OBJECTIVE

To investigate the role and possible mechanism of α-Klotho in the calcification and the osteogenic transition of cultured VSMCs.

METHODS

VSMCs were cultured in vitro and divided into 5 groups, each using a different medium: (1) control; (2) β-GP; (3) β-GP + Klotho; (4) β-GP + LiCl; (5) β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 and β-catenin levels were estimated by western blotting, and the level of α-SMA was determined by using immunofluorescence at day <em>12</em>.

RESULTS

β-GP induced an increase in the expression of BMP2, Runx2, and β-catenin. The calcium content increased, and the expression of α-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.

CONCLUSIONS

Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced by β-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway.

The <em>Wnt</em> signaling pathway plays a crucial role during embryogenesis in vertebrates. In this study, <em>12</em>4 SNP in 31 <em>Wnt</em> signaling pathway genes were selected to genotype 764 individuals in an F(2) resource population by reciprocally crossing Silkie fowls and Cornish broilers, and 102 SNP were polymorphic. Pairwise linkage disequilibrium among the SNP within each gene was calculated. Haplotypes were reconstructed from the SNP in strong linkage disequilibrium. The associations of SNP and haplotypes with carcass traits were analyzed respectively, and the SNP contributions to phenotypic variance were estimated. The present study showed that 58 SNP in 24 genes and 8 haplotype blocks within 7 genes were significantly (P < 0.05) associated with at least one carcass trait. Fourteen SNP (among the 58 SNP) explained >2% phenotypic variance, <em>12</em> of which had significantly (P < 0.01) additive or dominant effects. Furthermore, both rs15865526 (<em>Wnt</em>9A) and rs14066777 (MAPK9) as well as their corresponding haplotype blocks were significantly associated with shank circumference and wing weight, respectively. In addition, 5 muscle-weight-related SNP explained >7% phenotypic variance, which was much higher than those of others. It was found that the <em>Wnt</em> signaling pathway was strongly associated with chicken carcass traits, and 7 genes were particularly important, namely RHOA and CHP for breast muscle weight, <em>Wnt</em>3A for breast muscle weight percentage over carcass weight, RAC1 for thigh weight percentage and thigh muscle weight percentage over carcass weight, <em>Wnt</em>11 for thigh weight percentage over carcass weight, <em>Wnt</em>9A for shank length, and MAPK9 for shank circumference. It is evident that <em>Wnt</em> signaling plays a major role in regulating carcass characteristics important for production traits in chickens.

Although retinoic acid (RA), the active form of vitamin A, is required for normal embryonic growth and development, it is also a powerful teratogen. Infants born to mothers exposed to retinoids during pregnancy have a 25-fold increased risk for malformations, nearly exclusively of cranial neural crest-derived tissues. To characterize neural crest cell responses to RA, we exposed murine crest cultures to teratogenic levels of RA and subjected their RNA to microarray-based gene expression profile analysis using Affymetrix MG-U74Av2 GeneChips. RNAs were isolated from independent cultures treated with 10(-6) M RA for 6, <em>12</em>, 24, or 48 h. Statistical analyses of gene expression profile data facilitated identification of the 205 top-ranked differentially regulated genes whose expression was reproducibly changed by RA over time. Cluster analyses of these genes across the independently treated sample series revealed distinctive kinetic patterns of altered gene expression. The largest group was transiently affected within the first 6 h of exposure, representing early responding genes. Group 2 showed sustained induction by RA over all times, whereas group 3 was characterized by the suppression of a time-dependent expression increase normally seen in untreated cells. Additional patterns demonstrated time-dependent increased or decreased expression among genes not normally regulated to a significant extent. Gene function analysis revealed that more than one-third of all RA-regulated genes were associated with developmental regulation, including both canonical and noncanonical <em>Wnt</em> signaling pathways. Multiple genes associated with cell adhesion and cell cycle regulation, recognized targets for the biological effects of RA, were also affected. Taken together, these results support the hypothesis that the teratogenic effects of RA derive from reprogramming gene expression of a host of genes, which play critical roles during embryonic development regulating pathways that determine subsequent differentiation of cranial neural crest cells.

