Microsatellite instability (MSI) is a molecular phenotype present in approximately 25% of endometrial cancers. We examined the global gene expression profiles of early-stage endometrioid endometrial cancers with and without the MSI phenotype to test the hypothesis that MSI phenotype may determine a unique molecular signature among otherwise similar cancers. Unsupervised principal component analysis of the expression data from these cases indicated two distinct groupings of cancers based on MSI phenotype. A relatively small number of array features (392) at high statistical value (P < 0.001) were identified that drive the instability signature in these cancers; 109 of these transcripts differed by at least 2-fold. These data identify distinct gene expression profiles for MSI and microsatellite stable (MSS) cancers, which suggest that cancers with MSI develop in part by different mechanisms from their similar stable counterparts. In particular, we found evidence that two members of the secreted frizzled related protein family (SFRP1 and SFRP4) were more frequently down-regulated in MSI cancers as compared with MSS cancers. Down-regulation was accompanied by promoter hypermethylation for SFRP1. SFRP1 was hypermethylated in 8 of <em>12</em> MSI cancers whereas only 3 of 16 MSS cancers were methylated. The <em>WNT</em> target fibroblast growth factor 18 was found to be up-regulated in MSI cancers. These data classify histologically similar endometrioid endometrial cancers into two distinct groupings with implications affecting therapy and prevention.

WNT signaling is fundamental to bone health, and its aberrant activation leads to skeletal pathologies. The heterozygous missense mutation p.C218G in WNTWNT pathway ligand, leads to severe early-onset and progressive osteoporosis with multiple peripheral and spinal fractures. Despite the severe skeletal manifestations, conventional bone turnover markers are normal in mutation-positive patients.

This study sought to explore the circulating microRNA (miRNA) pattern in patients with impaired WNT signaling.

A cross-sectional cohort study at a university hospital.

Altogether, 12 mutation-positive (MP) subjects (median age, 39 years; range, 11 to 76 years) and 12 mutation-negative (MN) subjects (35 years; range, 9 to 59 years) from two Finnish families with <em>WNT</em>1 osteoporosis due to the heterozygous p.C218G <em>WNT</em>1 mutation.

Serum samples were screened for 192 miRNAs using quantitative polymerase chain reaction. Findings were compared between <em>WNT</em>1 MP and MN subjects.

The pattern of circulating miRNAs was significantly different in the MP subjects compared with the MN subjects, with two upregulated (miR-18a-3p and miR-223-3p) and six downregulated miRNAs (miR-22-3p, miR-31-5p, miR-34a-5p, miR-143-5p, miR-423-5p, and miR-423-3p). Three of these (miR-22-3p, miR-34a-5p, and miR-31-5p) are known inhibitors of <em>WNT</em> signaling: miR-22-3p and miR-34a-5p target <em>WNT</em>1 messenger RNA, and miR-31-5p is predicted to bind to <em>WNT</em>1 3'UTR.

The circulating miRNA pattern reflects WNTWNTWNTWNT-related bone disorders. These miRNAs may have potential in the diagnosis and treatment of osteoporosis.

OBJECTIVE

The onset and progression of colorectal cancer (CRC) involves a cascade of genetic and/or epigenetic events. The aim of the present study was to address the DNA methylation status of genes relevant in colorectal carcinogenesis and its progression, such as genes frequently mutated in CRC, genes involved in the DNA repair and Wnt signaling pathway.

METHODS

We analyzed methylation status in totally 160 genes in 12 paired colorectal tumors and adjacent healthy mucosal tissues using the Illumina Infinium Human Methylation 450 BeadChip.

RESULTS

We found significantly aberrant methylation in 23 genes (NEIL1, NEIL3, DCLRE1C, NHEJ1, GTF2H5, CCNH, CTNNB1, DKK2, DKK3, FZD5 LRP5, TLE3, WNT2, WNT3A, WNT6, TCF7L1, CASP8, EDNRB1, GPC6, KIAA1804, MYO1B, SMAD2 and TTN). External validation by mRNA expression showed a good agreement between hypermethylation in cancer and down-regulated mRNA expression of the genes EDNRB1, GPC6 and SMAD2, and between hypomethylation and up-regulated mRNA expression of the CASP8 and DCLRE1C genes.

