Although small and large intestines possess seemingly similar <em>Wnt</em>-driven leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)+ adult epithelial stem cells, we report here that the two organs exhibit distinct mechanisms of tissue response to ionizing radiation. Employing Lgr5-lacZ transgenic mice and Lgr5 in situ hybridization, we found colonic epithelial stem cells (CESC) markedly more radioresistant in vivo than small intestinal crypt base columnar stem cells (CBC; D0 = 6.0 ± 0.3 Gy vs. 1.3 ± 0.1, respectively; P < 0.01). Accordingly, CESCs survived 30 Gy exposure, while CBCs were completely depleted after 15 Gy. EdU incorporation studies indicated that after 19 Gy, CBCs exited growth arrest at <em>12</em> hours, resuming normal mitotic activity despite 60% of this population displaying residual γH2AX foci, indicative of persistent unrepaired DNA damage. Checkpoint recovery before complete double-strand break (DSB) repair represents the sine qua non of a newly defined potentially lethal pathophysiology termed checkpoint adaptation. In the small intestinal mucosa, checkpoint adaptation resulted in CBCs succumbing to an 8-fold increase in the incidence of highly lethal chromosomal aberrations and mitotic catastrophe by 48 hours postradiation. In contrast, Lgr5+ CESCs displayed delayed checkpoint recovery at 48 hours post-19 Gy, coordinated with complete DSB repair and regeneration of colonic mucosa originating, at least in part, from surviving CESCs. The discovery that small intestinal CBCs succumb to checkpoint adaptation is the first demonstration that this aberrant cell-cycle response may drive mammalian tissue radiosensitivity. Cancer Res; 77(8); 2<em>12</em>4-33. ©2017 AACR.

BACKGROUND

Inositol hexakisphosphate (IP6), a naturally occurring polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumors. However, the molecular mechanism of the anticancer effect of IP6 has not been fully elucidated.

METHODS

Using K-562 human leukemia cells we analysed the induction of the erythroid differentiation program, as well as modulation of the gene expression profile of K-562 leukemia cells treated with IP6.

RESULTS

A single treatment with IP6 (0.75 or 5.0 mM) resulted in a time- and dose-dependent growth inhibition of K-562 cells and also activation of the erythroid differentiation program. K-562 cells expressed a concomitant differentiation after <em>12</em> hours of exposure. Possible molecular mechanisms and key signaling pathways, as well as gene expression behind this anticancer effect were examined using oligonucleotide microarrays and quantitative real-time PCR. Treatment with IP6 (750 microM, 5 mM) had a marked impact, resulting in early (60 min) and late (<em>12</em> h) modulation of expression of about 1800 and <em>12</em>00 transcripts (at p<0.05). Through microarray analysis, the anticancer effect of IP6 in K-562 was found to be associated with the modulation of multiple genes involved in immunity, <em>Wnt</em> and IGF pathways, PI3 kinase signaling and apoptosis. Using selected subsets of genes, the microarray hits could be validated by Q-PCR. A 2-fold upregulation of the apoptosis pathway, measured using the BAX/BCL-2 ratio was observed for <em>12</em> hours. IP6 (5 mM) induced up to 6-fold increases in differentiation measured by hemoglobin synthesis, yielding up to 70% of benzidine-positive cells at <em>12</em>0 hours.

CONCLUSIONS

The results of this study show that IP6 is a strong inducer of differentiation (cytostatic effect) and a moderately strong inducer of apoptosis (cytotoxic effect). Evidence has been provided to show that the growth inhibitory effects of IP6 are mediated through the modulation of key signaling pathways.

Squamous metaplasia is a common pathological process that occurs in the ocular surface epithelium. At present, there is no effective treatment for this abnormality. In the current study, we established an ex vivo conjunctival squamous metaplasia model by culturing human conjunctival tissues at an air-liquid interface for durations of up to <em>12</em> days. We then investigated the effects of amniotic membrane (AM) on squamous metaplasia through coculture of conjunctival tissues with AM or AM extract. We found that metaplasia features such as hyperproliferation and abnormal epidermal differentiation of conjunctival epithelium could be inhibited by AM or its extract. In addition, existing squamous metaplasia of conjunctival epithelium could be reversed to a nearly normal phenotype by AM. The mechanism by which AM prevents squamous metaplasia may involve downregulation of p38 mitogen-activated protein kinase and <em>Wnt</em> signaling pathways, which were activated in conjunctival explants cultured with an airlift technique. In conclusion, AM can inhibit and reverse squamous metaplasia of conjunctival epithelium. This finding may shed new light on prevention and treatment of diseases that involve epithelial squamous metaplasia.