Insect and vertebrate Porcupine genes encode multi-pass endoplasmic reticulum proteins involved in the processing of <em>Wnt</em> (wingless and int homologue) proteins, a class of secreted glycoprotein factors homologous to the Drosophila melanogaster segment polarity gene Wingless (Wg). Here we report the cloning of cDNAs encoding the human homologue of the Drosophila gene Porcupine (Porc), the characterization of its genomic structure and the quantitative analysis of its expression in a comprehensive panel of human tissues. The human Porcupine locus (MG61/PORC) spans 15 exons over approximately <em>12</em> kb of genomic sequence on Xp11.23. Real-time quantitative expression analysis reveals that MG61/PORC transcripts are expressed in multiple tissues, but are particularly abundant in the brain. Like its mouse and Xenopus homologues, MG61/PORC encodes four protein isoforms (A-D) generated through alternative splicing and expressed in a tissue-specific fashion. Finally, we present evidence indicating that MG61/PORC can influence the activity of a human <em>Wnt</em>7A expression construct in a T-cell factor-responsive reporter assay.

Methylation of secreted frizzle-related protein (SFRP) genes activates <em>Wnt</em>/ß-catenin signaling and promotes tumor development. This study investigated whether SFRP5 gene methylation causes multidrug resistance (MDR) in leukemia through the <em>Wnt</em>/ß-catenin signaling, leading to the upregulation of the mdr1 gene and its product, P-glycoprotein (P-gp). Methylation-specific PCR identified SFRP5 gene methylation in cultured bone mononuclear cells from 7/<em>12</em> patients with acute leukemia and in four human leukemia cell lines (HL-60, Raji, U937 and KG1a). Western blotting revealed absent SFRP5 protein expression in cells from 5/7 patients with SFRP5 gene methylation and in all cell lines. Treatment with a demethylation agent (DAC) rescued SFRP5 expression. mdr1 mRNA and P-gp protein were detected in cells from 3/5 patients with absent SFRP5, and in the KG1a cell line; these cells also had the highest levels of activated ß-catenin. In cells from these three patients, DAC rescued SFRP5 expression and downregulated mdr1 and P-gp. SFRP5 protein expression was rescued in transgenic KG1a/SFRP5 cells, compared with KG1a/eGFP or untransfected KG1a cells. mdr1 and P-gp in KG1a/SFRP5 cells were downregulated. Doxorubicin IC50 values were significantly lower in KG1a/SFRP5 (0.573±0.131 μM) than in KG1a (0.963±0.115) or KG1a/eGFP (0.917±0.138) cells (P<0.05). We conclude that SFRP5 gene methylation in leukemia cells activates <em>Wnt</em>/ß-catenin signaling to upregulate mdr1/P-gp expression and cause MDR. Recovery of SFRP5 expression reversed MDR in the KG1a leukemia cell line. Our results suggest that modulating SFRP5 methylation could decrease MDR in leukemia patients.

FAP is characterized by 100-1000s of adenomatous polyps in colon and rectum, and is in 70% of the patients associated with extracolonic manifestations. Attenuated FAP (AFAP) is a less severe form of FAP, marked by the presence of < 100 polyps and a later onset of colorectal cancer (CRC). (A)FAP is caused by autosomal dominantly inherited mutations in the APC (Adenomatous polyposis coli) gene, a tumour suppressor gene that controls beta-catenin turnover in the <em>Wnt</em> pathway. De novo occurrence is reported in 30-40% of the patients. Mutations are detected in 85% of classical FAP families, while only 20%-30% of AFAP cases will exhibit a germline APC mutation. MUTYH is the second (A)FAP-related gene and is involved with base-excision repair of DNA damaged by oxidative stress. MUTYH mutations are inherited in an autosomal recessive way and account for 10%-20% of classical FAP cases without an APC mutation and for 30% of AFAP cases. Genotype-phenotype correlations exist for mutations in the APC gene, however, contradictions in the literature caution against the sole use of the genotype for decisions regarding clinical management. Once the family's specific APC mutation is identified in the proband, predictive testing for first degree relatives is possible from the age of 10 to <em>12</em> years on. For AFAP, relatives are tested at age 18 and older. Opinions about the appropriate ages at which to initiate genetic testing may vary. Physicians must have a discussion about prenatal testing with patients in childbearing age. They may either opt for conventional prenatal diagnosis (amniocentesis or chorionic villous sampling) or for preimplantation genetic diagnosis (PGD).

OBJECTIVE

During mammalian eye development, the restriction of Wnt/β-catenin signaling at the junction of the neural retina and the retinal pigment epithelium in the peripheral eyecup is required for the development of the ciliary margin, a non-neural region of the eyecup that is the precursor of the ciliary body and iris of the adult eye.