CONCLUSIONS

Aberrant methylation of the DCLRE1C and GPC6 genes are presented here for the first time and are therefore of special interest for further validation as novel candidate biomarker genes in CRC, and merit further validation with specific assays.

The egg-laying system of Caenorhabditis elegans hermaphrodites requires development of the vulva and its precise connection with the uterus. This process is regulated by LET-23-mediated epidermal growth factor signaling and LIN-<em>12</em>-mediated lateral signaling pathways. Among the nuclear factors that act downstream of these pathways, the LIM homeobox gene lin-11 plays a major role. lin-11 mutant animals are egg-laying defective because of the abnormalities in vulval lineage and uterine seam-cell formation. However, the mechanisms providing specificity to lin-11 function are not understood. Here, we examine the regulation of lin-11 during development of the egg-laying system. Our results demonstrate that the tissue-specific expression of lin-11 is controlled by two distinct regulatory elements that function as independent modules and together specify a wild-type egg-laying system. A uterine pi lineage module depends on the LIN-<em>12</em>/Notch signaling, while a vulval module depends on the LIN-17-mediated <em>Wnt</em> signaling. These results provide a unique example of the tissue-specific regulation of a LIM homeobox gene by two evolutionarily conserved signaling pathways. Finally, we provide evidence that the regulation of lin-11 by LIN-<em>12</em>/Notch signaling is directly mediated by the Su(H)/CBF1 family member LAG-1.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate <em>WNT</em>-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as <em>12</em>-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted <em>WNT</em> as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

Dickkopf-1 is an inhibitor of <em>Wnt</em> signaling, which is crucial for osteoblast differentiation. We evaluated serum levels of Dickkopf-1 in 66 patients with thalassemia-induced osteoporosis who received therapy with zoledronic acid in a placebo-controlled, randomized trial. At baseline, thalassemia patients had increased serum levels of Dickkopf-1 that correlated with reduced bone mineral density of the lumbar spine and the distal radius. High Dickkopf-1 also correlated with increased bone resorption and reduced bone formation markers. Zoledronic acid produced a reduction in serum Dickkopf-1, which was associated with bone mineral density increase after <em>12</em> months of therapy. On the contrary, placebo group showed a borderline increase of Dickkopf-1, which was higher in patients who showed deterioration in pain scores. These results suggest that Dickkopf-1 is implicated in the pathogenesis of osteoporosis in thalassemia and reveal Dickkopf-1 as a possible target for the development of novel agents for the management of thalassemia-induced osteoporosis.

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -<em>12</em> and -13, <em>Wnt</em>-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses <em>Wnt</em>-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of <em>Wnt</em>-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.

OBJECTIVE

Aberrant activation of the Wnt/beta-catenin signaling pathway is common in human cancers. Recently, we have shown that secreted frizzled-related proteins (SFRPs) are frequently methylated in cervical squamous cell carcinoma (SCC). Furthermore, reexpression of SFRP1 and SFRP2 could suppress tumor cell transformation and invasion. Here, we want to further investigate the methylation status and function of SFRPs in adenocarcinoma of uterine cervix.

METHODS

The methylation status of SFRPs was assessed in 23 adenocarcinomas (AC), and 45 normal control swabs by methylation-specific polymerase chain reaction and bisulfite sequencing. Then, we used reexpression of SFRP5 in cervical cancer cell lines, HeLa3rd and CaSki, to study the role of SFRP5 in cervical adenocarcinoma by colony formation and invasion assays. Finally, we checked whether SFRP5 could repress the expression of Wnt/beta-catenin downstream genes by quantitative reverse transcription-polymerase chain reaction.

RESULTS

The frequency of SFRP genes promoter hypermethylation in adenocarcinoma of cervix samples was 52.2% (12/23), 82.6% (19/23), 65.2% (15/23), and 73.9% (17/23), for SFRP1, SFRP2, SFRP4, and SFRP5, respectively. The frequency of SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in adenocarcinoma was significantly higher than in normal control samples (P < 0.001). Restoration of SFRP5 suppressed colony formation and invasive ability and inhibited expression of Wnt/beta-catenin downstream genes.