Tcf family transcription factors are the downstream effectors of the <em>Wnt</em> signal transduction pathway that regulates developmental and oncogenetic processes among species. The alternative splicing of consecutive exons in the 3' part of the Tcf-4 gene and an error-prone A9 repeat in exon 17 have recently been examined in several tumors. To further understand the roles played by differential expression and splicing isoforms of human Tcf-4 in brain tumorigenesis, the expression of the 3' part of the Tcf-4 gene (exons 10-17) was analyzed by RT-PCR, nested RT-PCR and DNA sequencing. The results showed that at least 13 of the Tcf-4 alternative splicing isoforms were found and most of them were overexpressed in various brain tumors. L3 isoform was particularly dominant in metastasis. Several novel splicing isoforms were identified. One that contains different combinations of exon 16 (10-11-<em>12</em>-16-17, S5) was found in normal brain and pituitary adenoma but not in astrocytoma, meningioma or metastasis, whereas the other that contains part of intron 16, designated exon-16', was found in metastasis. Overall 23% of sequencing analysis in brain tumors exhibited frameshift mutations in an A9 repeat region of exon 17. These mutants exhibited 2-base or 1-base deletion (A7, A8) and 1-base insertion (A10). Nonetheless, in vitro functional assay showed that these mutants did not affect the transactivity of Tcf-4 comparing to wild-type Tcf-4. Collectively, our data suggest that a large number of alternative splicing isoforms may together with variable mutations of A9 repeat region maintain balanced pools of Tcf-4 isoforms during brain tumorigenesis.

Coordinated movement depends on the creation of synapses between specific neurons in the motor circuit. In C. elegans, this important decision is regulated by the UNC-4 homeodomain protein. unc-4 mutants are unable to execute backward locomotion because VA motor neurons are mis-wired with inputs normally reserved for their VB sisters. We have proposed that UNC-4 functions in VAs to block expression of VB genes. This model is substantiated by the finding that ectopic expression of the VB gene ceh-<em>12</em> (encoding a homolog of the homeodomain protein HB9) in unc-4 mutants results in the mis-wiring of posterior VA motor neurons with VB-like connections. Here, we show that VA expression of CEH-<em>12</em> depends on a nearby source of the <em>Wnt</em> protein EGL-20. Our results indicate that UNC-4 prevents VAs from responding to a local EGL-20 cue by disabling a canonical <em>Wnt</em> signaling cascade involving the Frizzled receptors MIG-1 and MOM-5. CEH-<em>12</em> expression in VA motor neurons is also opposed by a separate pathway that includes the <em>Wnt</em> ligand LIN-44. This work has revealed a transcriptional mechanism for modulating the sensitivity of specific neurons to diffusible <em>Wnt</em> ligands and thereby defines distinct patterns of synaptic connectivity. The existence of comparable <em>Wnt</em> gradients in the vertebrate spinal cord could reflect similar roles for <em>Wnt</em> signaling in vertebrate motor circuit assembly.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549), was exposed to cigarette smoke extract (CSE) for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-β and <em>Wnt</em>. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-<em>12</em>) and rat (RLE-6TN) epithelial cells. The secretion of activated TGF-β1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-β1, which may explain at least partially, the increased risk of developing IPF in smokers.