METHODS

To identify genes that are modulated by β-catenin activity in the embryonic retina, we performed gene expression profiling in Li(+)-treated retinal explants, a pharmacological model of β-catenin activation. The Li(+)-modulated gene data set was searched for β-catenin/T-cell specific transcription factor binding sites.

RESULTS

Functional annotations of this data set revealed significant enrichments for genes involved in chromatin organization, neurogenesis, and cell motion/migration. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the modulation of 12 genes in Li(+)-treated explants and retinas of mice with Cre-mediated induction of constitutively active β-catenin (β-cat(act)). In situ hybridization revealed β-catenin-specific upregulation of cyclin-dependent kinase inhibitor 1A (P21) [Cdkn1a] and tumor necrosis factor receptor superfamily, member 19 (Tnfrsf19) in the developing retina consistent with the antineurogenic and proliferation changes associated with ectopic Wnt/β-catenin signaling in the eyecup.

CONCLUSIONS

This data set of Li(+)-modulated genes provides a valuable resource for characterizing the Wnt/ β-catenin regulated gene network in eyecup patterning.

p14ARF is inactivated by deletions/mutations in many cancer types and can suppress cell growth by both p53-dependent and p53-independent mechanisms. To identify novel downstream effectors of p14ARF, we used gene expression profiling as a primary screening tool to select candidates for follow up validation studies using in vitro cell-based assays. Gene expression profiles of a panel of 35 melanoma cell lines with either wild-type (n = <em>12</em>) or mutant (n = 23) p14ARF were compared to identify genes associated with inactivation of p14ARF. Analysis of the microarray data identified 1,316 probe sets that were significantly (p < 0.01) differentially expressed between the p14ARF wild-type and mutant cell lines. Pathway analysis of these genes showed an overrepresentation of many receptor-mediated signal transduction pathways, e.g. TGFbeta, EGF, HGF, PDGF, MAPK, <em>Wnt</em> and integrin pathways. A number of components of these pathways, including FLRT3, RUNX2, MIG-6 and SMURF2 were confirmed as downstream targets of p14ARF using p14ARF-inducible cell lines and RNAi. We propose that regulation of these genes may contribute to melanoma development when p14ARF function is lost.

A tumor suppressor gene, Adenomatous polyposis coli (Apc), is expressed in the nervous system from embryonic to adulthood stages, and transmits the <em>Wnt</em> signaling pathway in which schizophrenia susceptibility genes, including T-cell factor 4 (TCF4) and calcineurin (CN), are involved. However, the functions of Apc in the nervous system are largely unknown. In this study, as the first evaluation of Apc function in the nervous system, we have investigated the behavioral significance of the Apc gene, applying a battery of behavioral tests to Apc heterozygous knockout (Apc(+/-)) mice. Apc(+/-) mice showed no significant impairment in neurological reflexes or sensory and motor abilities. In various tests, including light/dark transition, open-field, social interaction, eight-arm radial maze, and fear conditioning tests, Apc(+/-) mice exhibited hypoactivity. In the eight-arm radial maze, Apc(+/-) mice 6-7 weeks of age displayed almost normal performance, whereas those 11-<em>12</em> weeks of age showed a severe performance deficit in working memory, suggesting that Apc is involved in working memory performance in an age-dependent manner. The possibility that anemia, which Apc(+/-) mice develop by 17 weeks of age, impairs working memory performance, however, cannot be excluded. Our results suggest that Apc plays a role in the regulation of locomotor activity and presumably working memory performance.

BACKGROUND

Gliomas represent the most common primary malignant brain tumors, yet little is known about the molecular pathogenesis of these tumors. The highly-regulated Wnt signal transduction pathway is essential for normal developmental processes, and defects in the pathway are closely linked to oncogenesis. Dickkopf-1 (DKK-1) is a secreted protein that acts as a potent inhibitor of the Wnt pathway. The aim of this study was to examine the expression profile of DKK-1 gene in human glioma and its association with tumor malignancy.

METHODS

We determined the expression levels of DKK-1 transcript and protein in 12 glioblastoma cell lines, medulloblastoma cells, low-grade glioma cells, and human astrocyte cells by semiquantitative RT-PCR and ELISA. A total of 47 tumor biopsy specimens and 11 normal brain tissue samples from patients with cerebral trauma internal decompression were embedded in paraffin blocks and used for immunostaining. Twenty-six primary tumors and 7 corresponding brain samples were stored in liquid nitrogen and used for RT-PCR. We further examined serologic concentrations and cerebral fluid levels of DKK-1 in patients with tumors.