CONCLUSIONS

Our data suggest that promoter hypermethylation of SFRPs is associated with cervical adenocarcinoma, which could be used for molecular screening of cervical adenocarcinoma in the future. Moreover, SFRP5 inhibits cervical tumorigenesis through interfering Wnt pathway in vitro.

Transforming growth factor-beta 1 (TGF-beta1) is a potent inhibitor of epithelial cell proliferation. During the development of cervical carcinoma however, an increase in production of TGF-beta1 is accompanied by decreased sensitivity for the growth-limiting effect of TGF-beta1. TGF-beta1 has an anti-proliferative effect on cells of the immune system and thus can be advantageous for tumor progression. The aim of the present study was to determine the effect of TGF-beta1 on mRNA expression profile of genes in pathways involved in cell growth and cell death, in cervical carcinoma cell lines with different sensitivity to TGF-beta1. For this purpose, we have investigated changes in gene expression in TGF-beta1 stimulated cervical cancer cell lines with high (CC10B), intermediate (SiHa) and low (HeLa) sensitivity to the anti-proliferative effect of TGF-beta1, at timepoints 0, 6, <em>12</em> and 24 h. Microarray analysis, using Affymetrics focus arrays, representing 8973 genes, was used to measure gene expression. In our study novel target genes involved in tumor necrosis factor alpha (TNFalpha), mitogen-activated protein kinase (MAPK) and wingless type (<em>Wnt</em>) pathways in response to TGF-beta1 were found. Substantial differences in gene expression between TGF-beta1 sensitive and insensitive cell lines were observed involving genes in TNFalpha, MAPK, <em>Wnt</em> and Smad pathways. Since these pathways are implicated in cell proliferation and cell death, these pathways may play a role in determining the overall sensitivity of a cell to TGF-beta1 induced cell growth inhibition. The results were subsequently validated by quantitative real-time PCR. Increased resistance to TGF-beta1 induced cell growth inhibition was correlated with an elevated production of TGF-beta1 by the cell lines, as measured by enzyme linked immunosorbent assay. TGF-beta1 production did not inhibit cell growth, since blocking TGF-beta1 protein by anti-TGF-beta had no effect on cell proliferation. TGF-beta1 excretion by tumor cells more likely contributes to paracrine stimulation of tumor development.

Beta-catenin plays significant roles in cell-to-cell adhesion and the <em>Wnt</em>/Wg signal transduction pathway. Accumulation of this protein in the cytoplasm and nucleus as a result of mutations of the adenomatous polyposis coli tumor suppressor gene or of the beta-catenin gene itself is often seen in a wide variety of tumors including carcinomas of the colon, liver, uterus, and brain. Interaction of accumulated beta-catenin with Tcf/Lef transcription factors is known to deregulate expression of some downstream genes, but the precise mechanisms whereby beta-catenin contributes to carcinogenesis remain to be disclosed. Here we report isolation of a novel murine gene, Drctnnb1a (down-regulated by Ctnnb1, a), the expression of which was experimentally down-regulated in response to the activated form of beta-catenin. To investigate a possible role of DRCTNNB1A in cancers, we also isolated the human homologue, DRCTNNB1A, the deduced product of which was 91% identical to the murine protein. The transcript was expressed in all human tissues examined, and we assigned the genomic location of DRCTNNB1A to chromosomal band 7p15.3 by in situ hybridization. Expression of DRCTNNB1A in SW480 colon cancer cells was significantly increased in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of the beta-catenin-binding domain of the adenomatous polyposis coli gene into the cells. Furthermore, we documented reduced expression of DRCTNNB1A in <em>12</em> of 15 primary colorectal cancers examined, compared with corresponding adjacent noncancerous mucosae. Our results implied that DRCTNNB1A is one of the genes involved in the beta-catenin-Tcf/Lef signaling pathway, and that reduced expression of DRCTNNB1A may have some role in colorectal carcinogenesis.

