlated with beta-catenin expression (p < 0.001), but not with patients' ages, tumor sizes, nodal status, TNM stages, and p53 expression. Different expression of Lgr5 between tumor centre and invasive margins was not found (p>> 0.05).

CONCLUSIONS

The results suggest that up-regulation of Lgr5 expression, especially in female patients, may play an important role in colorectal carcinogenesis, probably through the WNT/beta-catenin pathway, but not involve the progression of the CRC.

The aim of this work was to study gene expression patterns of cultured cumulus cells from lean and overweight-obese polycystic ovary syndrome (PCOS) patients using genome-wide oligonucleotide microarray. The study included 25 patients undergoing in vitro fertilization and intra-cytoplasmic sperm injection: <em>12</em> diagnosed with PCOS and 13 matching controls. Each of the groups was subdivided into lean (body mass index (BMI) < 24) and overweight (BMI>> 27) subgroups. The following comparisons of gene expression data were made: lean PCOS versus lean controls, lean PCOS versus overweight PCOS, all PCOS versus all controls, overweight PCOS versus overweight controls, overweight controls versus lean controls and all overweight versus all lean. The largest number of differentially expressed genes (DEGs), with fold change (FC) |FC|>>or= 1.5 and P-value < 0.01, was found in the lean PCOS versus lean controls comparison (487) with most of these genes being down-regulated in PCOS. The second largest group of DEGs originated from the comparison of lean PCOS versus overweight PCOS (305). The other comparisons resulted in a much smaller number of DEGs (174, 109, <em>12</em>5 and <em>12</em>, respectively). In the comparison of lean PCOS with lean controls, most DEGs were transcription factors and components of the extracellular matrix and two pathways, <em>Wnt</em>/beta-catenin and mitogen-activated protein kinase. When comparing overweight PCOS with overweight controls, most DEGs were of pathways related to insulin signaling, metabolism and energy production. The finding of unique gene expression patterns in cumulus cells from the two PCOS subtypes is in agreement with other studies that have found the two to be separate entities with potentially different pathophysiologies.