Neuropeptide Y (NPY) is a neuropeptide secreted by sensory nerve fibers distributed in the marrow and vascular canals of bone tissue. However, the effect of NPY on the osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) remains controversial and has not been thoroughly investigated. To explore the osteogenic activity and the migration and VEGF expression capabilities of BMSCs affected by NPY, as well as the underlying mechanisms, we investigated the potential relationships among NPY, osteoblastic differentiation, angiogenesis and canonical <em>Wnt</em> signaling in BMSCs. NPY was observed to regulate osteoblastic differentiation at concentrations ranging from 10(-8) to 10(-<em>12</em>)mol/L, and the effects of NPY on the levels of <em>Wnt</em> signaling proteins were detected using Western blotting. To unravel the underlying mechanism, BMSCs were treated with NPY after pretreatment with the NPY-1R antagonist PD160170 or the <em>Wnt</em> pathway antagonist DKK1, and gene expression levels of <em>Wnt</em> signaling molecules and osteoblastic markers were determined by qPCR. Our results indicated that NPY significantly promoted osteoblastic differentiation of BMSCs in a concentration-dependent manner and up-regulated the expression levels of proteins including β-catenin and p-GSK-3β and the mRNA level of β-catenin. Moreover, NPY promoted the translocation of β-catenin into nucleus. The effects of NPY were inhibited by PD160170 or DKK1. Additionally, NPY enhanced the ability of BMSCs to migrate and promoted the expression of vascular endothelial growth factor (VEGF) as measured by immunocytochemical staining, qPCR and Western blot. These results suggested that NPY may stimulate osteoblastic differentiation via activating canonical <em>Wnt</em> signaling and enhance the angiogenic capacity of BMSCs.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at <em>12</em> mo of age. These tumors demonstrated pAKT and nuclear β-catenin staining, indicating simultaneous activation of the PI3K/AKT and <em>Wnt</em> signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of β-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

Transgenic mice that express dominant-negative RhoA (RhoA(DN) ) in ameloblasts have hypoplastic enamel with defects in molar cusps. β-catenin and <em>Wnt</em>5a were up-regulated in enamel organs of RhoA(DN) transgenic mice, which indicated that both canonical and non-canonical <em>Wnt</em> pathways are implicated in the process of enamel defect formation. It was hypothesized that expression of RhoA(DN) in ameloblasts interfered with normal enamel development through the pathways that were induced by fluoride. The <em>Wnt</em> and RhoA pathways were further investigated in an ameloblast-lineage cell line (ALC) by treatment with sodium fluoride (NaF). The activities of RhoA and Rho-associated protein kinase (ROCK) II decreased significantly by 8-<em>12</em> hours, similar to decreased activity in RhoA(DN) transgenic mice. Both canonical and non-canonical <em>Wnt</em> pathways were activated by treatment with NaF, which was verified by western blotting and the β-catenin-TCF/LEF (T cell factor lymphanoid/enhancer factor) reporter gene (TOPflash) assay. β-catenin localization to both cytoplasm and nucleus was up-regulated in NaF-treated ALC, while Gsk-3β, the negative regulator of the <em>Wnt</em> pathway, showed a decreased pattern of expression. The current results indicate that both <em>Wnt</em> and RhoA pathways are implicated in fluoride-induced signaling transductions in the ALC as well as in the development of enamel defects in RhoA(DN) transgenic mice.

BACKGROUND

Spinal cord injury (SCI) results in muscle atrophy and a shift of slow oxidative to fast glycolytic fibers. Electrical stimulation (ES) at least partially restores muscle mass and fiber type distribution. The objective of this study was to was to characterize the early molecular adaptations that occur in rat soleus muscle after initiating isometric resistance exercise by ES for one hour per day for 1, 3 or 7 days when ES was begun 16 weeks after SCI. Additionally, changes in mRNA levels after ES were compared with those induced in soleus at the same time points after gastrocnemius tenotomy (GA).

RESULTS

ES increased expression of Hey1 and Pitx2 suggesting increased Notch and <em>Wnt</em> signaling, respectively, but did not normalize RCAN1.4, a measure of calcineurin/NFAT signaling, or PGC-1ß mRNA levels. ES increased PGC-1α expression but not that of slow myofibrillar genes. Microarray analysis showed that after ES, genes coding for calcium binding proteins and nicotinic acetylcholine receptors were increased, and the expression of genes involved in blood vessel formation and morphogenesis was altered. Of the 165 genes altered by ES only 16 were also differentially expressed after GA, of which <em>12</em> were altered in the same direction by ES and GA. In contrast to ES, GA induced expression of genes related to oxidative phosphorylation.

CONCLUSIONS

Notch and Wnt signaling may be involved in ES-induced increases in the mass of paralyzed muscle. Molecular adaptations of paralyzed soleus to resistance exercise are delayed or defective compared to normally innervated muscle.