The infiltration of adipocytes in osteoporotic patients' bone marrow suggests an important regulatory function of bone marrow fat on the development of aged bone. Therefore, we have examined the effects of adipocytes derived from bone mesenchymal stem cell (MSC) on osteoblast differentiation using two different co-culture modes (direct mode and indirect mode). Alkaline phosphatase (ALP)-positive areas and mineralized areas of MSC-derived osteoblasts decrease similarly in the two co-culture modes as the amount of MSC-derived adipocytes increases, suggesting that the crosstalk between adipocytes and osteoblasts may be mainly through secretory factors in the medium. To further understand the molecular mechanisms, both mRNA and protein expressions in osteoblasts in the lower layer of the indirect mode were analyzed, leading to identification of <em>12</em> differential genes/proteins. Among them, S100A6 and calreticulin are possibly related to bone formation. S100A6 was down-regulated and calreticulin was up-regulated as MSC-derived adipocytes increased. Similarly, differential expression of these proteins was also observed in bone tissue slides from young (1-month-old) and old (6-month-old) mice. The expression level of β-catenin in osteoblasts of bone tissues was lower in 6-month-old mice compared to 1-month-old mice. Total TGF-β analyzed with antibody-based protein microarray and active TGF-β analyzed with ELISA in the co-cultured cell medium increased consistently as the amount of adipocytes increased. Taken together, our results suggest that MSC-derived adipocytes may regulate osteoblast differentiation in the aged bone through TGF-β-mediated canonical <em>Wnt</em> signaling.

The C. elegans hermaphrodite vulva serves as a paradigm for understanding how signaling pathways control organ formation. Previous studies have shown that <em>Wnt</em> signaling plays important roles in vulval development. To understand the function and evolution of <em>Wnt</em> signaling in Caenorhabditis nematodes we focused on C. briggsae, a species that is substantially divergent from C. elegans in terms of the evolutionary time scale yet shares almost identical morphology. We isolated mutants in C. briggsae that display multiple pseudo-vulvae resulting from ectopic VPC induction. We cloned one of these loci and found that it encodes an Axin homolog, Cbr-PRY-1. Our genetic studies revealed that Cbr-pry-1 functions upstream of the canonical <em>Wnt</em> pathway components Cbr-bar-1 (beta-catenin) and Cbr-pop-1(tcf/lef) as well as the Hox target Cbr-lin-39 (Dfd/Scr). We further characterized the pry-1 vulval phenotype in C. briggsae and C. elegans using 8 cell fate markers, cell ablation, and genetic interaction approaches. Our results show that ectopically induced VPCs in pry-1 mutants adopt 2° fates independently of the gonad-derived inductive and LIN-<em>12</em>/Notch-mediated lateral signaling pathways. We also found that Cbr-pry-1 mutants frequently show a failure of P7.p induction. A similar, albeit low penetrant, defect is also observed in C. elegans pry-1 mutants. The genetic analysis of the P7.p induction defect revealed that it was caused by altered regulation of lin-<em>12</em> and its transcriptional target lip-1 (MAP kinase phosphatase). Thus, our results provide evidence for LIN-<em>12</em>/Notch-dependent and independent roles of <em>Wnt</em> signaling in promoting 2 degrees VPC fates in both nematode species.

The adaptive response of bone to load is dependent on molecular factors, including growth factor signaling, which is involved in the regulation of proliferation, differentiation and function of osteoblasts and osteoclasts. Based on a recent study, which has shown that the deficiency of growth factor midkine (Mdk) in mice at <em>12</em> and 18 months of age resulted in increased trabecular bone formation, we hypothesized that mechanically-induced bone remodeling may, at least in part, be dependent on Mdk expression. To investigate this, we loaded the ulnae of Mdk-deficient mice and appropriate wild-type mice at the age of <em>12</em> months using the in vivo ulna loading model. Histomorphometric quantification of the periosteal bone demonstrated an increased mineralizing surface, mineral apposition rate, and bone formation rate in ulnae of Mdk-deficient mice compared to wild-type mice in response to loading. Because Mdk has been shown to bind to a complex of receptor-type protein tyrosine phosphatase zeta (Ptprz) and low density lipoprotein receptor-related protein-6 (Lrp-6) together with the α4β1- and α6β1-integrins, we performed in vitro studies using osteoblastic cells, transiently over-expressing Mdk, <em>Wnt</em>-3a, and Ptprz to evaluate whether Mdk has a role in regulating bone formation by modulating <em>Wnt</em> signaling. We observed a negative effect of Mdk on <em>Wnt</em> signaling, the extent of which appeared to be dependent on Ptprz expression. Moreover, we performed in vitro loading studies with osteoblasts treated with recombinant Mdk and observed a negative effect on the expression of <em>Wnt</em> target genes, which play a critical role in osteoblast proliferation. In summary, our data demonstrate that Mdk-deficiency in mice has an anabolic effect on mechanically induced cortical bone formation. This could be due to an improved osteoblast function based on an enhancement of β-catenin-dependent <em>Wnt</em> signaling by both Mdk-deficiency and mechanical loading.