RESULTS

DKK-1 could only be detected in 12 human glioblastoma cell lines, not in a panel of other tumor and normal cell lines. The difference between glioma patients and healthy individuals was significant. Kendall's tau-c association analysis also revealed the increased DKK-1 protein expression in tumor tissues of higher pathologic classification. The levels of cerebral fluid DKK-1 protein were significantly higher in glioma patients than in healthy donors or in neuronal benign tumor patients, suggesting that the DKK-1 molecule in cerebral fluids can be applicable to detect the presence of glioma and be developed as a novel prognostic treatment.

CONCLUSIONS

The Wnt antagonist DKK-1 gene may have important roles in glioma tumorigenesis and act as a novel biomarker in human malignant glioblastoma.

OBJECTIVE

To assess the ability of sclerostin antibody therapy to blunt the negative effects of polyethylene particles on implant fixation and peri-implant bone structure in a rat implant fixation model.

METHODS

Thirty-six adult male rats received intramedullary titanium implants; <em>12</em> rats received vehicle injections only (control), and 24 rats received intraarticular injections of lipopolysaccharide-doped polyethylene particles. Twelve of the rats that received particles also received sclerostin antibody treatment. The 3 groups of rats were maintained for <em>12</em> weeks in a pathogen-free environment, at which time mechanical, micro-computed tomography, and dynamic and static histomorphometry end points were assessed.

RESULTS

Sclerostin antibody treatment completely blocked the negative effect of the lipopolysaccharide-doped polyethylene particles on implant fixation and peri-implant bone volume by increasing the bone formation rate and depressing bone resorption.

CONCLUSIONS

Anabolic agents targeting the Wnt signaling pathway are a promising new alternative for the prevention of periprosthetic osteolysis and aseptic loosening.

Connexins (Cxs) are critical for normal tissue development, differentiation, and cell proliferation. Normal expression and function of Cxs are considered to play a role in tumor suppression, but abnormal localization and abnormally increased expression of Cxs have been found in a variety of carcinomas. Of the Cx family, Cx43 is a most prevalent member and has been known as a downstream target of β-catenin, a key component of <em>Wnt</em> signaling pathway. We evaluated the expression of Cx43 in the colonic neoplasia progression sequence with additional attention to the stromal component. Resections of 50 colonic adenocarcinomas were stained immunohistochemically for Cx43 on paraffin embedded sections. Cx43 cytoplasmic expression increased progressively in the colonic adenocarcinoma sequence in both the epithelial [normal (4 ± 1), adenomatous (20 ± 2), cancerous (<em>12</em>4 ± 10) (P < 0.01)], and stromal [normal (19 ± 1), cancerous (45 ± 4) (P < 0.01)] components. In the epithelial component, Cx43 was expressed lower in stage I adenocarcinomas (69 ± <em>12</em>) compared to stage III/IV (158 ± 10, P < 0.01). Additionally, Cx43 was relatively increased in the adenocarcinoma at the invasive tumor front in all stages. Cx43 may play a critical role in the pathogenesis of colon cancer via gap junction or other gap junction independent mechanisms such as the <em>Wnt</em>/β-catenin pathway.

OBJECTIVE

Sex disparities in rheumatoid arthritis (RA) are well documented despite the lack of any known major RA susceptibility genes mapped to sex chromosomes. Murine chromosome 15 carries the sex-affected Pgia8 locus that mediates proteoglycan-induced arthritis, and homologous human loci are associated with RA. This study was undertaken to identify genes/mechanisms implicated in sex disparities in arthritis.

METHODS

Gene expression analysis was performed using RNA isolated from the paws of male and female Pgia8-congenic mice with collagen antibody-induced arthritis. Results were corroborated by reverse transcription-polymerase chain reaction, and mice were also studied prior to disease onset. Ingenuity Pathways Analysis of the expression patterns and gene functions was used to discover locus-specific and sex-affected signature transcripts.