There is current evidence implicating the <em>Wnt</em>/beta-catenin/TCF pathway in breast cancer. We investigated the effect of para- and meta-positional isomers of nitric oxide-releasing aspirin (NO-ASA), and aspirin (ASA) on MCF-7 human breast cancer cell growth and beta-catenin/TCF signaling. The p- and m-NO-ASA isomers strongly inhibited cell growth and beta-catenin/TCF transcriptional activity compared to ASA; the IC50s for growth inhibition were 57+/-4, 193+/-10 and >5000microM, and for transcriptional inhibition they were <em>12</em>+/-1.8, 75+/-6.5 and >5000microM for p-, m-NO-ASA and ASA, respectively. p-NO-ASA reduced the expression of <em>Wnt</em>/beta-catenin downstream target gene cyclin D1, and total cellular beta-catenin levels. COX-2 expression was induced by p-NO-ASA, protein kinase C inhibitors reversed this induction. p-NO-ASA blocked the cell cycle transition at S to G2/M phase. These studies suggest a targeted chemopreventive/chemotherapeutic potential for NO-ASA against breast cancer.

Dishevelled (DVL) proteins are central mediators of the <em>Wnt</em> signalling pathway and are versatile regulators of several cellular processes, yet little is known about their post-translational regulation. Acetylation is a reversible post-translational modification (PTM) which regulates the function of several non-histone proteins involved in tumorigenesis. Since we previously demonstrated that lysine deacetylase, SIRT-1, regulates DVL protein levels and its function, we reasoned that DVL could potentially be a substrate for SIRT-1 mediated deacetylation. To further examine the potential role of multiple families of lysine deacetylases in the post-translational regulation of DVL, we screened for novel acetylation sites using liquid chromatography mass-spectrometry (LC-MS/MS) analysis. Herein, we report <em>12</em> DVL-1 lysine residues that show differential acetylation in response to changes in oxygen tension and deacetylase inhibition in triple-negative breast cancer (TNBC). PTMs are well documented to influence protein activity, and cellular localization. We also identify that acetylation of two key lysine residues, K69 and K285, present on the DIX and PDZ domains respectively, promote nuclear over cytoplasmic localization of DVL-1, and influences its promoter binding and regulation of genes implicated in cancer. Collectively, these findings for the first time, uncover acetylation as a novel layer of regulation of DVL-1 proteins.

BACKGROUND

Biallelic mutations in WNT1 can give rise to a rare form of moderate to severe OI. Here we report on <em>12</em> children (age 2 to 16 years; 5 girls) with biallelic WNT1 mutations.

METHODS

Genomic DNA was analyzed either by targeted next-generation sequencing or Sanger sequencing. Mutations were modeled on the WNT1 protein structure. The in vitro functional effect of WNT1 mutations on WNT signaling was assessed in HEK293 cells using the topflash reporter assay system.

RESULTS

All patients had lower extremity deformities and vertebral compression fractures. Seven individuals had upper extremity deformities. Intellectual development appeared normal in 11 children, but was clearly impaired in a 3-year old boy. Ptosis was noted in 7 patients. Height z-scores varied widely, from -7.2 to +1.5. A total of 11 disease-causing WNT1 variants (7 missense mutations, 4 mutations leading to premature termination codons) were identified, of which 9 were novel. Three-dimensional protein modeling suggested that each of the missense mutations led to structural modifications. Functional in vitro studies revealed that all observed missense mutations led to decreased ability of WNT1 to induce WNT signaling via the canonical WNT pathway.

CONCLUSIONS

The reported biallelic WNT1 variants cause loss of WNT1 function and lead to a severe bone fragility phenotype with conspicuous involvement of the spine.

OBJECTIVE

This study investigates for the first time the effect of Porphyromonas gingivalis lipopolysaccharides (Pg-LPS) on proliferative/regenerative aptitudes of gingival stem/progenitor cells (G-MSCs).

METHODS

G-MSCs (n = 5) were treated by 0, 10 ng/ml, 100 ng/ml, 1 μg/ml or 10 μg/ml Pg-LPS. At 1 hour, Toll-like receptor 4 (TLR-4) expression and NF-κB and <em>Wnt</em>/β-catenin signalling pathways were examined. Colony-forming unit assay was conducted at day <em>12</em>. At 24 and 48 hours, MTT test, ALP activity, mRNA for tumour necrosis factor-α (TNF-α), interleukin-6, collagen-I (Col-I), collagen-III, RUNX-2, alkaline phosphatase (ALP), osteonectin and protein expression of interleukin-6 and TNF-α were analysed.