Tumor Endothelial Marker 8/Anthrax Toxin Receptor 1 (TEM8/ANTXR1) expression is induced in the vascular compartment of multiple tumors and therefore, is a candidate molecule to target tumor therapies. This cell surface molecule mediates anthrax toxin internalization, however, its physiological function in blood vessels remains largely unknown. We identified the chicken chorioallantoic membrane (CAM) as a model system to study the endogenous function of TEM8 in blood vessels as we found that TEM8 expression was induced transiently between day 10 and <em>12</em> of embryonic development, when the vascular tree is undergoing final development and growth. We used the cell-binding component of anthrax toxin, Protective Antigen (PA), to engage endogenous TEM8 receptors and evaluate the effects of PA-TEM8 complexes on vascular development. PA applied at the time of highest TEM8 expression reduced vascular density and disrupted hierarchical branching as revealed by quantitative morphometric analysis of the vascular tree after 48h. PA-dependent reduced branching phenotype was partially mimicked by <em>Wnt</em>3a application and ameliorated by the <em>Wnt</em> antagonist, Dikkopf-1. These results implicate TEM8 expression in endothelial cells in regulating the canonical <em>Wnt</em> signaling pathway at this day of CAM development. Consistent with this model, PA increased beta catenin levels acutely in CAM blood vessels in vivo and in TEM8 transfected primary human endothelial cells in vitro. TEM8 expression in Hek293 cells, which neither express endogenous PA-binding receptors nor <em>Wnt</em> ligands, stabilized beta catenin levels and amplified beta catenin-dependent transcriptional activity induced by <em>Wnt</em>3a. This agonistic function is supported by findings in the CAM, where the increase in TEM8 expression from day 10 to day <em>12</em> and PA application correlated with Axin 2 induction, a universal reporter gene for canonical <em>Wnt</em> signaling. We postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical <em>Wnt</em> signaling to regulate vessel patterning and density.

BACKGROUND

Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5) collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon) which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM) from ambient air particles, human embryonic lung fibroblasts (HEL<em>12</em>469) were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls.

METHODS

For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina). The expression of selected genes was verified by quantitative real-time PCR.

RESULTS

Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between <em>12</em> to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair), the <em>Wnt</em> and TGF-β signaling pathways (associated particularly with tumor promotion and progression), Steroid hormone biosynthesis (involved in the endocrine-disrupting activity of chemicals), and Glycerolipid metabolism (pathways involving the lipids with a glycerol backbone including lipid signaling molecules).

CONCLUSIONS

The microarray data suggested a prominent role of activation of aryl hydrocarbon receptor-dependent gene expression.