RESULTS

We found that the Pgia8 locus regulates antibody-mediated inflammatory arthritis differently in males and females. In Pgia8-congenic males, arthritis severity was 30% less (P < 0.005) than in wild-type males, but the antiinflammatory effect was similar in wild-type and congenic females. Transcriptome analysis indicated that <em>12</em> genes within the locus were significantly dysregulated in arthritic joints of congenic mice; expression of these genes was also sex specific. The genes that correlated most highly with arthritis severity included those for collagen triple-helix repeat-containing 1 (Cthrc1), metalloproteinase (Adamts<em>12</em>), R-spondin (Rspo2), and syndecan (Sdc2) (r = 0.87-0.91). The level of Cthrc1 message also correlated with that of the genes for the proinflammatory cytokines interleukin-1β and interleukin-6.

CONCLUSIONS

These results indicate that sex-specific disparities in RA are linked to transcriptional regulation of genes involved in cartilage degradation (Adamts<em>12</em>) and canonical and noncanonical Wnt signaling (Cthrc1, Rspo2, Sdc2).

We reported previously that the expression of <em>Wnt</em>-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic and epigenetic mechanisms involved. β-Catenin gene expression and nuclear levels were analyzed by real time PCR and confocal immunofluorescence. Increased nuclear β-catenin was found in osteoblasts isolated from patients with osteoarthritis (99 ± 4 units vs. 76 ± <em>12</em>, p=0.01, n=10), without differences in gene transcription, which is consistent with a post-translational down-regulation of β-catenin and decreased <em>Wnt</em> pathway activity. Twenty four single nucleotide polymorphisms (SNPs) of genes showing differential expression between fractures and osteoarthritis (WNT4, WNT10A, WNT16 and SFRP1) were analyzed in DNA isolated from blood of 853 patients. The genotypic frequencies were similar in both groups of patients, with no significant differences. Methylation of <em>Wnt</em> pathway genes was analyzed in bone tissue samples (15 with fractures and 15 with osteoarthritis) by interrogating a CpG-based methylation array. Six genes showed significant methylation differences between both groups of patients: FZD10, TBL1X, CSNK1E, WNT8A, CSNK1A1L and SFRP4. The DNA demethylating agent 5-deoxycytidine up-regulated 8 genes, including FZD10, in an osteoblast-like cell line, whereas it down-regulated other 16 genes. In conclusion, <em>Wnt</em> activity is reduced in patients with hip fractures, in comparison with those with osteoarthritis. It does not appear to be related to differences in the allele frequencies of the <em>Wnt</em> genes studied. On the other hand, methylation differences between both groups could contribute to explain the differences in <em>Wnt</em> activity.

Craniopharyngiomas (CP) are benign epithelial tumors of the sellar region and can be clinicopathologically distinguished into adamantinomatous (adaCP) and papillary (papCP) variants. Both subtypes are classified according to the World Health Organization grade I, but their irregular digitate brain infiltration makes any complete surgical resection difficult to obtain. Herein, we characterized the cellular interface between the tumor and the surrounding brain tissue in 48 CP (41 adaCP and seven papCP) compared to non-neuroepithelial tumors, i.e., <em>12</em> cavernous hemangiomas, 10 meningiomas, and 14 metastases using antibodies directed against glial fibrillary acid protein (GFAP), vimentin, nestin, microtubule-associated protein 2 (MAP2) splice variants, and tenascin-C. We identified a specific cell population characterized by the coexpression of nestin, MAP2, and GFAP within the invasion niche of the adamantinomatous subtype. This was especially prominent along the finger-like protrusions. A similar population of presumably astroglial precursors was not visible in other lesions under study, which characterize them as distinct histopathological feature of adaCP. Furthermore, the outer tumor cell layer of adaCP showed a distinct expression of MAP2, a novel finding helpful in the differential diagnosis of epithelial tumors in the sellar region. Our data support the hypothesis that adaCP, unlike other non-neuroepithelial tumors of the central nervous system, create a tumor-specific cellular environment at the tumor-brain junction. Whether this facilitates the characteristic infiltrative growth pattern or is the consequence of an activated <em>Wnt</em> signaling pathway, detectable in 90% of these tumors, will need further consideration.