RESULTS

With increasing Pg-LPS, TLR-4 was upregulated, pNF-κB-p65 rose from median (Q25/Q75) 6.56% (4.19/7.90) to 13.02% (8.90/16.50; p = 0.002) and pNF-κB-p65/tNF-κB-p65 from 0.14(0.10/0.17) to 0.30(0.21/0.42; p = 0.002). pβ-Catenin, tβ-catenin and pβ-catenin/tβ-catenin showed no differences. Increasing Pg-LPS concentration increased cell numbers from 288.00(72.98/484.32) to 861.39 (540.41/1599.94; p = 0.002), ALP mRNA from 0.00(0.00/0.01) to 0.56(0.00/1.90; p = 0.004) and TNF-α from 32.47(<em>12</em>.11/38.57) to 45.32(28.68/48.65; p = 0.036). Over time, ALP activity increased from 0.89(0.78/0.95) to 1.90(1.83/2.09; p < 0.001), mRNA for TNF-α from 0.00(0.00/0.<em>12</em>) to 0.01(0.00/0.06; p = 0.007), mRNA for Col-I from 82.70(0.03/171.50) to <em>12</em>4.00(52.85/232.50; p = 0.019), while mRNA for RUNX-2 decreased from 1.73(0.92/3.20) to 0.84(0.48/1.47; p = 0.005).

CONCLUSIONS

Pg-LPS upregulated G-MSCs' proliferation, without attenuation of their regenerative potential. The effects were NF-κB, but not Wnt/β-catenin, pathway dependent.

UNASSIGNED

We aimed to explore the potential molecular mechanism and independent prognostic genes for colon cancer (CC).

METHODS

Microarray datasets GSE17536 and GSE39582 were downloaded from Gene Expression Omnibus. Meanwhile, the whole CC-related dataset were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNA (DEMs) were identified between cancer tissue samples and para-carcinoma tissue samples in TCGA dataset, followed by the KEGG pathway and GO function analyses. Furthermore, the clinical prognostic analysis including overall survival (OS) and disease-free survival (DFS) were performed in all three datasets.

RESULTS

A total of 633 up- and 321 down-regulated mRNAs were revealed in TCGA dataset. The up-regulated mRNAs were mainly assembled in functions including extracellular matrix and pathways including <em>Wnt</em> signaling. The down-regulated mRNAs were mainly assembled in functions like Digestion and pathways like Drug metabolism. Furthermore, up-regulation of UL16-binding protein 2 (ULBP2) was associated with OS in CC patients. A total of <em>12</em> DEMs including Surfactant Associated 2 (SFTA2) were potential DFS prognostic genes in CC patients. Meanwhile, the GRP and Transmembrane Protein 37 (TMEM37) were two outstanding independent DFS prognostic genes in CC.

CONCLUSIONS

ULBP2 might be a potential novel OS prognostic biomarker in CC, while GRP and TMEM37 could be served as the independent DFS prognostic genes in CC. Furthermore, functions including extracellular matrix and digestion, as well as pathways including Wnt signaling and drug metabolism might play important roles in the process of CC.

<i>TREM2</i> is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of <i>Trem2</i> in a mouse model of β-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking <i>Trem2</i> (PS2APP;Trem2^ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2^ko mice, with <i>Trem2</i>-deficient microglia showing compromised expression of proliferation/<em>Wnt</em>-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2^ko females at 6-7 months, but by <em>12</em> or 19-22 months of age it was notably diminished in female and male PS2APP;Trem2^ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2^ko brains, and the Aβ42:Aβ40 ratio was elevated. The amount of soluble, fibrillar Aβ oligomers also increased in PS2APP;Trem2^ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6-7 months onward in PS2APP;Trem2^ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2^ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in cerebrospinal fluid. Thus, aggravated neuritic dystrophy is a more consistent outcome of <i>Trem2</i> deficiency than amyloid plaque load, suggesting that the microglial packing of Aβ into dense plaque is an important neuroprotective activity.<b>SIGNIFICANCE STATEMENT</b>Genetic studies indicate that <i>TREM2</i> gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of <i>Trem2</i> deletion in the PS2APP mouse AD model, in which overproduction of Aβ peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of <i>Trem2</i>-deficient mice, despite these mice displaying reduced plaque accumulation at later ages (<em>12</em>-22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aβ42:Aβ40 ratio and amount of soluble, fibrillar Aβ oligomers were elevated in <i>Trem2</i>-deficient brains. These results suggest that the Trem2-dependent compaction of Aβ into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aβ species.