Kaiso is a dual-specificity POZ-ZF transcription factor that regulates gene expression by binding to sequence-specific Kaiso binding sites (KBS) or methyl-CpG dinucleotide pairs. Kaiso was first identified as a binding partner for the epithelial cell adhesion regulator p<em>12</em>0(ctn). The p<em>12</em>0(ctn)/Kaiso interaction is reminiscent of the beta-catenin/TCF interaction and several studies have suggested that Kaiso is a negative regulator of the <em>Wnt</em>/beta-catenin TCF signaling pathway. To gain further insight into Kaiso's function, we performed a yeast two-hybrid screen using the Kaiso POZ domain as bait. This screen identified the POZ-ZF protein, Znf131, as a Kaiso-specific binding partner. GST pull-down assays confirmed that the interaction is mediated via the POZ domain of each protein, and co-immunoprecipitation experiments further supported an in vivo Kaiso-Znf131 interaction. Using a Cyclic Amplification and Selection of Targets (CAST) approach, we identified the <em>12</em>-base pair DNA palindrome sequence GTCGCR-(X)(n)-YGCGAC as a potential Znf131 binding element (ZBE). In vitro studies using electrophoretic mobility shift assay (EMSA) demonstrated that Znf131 binds the ZBE via its zinc finger domain. Znf131 DNA-binding specificity was confirmed using competition assays and ZBE mutational analyses. An artificial promoter-reporter construct containing four tandem copies of the ZBE was constructed and used to assess Znf131 transcriptional properties. We observed dose-dependent transcriptional activation of this artificial promoter-reporter by Znf131 in both epithelial and fibroblast cells, suggesting that Znf131 is a transcriptional activator. Kaiso overexpression significantly decreased the Znf131-mediated transcriptional activation, and interestingly, co-expression of the Kaiso-specific interaction partner p<em>12</em>0(ctn) relieved Kaiso's inhibition of Znf131-mediated transcriptional activation. These findings indicate that Znf131 is a transcriptional activator, a less common function of POZ-ZF proteins, that is negatively regulated by its heterodimerization partner Kaiso.

BACKGROUND

Lead (Pb) exposure and obesity are co-occurring risk factors for decreased bone mass in the young, particularly in low socioeconomic communities.

OBJECTIVE

The goal of this study was to determine whether the comorbidities of Pb exposure and high-fat diet-induced obesity amplify skeletal deficits independently associated with each of these risk factors, and to explore associated mechanisms of the observed deficiencies.

METHODS

Five-week-old male C57BL/6J mice were placed on low-fat (10% kcal, LFD) or high-fat (60% kcal, HFD) diets for <em>12</em> weeks. Mice were exposed to lifetime Pb (50 ppm) through drinking water.

RESULTS

HFD was associated with increased body mass and glucose intolerance. Both HFD and Pb increased fasting glucose and serum leptin levels. Pb and HFD each reduced trabecular bone quality and together had a further detrimental effect on these bone parameters. Mechanical bone properties of strength were depressed in Pb-exposed bones, but HFD had no significant effect. Both Pb and HFD altered progenitor cell differentiation, promoting osteoclastogenesis and increasing adipogenesis while suppressing osteoblastogenesis. In support of this lineage shift being mediated through altered Wnt signaling, Pb and non-esterified fatty acids in MC3T3 cells increased in vitro PPAR-γ activity and inhibited β-catenin activity. Combining Pb and non-esterified fatty acids enhanced these effects.

CONCLUSIONS

Pb and HFD produced selective deficits in bone accrual that were associated with alterations in progenitor cell activity that may involve reduced Wnt signaling. This study emphasizes the need to assess toxicants together with other risk factors relevant to human health and disease.

OBJECTIVE

Sublesional osteoporosis predisposes individuals with spinal cord injury (SCI) to an increased risk of low-trauma fracture. The aim of the present work was to investigate the effect of treatment with resveratrol (RES) on sublesional bone loss in spinal cord-injured rats.

METHODS

Complete SCI was generated by surgical transaction of the cord at the T10-<em>12</em> level. Treatment with RES (400 mg·kg(-1) body mass per day(-1) , intragastrically) was initiated <em>12</em> h after the surgery for 10 days. Then, blood was collected and femurs and tibiae were removed for evaluation of the effects of RES on bone tissue after SCI.

RESULTS

Treatment of SCI rats with RES prevented the reduction of bone mass including bone mineral content and bone mineral density in tibiae, preserved bone structure including trabecular bone volume fraction, trabecular number, and trabecular thickness in tibiae, and preserved mechanical strength including ultimate load, stiffness, and energy in femurs. Treatment of SCI rats with RES enhanced femoral total sulfhydryl content, reduced femoral malondialdehyde and IL-6 mRNA levels. Treatment of SCI rats with RES suppressed the up-regulation of mRNA levels of PPARγ, adipose-specific fatty-acid-binding protein and lipoprotein lipase, and restored mRNA levels of Wnt1, low-density lipoprotein-related protein 5, Axin2, ctnnb1, insulin-like growth factor 1 (IGF-1) and receptor for IGF-1 in femurs and tibiae.