We evaluated the role of the CXCL<em>12</em>/CXCR4 (C-X-C motif chemokine ligand <em>12</em>/C-X-C chemokine receptor type 4) axis in aggrecanase-mediated cartilage degradation, and explored the underlying mechanism in a post-traumatic osteoarthritis rat model. Expression of CXCL<em>12</em>/CXCR4 and ADAMTS-5 was analyzed in the knees of osteoarthritic and non-arthritic rats using Western blot, ELISA, immunohistochemistry and immunofluorescence. Rodent studies were performed using Sprague-Dawley rats, with animals divided into three groups: Destabilization of the medial meniscus/AMD3100-treated (DMM/AMD3100-treated), DMM/PBS-treated, and sham controls. Rats were sacrificed after eight weeks, and samples were collected for histology and immunohistochemistry analyses. IL-1-pretreated primary chondrocytes were cultured with untreated control, CXCL<em>12</em>a, siNC + CXCL<em>12</em>a, or siRNA CXCR4 + CXCL<em>12</em>a, and analyzed for expression of relevant markers and cellular pathways. Higher levels of CXCL<em>12</em> were detected in the knee fluid of osteoarthritic subjects, with strong staining for CXCR4 in chondrocytes and CXCL<em>12</em> in synoviocytes together with enhanced expression of ADAMTS-5. DMM/AMD3100-treated rats showed a significantly reduced immunological response, with minimal evidence of pathology in both histological and immunohistochemical analyses. Treatment with CXCL<em>12</em>a increased the expression of ACAN, RUNX-2, and ADAMTS-4/5 in IL-1-pretreated primary chondrocytes, together with a decrease in the expression of SOX-9. Molecular analyses revealed strong induction of NF-κB activation, along with phosphorylation of MAPKs, and activation of canonical <em>Wnt</em>/β-catenin signaling. In conclusion, inhibition of SDF-1α/CXCR4 signaling axis was able to inhibit aggrecanase expression and lessen cartilage degeneration in post-traumatic osteoarthritis rats.

Improvement in litter size has become of great interest in the pig industry because fecundity is directly related to sow reproductive life. Improved reproduction has thus been achieved by elucidating the molecular functions of genes associated with fecundity. In the present study, we identified differentially expressed genes (DEGs) via transcriptomic analysis using RNA-sequencing (RNA-Seq) in Berkshire pig placentas from larger (LLG, mean litter size>><em>12</em>) and smaller (SLG, mean litter size < 6.5) litter size groups. In total 588 DEGs were identified (p < 0.05,>> 1.5-fold change), of which 98 were upregulated, while 490 were downregulated in the LLG compared with the SLG. Gene Ontology (GO) enrichment was also performed. We concluded that <em>12</em>9 of the 588 DEGs were closely related to litter size according to reproduction related genes selected based on previous reports, as 110 genes were downregulated and 19 upregulated in the LLG compared with the SLG. RT-qPCR utilizing specific primers targeting the early growth response 2 (EGR2), pheromaxein c subunit (PHEROC) and endothelial lipase (LIPG) genes showed high accordance with RNA-Seq results. Furthermore, we investigated the upstream regulators of these three genes in the placenta. We found that WNT9B, a <em>Wnt</em> signaling pathway molecule, and IL-6, known inducers of EGR2 and LIPG, respectively, were significantly increased in LLG compared with SLG. We believe that the induction of IL-6 and LIPG may play an important role in increasing nutrition supply through the placenta from the sow to the piglet during gestation. These results provide novel molecular insights into pig reproduction.

Dental follicle cells (DFCs) are reported to contain stem cells. The canonical <em>Wnt</em> signaling pathway plays an important role in stem cell self-renewal and tooth development through β-catenin expression. The objective of this study was to investigate whether <em>Wnt</em>/β-catenin signaling pathway participates in the cementoblast/osteoblast differentiation of rat DFCs. Immunohistochemistry was used to compare the expression of β-catenin in rat mandibular first molars from postnatal days 1-13. The effects of <em>Wnt</em>/β-catenin signaling on DFCs in vitro were examined by lithium chloride (LiCl) treatment by immunofluorescence, cell counting, dual-luciferase reporter assays, western blotting, and alkaline phosphatase activity analysis. β-Catenin expression was absent in the dental follicles on days 1 and 3 in vivo. It then progressively increased from days 5 to 13. In vitro studies of the DFCs showed that LiCl stimulation caused β-catenin, which was mainly located in the cell membrane and cytoplasm of DFCs, to be immediately transferred to the nucleus and led to the inhibition of proliferation at <em>12</em> and 24 hr. LiCl treatment also downregulated the levels of phosphorylated-β-catenin, while upregulating the levels of total β-catenin, nuclear β-catenin, osteocalcin, runt-related transcription factor 2, and collagen type I. In addition, LiCl enhanced the β-catenin/T-cell factor luciferase activity and alkaline phosphatase activity. These results suggest that <em>Wnt</em>/β-catenin signaling pathway positively regulates the cementoblast/osteoblast differentiation of the DFCs.