<em>WNTs</em> (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of <em>WNTs</em> action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E(2)) and progesterone (P(4)) on <em>WNTs</em>, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(4) level in uterine luminal flushings (ULFs) on Day <em>12</em> of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day <em>12</em> of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.

The <em>Wnt</em>/β-catenin response pathway is central to many developmental processes. Here, we assessed the role of <em>Wnt</em> signaling in early eye development using the mouse as a model system. We showed that the surface ectoderm region that includes the lens placode expressed <em>12</em> out of 19 possible <em>Wnt</em> ligands. When these activities were suppressed by conditional deletion of wntless (Le-cre; Wls(fl/fl)) there were dramatic consequences that included a saucer-shaped optic cup, ventral coloboma, and a deficiency of periocular mesenchyme. This phenotype shared features with that produced when the <em>Wnt</em>/β-catenin pathway co-receptor Lrp6 is mutated or when retinoic acid (RA) signaling in the eye is compromised. Consistent with this, microarray and cell fate marker analysis identified a series of expression changes in genes known to be regulated by RA or by the <em>Wnt</em>/β-catenin pathway. Using pathway reporters, we showed that <em>Wnt</em> ligands from the surface ectoderm directly or indirectly elicit a <em>Wnt</em>/β-catenin response in retinal pigment epithelium (RPE) progenitors near the optic cup rim. In Le-cre; Wls(fl/fl) mice, the numbers of RPE cells are reduced and this can explain, using the principle of the bimetallic strip, the curvature of the optic cup. These data thus establish a novel hypothesis to explain how differential cell numbers in a bilayered epithelium can lead to shape change.

The <em>Wnt</em> genes encode secreted glycoprotein ligands that are key players during animal development. Previous studies revealed the presence of <em>12</em> classes of <em>Wnt</em> genes in protostomes, although lineage specific losses of <em>Wnt</em> genes are common. So far, the gene expression profile of only two complete sets of arthropod <em>Wnt</em> genes has been studied; these are the <em>Wnt</em> genes of the fly Drosophila melanogaster and the beetle Tribolium castaneum. Insects, however, do not represent good models for the understanding of <em>Wnt</em> gene evolution because several <em>Wnt</em> genes have been lost in the lineage leading to the insects, or within the different orders of insects. Comparative gene expression data from non-insect arthropods are rare and restricted to a subset of <em>Wnt</em> genes. This study aims to fill this gap and describes four newly detected <em>Wnt</em> genes from the millipede Glomeris marginata (Myriapoda: Diplopoda). Together with previous studies, now 11 Glomeris <em>Wnt</em> genes have been isolated and their expression has been studied. The only predicted but hitherto undetected <em>Wnt</em> gene is <em>Wnt</em>10. The new data provide a platform for the comparison of <em>Wnt</em> gene expression patterns in arthropods and reveal conserved as well as diverged aspects of <em>Wnt</em> gene expression in Arthropoda. Prominent expression of <em>Wnt</em>4 in dorsal tissue implies a role in dorsal segmentation and suggests that <em>Wnt</em>4 may be the predicted substitute for the previously reported missing expression of wg/<em>Wnt</em>1 in dorsal tissue.

<AbstractText>Cancer stem cells (CSCs) promote tumor progression and distant metastasis in breast cancer. Cadherin 11 (CDH11) is overexpressed in invasive breast cancer cells and implicated in distant bone metastases in several cancers. The <em>WNT</em> signalling pathway regulates CSC activity. Growing evidence suggest that cadherins play critical roles in <em>WNT</em> signalling pathway. However, CDH11 role in canonical <em>WNT</em> signalling and CSCs in breast cancer is poorly understood.</AbstractText><AbstractText>We investigated the functional association between CDH11 and <em>WNT</em> signalling pathway in triple negative breast cancer (TNBC), by analyzing their expression profile in the TCGA Breast Cancer (BRCA) cohort and immunohistochemical (IHC) staining of TNBC samples.</AbstractText><p><div><b>RESULTS</b></div>We observed a significant correlation between high CDH11 expression and poor prognosis in the basal and TNBC subtypes. Also, CDH11 expression positively correlated with β-catenin, wingless type MMTV integration site (<em>WNT</em>)2, and transcription factor (TCF)<em>12</em> expression. IHC results showed CDH11 and β-catenin expression significantly correlated in TNBC patients (<i>p</i> < 0.05). We also showed that siRNA-mediated loss-of-CDH11 (siCDH11) function decreases β-catenin, Met, c-Myc, and matrix metalloproteinase (MMP)7 expression level in MDA-MB-231 and Hs578t. Interestingly, immunofluorescence staining showed that siCDH11 reduced β-catenin nuclear localization and attenuated TNBC cell migration, invasion and tumorsphere-formation. Of translational relevance, siCDH11 exhibited significant anticancer efficacy in murine tumor xenograft models, as demonstrated by reduced tumor-size, inhibited tumor growth and longer survival time.</p><AbstractText>Our findings indicate that by modulating β-catenin, CDH11 regulates the canonical <em>WNT</em> signalling pathway. CDH11 inhibition suppresses the CSC-like phenotypes and tumor growth of TNBC cells and represents a novel therapeutic approach in TNBC treatment.</AbstractText>