CONCLUSIONS

Treatment with RES attenuated sublesional bone loss in spinal-cord-injured rats, associated with abating oxidative stress, attenuating inflammation, depressing PPARγ signalling, and restoring Wnt/β-catenin and IGF-1 signalling.

Progesterone is often used to protect the endometrium and prevent endometrial cancer. An intensive study on its molecular mechanism in endometrial cancer would contribute to the development of more promising therapies. Relevant lncRNAs and mRNAs expression data in endometrial cancer cell line Ishikawa pretreated and post-treated with progesterone were derived from Gene Expression Omnibus (accession no. GSE29435), and then we analyzed long noncoding RNAs and mRNAs with differential expressions in two different conditions. The Cytoscape software, TargetScan, miRanda, and Human microRNA Disease Database (HMDD) websites were employed. Gene set enrichment analysis (GSEA) was used to determine related Kyoto Encyclopedia of Genes and Genomes pathways alteration in Ishikawa cells treated with progesterone. In addition to bioinformatics analysis, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blot, and dual-luciferase reporter assays were performed. The impact of progesterone on cell propagation and cell cycle was testified by colony formation and flow cytometry analysis. LncRNA nuclear enriched abundant transcript 1 (NEAT1) was the most significantly downregulated lncRNA in endometrial cancer cells treated with progesterone. Lymphoid enhancing factor 1 (LEF1) was positively associated with NEAT1, and eventually hsa_miR-146b-5p was validated to target both LEF1 and NEAT1. <em>Wnt</em>/β-catenin signaling pathway was identified to involve in endometrial cancer. NEAT1 or LEF1 was overexpressed in endometrial cancer cells while downregulated following post-treatment with progesterone. Conversely, miR-146b-5p was notably decreased in Ishikawa cells while upregulated after treatment with progesterone. Downstream gene c-myc or MMP9 regulated by upstream gene LEF1 in <em>Wnt</em>/β-catenin signaling pathway was remarkably increased in Ishikawa cells and positively related with NEAT1. Progesterone inhibited cell cycle and viability through regulating NEAT1/miR-146b-5p axis via <em>Wnt</em>/β-catenin signaling pathway. Progesterone exerted suppressive influence on endometrial cancer progression via regulation of lncRNA NEAT1/miR-146b-5p-mediated <em>Wnt</em>/β-catenin signaling pathway, which might reveal new strategies for developing more effective therapeutics. © 2018 IUBMB Life, :1-<em>12</em>, 2018.

Research has shown the importance of follistatin, <em>Wnt</em> 7a, and wound healing growth factors on the stimulation of bulge cells and inter-follicular stem cells to induce hair growth. We have studied the effects of a bioengineered, non-recombinant, human cell-derived formulation, termed Hair Stimulating Complex (HSC), containing these factors to assess its hair growth activity in male pattern baldness. HSC showed in vitro <em>Wnt</em> activity and contained follistatin, KGF, and VEGF. The clinical study was a double-blind, placebo-controlled, randomized single site trial and was designed to evaluate safety of the HSC product and assess efficacy in stimulating hair growth. All 26 subjects tolerated the single, intradermal injection of HSC procedures well, and no signs of an adverse reaction were reported. Histopathological evaluation of the treatment site biopsies taken at 22 and 52 weeks post-treatment revealed no abnormal morphology, hamartomas, or other pathological responses. Trichoscan image analysis of HSC-treated sites at <em>12</em> and 52 weeks showed significant improvements in hair growth over the placebo. At the initial <em>12</em>-week evaluation period, HSC-treated sites demonstrated an increase in hair shaft thickness (6.3%±2.5% vs. -0.63%±2.1%; P=0.046), thickness density (<em>12</em>.8%±4.5% vs. -0.2%±2.9%; P=0.028), and terminal hair density (20.6±4.9% vs. 4.4±4.9%; P=0.029). At one year, a statistically significant increase in total hair count (P=0.032) continued to be seen. These results demonstrate that a single intradermal administration of HSC improved hair growth in subjects with androgenetic alopecia and is a clinical substantiation of previous preclinical research with <em>Wnts</em>, follistatin, and other growth factors associated with wound healing and regeneration.