Slit-Robo GTPase-activating protein 1 (SRGAP1) functions as a GAP for Rho-family GTPases and downstream of Slit-Robo signaling. We aim to investigate the biological function of SRGAP1 and reveal its regulation by deregulated microRNAs (miRNAs) in gastric cancer (GC). mRNA and protein expression of SRGAP1 were examined by quantitative reverse transcription PCR (qRT-PCR) and western blot. The biological role of SRGAP1 was demonstrated through siRNA-mediated knockdown experiments. The regulation of SRGAP1 by miR-340 and miR-<em>12</em>4 was confirmed by western blot, dual luciferase activity assays and rescue experiments. SRGAP1 is overexpressed in 9 out of <em>12</em> (75.0%) GC cell lines. In primary GC samples from TCGA cohort, SRGAP1 shows gene amplification in 5/258 (1.9%) of cases and its mRNA expression demonstrates a positive correlation with copy number gain. Knockdown of SRGAP1 in GC cells suppressed cell proliferation, reduced colony formation, and significantly inhibited cell invasion and migration. Luciferase reporter assays revealed that SRGAP1 knockdown significantly inhibited <em>Wnt</em>/β-catenin pathway. In addition, SRGAP1 was found to be a direct target of two tumor-suppressive miRNAs, miR-340 and miR-<em>12</em>4. Concordantly, these two miRNAs were downregulated in primary gastric tumors and these decreasing levels w5ere associated with poor outcomes. Expression of miR-340 and SRGAP1 displayed a reverse relationship in primary samples and re-expressed SRGAP1, rescued the anti-cancer effects of miR-340. Taken together, these data strongly suggest that, apart from gene amplification and mutation, the activation of SRGAP1 in GC is partly due to the downregulation of tumor-suppressive miRNAs, miR-340 and miR-<em>12</em>4. Thus SRGAP1 is overexpressed in gastric carcinogenesis and plays an oncogenic role through activating <em>Wnt</em>/β-catenin pathway.

BACKGROUND

High tidal volume ventilation of healthy lungs or exacerbation of existing acute lung injury (ALI) by more moderate mechanical ventilation (MTV) produces ventilator-induced lung injury. It is less clear whether extrapulmonary sepsis sensitizes the lung to MTV.

METHODS

We used a two-hit model of cecal ligation and puncture (CLP) followed <em>12</em> h later by MTV (10 ml/kg; 6 h) to determine whether otherwise noninjurious MTV enhances CLP-induced ALI by contrasting wildtype and TLR4-/- mice with respect to: alveolar-capillary permeability, histopathology and intrapulmonary levels of <em>WNT</em>-inducible secreted protein 1 (WISP1) and integrin β5; plasma levels of cytokines and chemokines (TNF-α, IL-6, MIP-2, MCP-1) and intrapulmonary neutrophil infiltration; and other inflammatory signaling via intrapulmonary activation of JNK, p38 and ERK. A separate cohort of mice was pretreated with intratracheal neutralizing antibodies to WISP1, integrin β5 or IgG as control and the presented phenotyping repeated in a two-hit model; there were 10 mice per group in these first three experiments. Also, isolated peritoneal macrophages (PM) from wildtype and TLR4-/-, MyD88-/- and TRIF-/- mice were used to identify a WISP1-TLR4-integrin β5 pathway; and the requisite role of integrin β5 in WISP1-induced cytokine and chemokine production in LPS-primed PM was examined by siRNA treatment.

RESULTS

MTV, that in itself did not cause ALI, exacerbated increases in alveolar-capillary permeability, histopathologic scoring and indices of pulmonary inflammation in mice that previously underwent CLP; the effects of this two-hit model were abrogated in TLR4-/- mice. Attendant with these findings was a significant increase in intrapulmonary WISP1 and integrin β5 in the two-hit model. Anti-WISP1 or anti-integrin β5 antibodies partially inhibited the two-hit phenotype. In PM, activation of TLR4 led to an increase in integrin β5 expression that was MyD88 and NF-κB dependent. Recombinant WISP1 increased LPS-induced cytokine release in PM that was inhibited by silencing either TLR4 or integrin β5.

CONCLUSIONS

These data show for the first time that otherwise noninjurious mechanical ventilation can exacerbate ALI due to extrapulmonary sepsis underscoring a potential interactive contribution of common events (sepsis and mechanical ventilation) in critical care, and that a WISP1-TLR4-integrin β5 pathway contributes to this phenomenon.