Secreted frizzled-related protein 5 (sFRP5) is a novel adipokine that functions as an inhibitor of <em>Wnt</em> signaling and is involved in embryonic development, proliferation, atherosclerosis, and apoptosis. Studies have shown that sFRP1-4 is expressed in cardiomyocytes, and sFRP3 and sFRP4 are elevated during heart failure. However, it is unclear whether sFRP5 is expressed in cardiomyocytes or cardiac hypertrophy, and as regards the effects of sFRP5 in the process. Here, we report the expression and the corresponding mechanisms of sFRP5 in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. Neonatal rat ventricular myocytes were exposed to increasing concentrations of Ang II for <em>12</em>-72 h. Y27632 was used to block ROCK signal. PD98059, SB203580, and SP600<em>12</em>5 were used to inhibit ERK1/2, p38 MAPK, and JNK signaling pathways, respectively, and anisomycin was used to activate JNK pathway. RT-PCR and Western-blot determined the expressions of sFRP5. BNP, TNF-α, ROCK1, ROCK2, MYPT1, and JNK were examined through Western-blot analysis. Ang II increased sFRP5 mRNA and protein levels in a time- and dose-dependent manner. Telmisartan, Y27632 and SP600<em>12</em>5 effectively suppressed the expression of sFRP5. sFRP5 downregulated BNP and TNF-α expressions in hypertrophic cardiomyocytes. sFRP5 is expressed in cardiomyocytes, and upregulated in Ang II-induced cardiomyocyte hypertrophy through the AT1 receptor/Rho/ROCK1/JNK signaling pathway. sFRP5 may play an important role during cardiomyocyte hypertrophy.

N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-<em>12</em> peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3β (GSK-3β), which phosphorylates β-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of β-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of β-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical <em>Wnt</em> signaling in hMSCs by reducing β-catenin nuclear translocation and the associated transcriptional activity of β-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.

Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and <em>12</em> pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the <em>Wnt</em>/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in <em>Wnt</em> signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.

BACKGROUND

ClinicalTrials.gov NCT02349113.

PPARdelta (peroxisome proliferator-activated receptor delta) is a member of the nuclear receptor superfamily. However, its function in tissues and cells is unknown, particularly as related to stem cell biology. We therefore investigated the PPARdelta effects on DNA synthesis in mouse embryonic stem cells (ES cells) and its related signal pathways. PPARdelta increased biphasic reactive oxygen species (ROS) production at 15 min and at <em>12</em>0 min incubation. PPARdelta significantly increased [(3)H] thymidine incorporation levels at various concentrations (10(-8) M to 10(-6) M) and incubation times (<em>12</em> to 48 hr), and this activity was blocked by antioxidants. In addition, PPARdelta increased protein kinase C (PKC), cytosolic phospholipase A(2) (cPLA(2)) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, and <em>Wnt</em>/beta-catenin activation. PPARdelta increased the protein levels of cell cycle regulators, and these levels were abolished by antioxidants, bisindolymaleimide I, SB203580 and beta-catenin specific siRNA. In addition, the effect of PPARdelta on increased [(3)H] thymidine incorporation was blocked by bisindolymaleimide I, SB203580 and beta-catenin specific siRNA. In conclusion, PPARdelta agonist enhanced mouse ES cells proliferation through ROS-mediated p38 MAPK and <em>Wnt</em>/beta-catenin activation.