We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and beta-catenin stabilization. Although it is well recognized that <em>Wnt</em> signaling leads to beta-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces beta-catenin stabilization independent of <em>Wnt</em>, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-beta-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Galpha(<em>12</em>) and Galpha(13) compared with age-matched control counterparts. However, only Galpha(13) was found to be actively involved in PAR1-induced beta-catenin stabilization. Indeed, a dominant negative form of Galpha(13) inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Galpha(13) association is followed by the recruitment of DVL (Dishevelled), an upstream <em>Wnt</em> signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased beta-catenin accumulation. It is of note that PAR1 also promotes the binding of beta-arrestin-2 to DVL, suggesting a role for beta-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the <em>Wnt</em> antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced <em>Wnt</em>3A-mediated beta-catenin accumulation, no effect was observed on PAR1-induced beta-catenin stabilization. Collectively, our data show that PAR1 mediates beta-catenin stabilization independent of <em>Wnt</em>. We propose here a novel cascade of PAR1-induced Galpha(13)-DVL axis in cancer and beta-catenin stabilization.

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from <em>12</em> patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of <em>WNT</em> signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and <em>WNT</em> activation in relapse samples. Activation of <em>WNT</em> signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased <em>WNT</em> activity. Overall, our results suggest <em>WNT</em> signaling activation as a mechanism of chemoresistance in relapsed SCLC.

OBJECTIVE

Sclerostin is secreted by osteocytes. As a circulating inhibitor of the Wnt-signaling pathway it inhibits bone formation and contributes to the development of osteoporosis. Sclerostin levels are elevated in patients with chronic kidney disease and end-stage renal disease. Since data for patients after kidney transplantation are scarce, we have prospectively measured sclerostin levels before and during the first year after renal transplantation and have examined the association of sclerostin with parameters of bone mineral metabolism and with bone mineral density.

METHODS

Sclerostin levels were measured by ELISA in 42 consecutive renal transplant recipients before and at defined intervals in the first year after transplantation. Bone mineral density was measured by dual energy X-ray absorptiometry.

RESULTS

Pre-transplant serum sclerostin levels were elevated in all patients (61.8 ± 32.3 pmol/l, normal range 20-30 pmol/l). Within 15 days after transplantation and correlating with the improvement of renal function, sclerostin levels dropped to 21.0 ± 14.7 pmol/l and subsequently increased to 23.8 ± 14.9 and 28.0 ± 16.8 pmol/l after 6 and 12 months, respectively (P<0.001). A linear mixed model indicated that pre-transplant sclerostin levels (P<0.001) and time after transplantation (P<0.001) were the most important predictors for the rise of post-transplant sclerostin levels. No correlation was found between post-transplant sclerostin levels and bone mineral density.

CONCLUSIONS

The rapid reduction of elevated serum sclerostin levels shortly after kidney transplantation parallels the improvement of renal function, but contrasts with the more delayed improvement of hyperparathyroidism. The normalization of both hormones could contribute to improved bone health after renal transplantation.

<em>Wnt</em> signaling regulates some aspect of development of nearly all endoderm-derived organs and <em>Wnts</em> mediate both differentiation and proliferation at different steps during visceral organogenesis. <em>Wnt</em>2b induces liver formation in zebrafish 1 and may combine with other inducers, Fibroblast Growth Factors 1 & 4 and Bone Morphogenetic Protein 4, to specify the mammalian liver.2-5 Later in development, <em>Wnts</em> are critical for liver expansion and, finally, for terminal hepatocyte differentiation,6-<em>12</em> as reviewed elsewhere in this issue (Monga). Likewise, in the pancreas, <em>Wnts</em> drive proliferation of exocrine and endocrine cells13,14 and promote acinar cell differentiation,13,15 as reviewed in the chapter by Murtaugh. Here we examine the intricate involvement of <em>Wnt</em> signaling in growth and differentiation of the digestive tract.