β-Catenin in hepatocytes, under the control of <em>Wnts</em>, regulates pericentral gene expression. It also contributes to liver regeneration (LR) after partial hepatectomy (PH) by regulating cyclin-D1 gene expression as shown in the β-catenin and <em>Wnt</em> coreceptors low-density lipoprotein receptor-related protein 5/6 conditional knockouts (KO). However, conditional deletion of <em>Wnt</em>less (Wls), required for <em>Wnt</em> secretion, in hepatocytes, cholangiocytes, or macrophages lacked any impact on zonation, while Wls deletion in macrophages only marginally affected LR. Here, we address the contribution of hepatic endothelial cells (ECs) in zonation and LR by characterizing EC-Wls-KO generated by interbreeding Wls-floxed and lymphatic vessel endothelial hyaluronan receptor (Lyve1)-cre mice. These mice were also used to study LR after PH. While Lyve1 expression in adult liver is limited to sinusoidal ECs only, Lyve1-cre mice bred to ROSA26-Stopflox/flox-enhanced yellow fluorescent protein (EYFP) mice showed EYFP labeling in sinusoidal and central vein ECs. EC-Wls-KO mice showed decreased liver weights; lacked glutamine synthetase, cytochrome P450 2e1, and cytochrome P450 1a2; and were resistant to acetaminophen-induced liver injury. After PH, EC-Wls-KO showed quantitative and qualitative differences in cyclin-D1 expression at 24-72 hours, which led to a lower hepatocyte proliferation at 40 hours but a rebound increase by 72 hours. ECs and macrophages isolated from regenerating livers at <em>12</em> hours showed significant up-regulation of <em>Wnt</em>2 and <em>Wnt</em>9b messenger RNA; these are the same two <em>Wnts</em> involved in baseline β-catenin activity in pericentral hepatocytes. Conclusion: At baseline, ECs secrete <em>Wnt</em> proteins essential for β-catenin activation in pericentral hepatocytes. During LR, sinusoidal and central vein ECs and secondarily macrophages secrete <em>Wnt</em>2, while predominantly central vein ECs and secondarily macrophages are the likely source of <em>Wnt</em>9b. This process spatiotemporally regulates β-catenin activation in hepatocytes to induce cell proliferation. (Hepatology Communications 2018;2:845-860).

Thymoma is the most commonly identified cancer in the anterior mediastinum. To date, the causal mechanism that drives thymoma progression is not clear. Here, we generated K5-∆N64Ctnnb1/ERT2 transgenic mice, which express an N-terminal deletion mutant of β-catenin fused to a mutated ligand-binding domain of estrogen receptor (ERT2) under the control of the bovine cytokeratin 5 (K5) promoter. The transgenic mouse lines named Tg1 and Tg4 were characterized. Forced expression of ∆N64Ctnnb1/ERT2 in the Tg1 and Tg4 mice developed small thymoma lesions in response to tamoxifen treatment. In the absence of tamoxifen, the Tg1 mice exhibited leaky activation of β-catenin, which activated the TOP-Gal transgene and <em>Wnt</em>/β-catenin-targeted genes. As the Tg1 mice aged in the absence of tamoxifen, manifest thymomas were found at 10-<em>12</em> months. Interestingly, we detected loss of AIRE and increase of p63 in the thymomas of Tg1 mice, similar to that observed in human thymomas. Moreover, the β5t protease subunit, which was reported as a differential marker for human type B3 thymoma, was expressed in the Tg1 thymomas. Thus, the Tg1 mice generated in this study accurately mimic the characteristics of human thymomas and may serve as a model for understanding thymoma pathogenesis.

The cholinergic system impairment observed in Alzheimer's disease (AD) patients leads to the cognitive, global and behavioral dysfunction commonly associated with dementia. The only treatment for AD has been the use of inhibitors of acetylcholinesterase (AChE) (E.C. 3.1.1.7), which is one of the several proteins associated with amyloid plaque deposits. Recently, novel dual inhibitors of AChE have been developed that target both the active site of the enzyme as well as the peripheral anionic site (PAS). Such inhibitors prevent the aggregation of amyloid-beta-peptide (Abeta) into Alzheimer's fibrils. The incorporation of AChE, as a "chaperone" into amyloid aggregates results in the modification of the biochemical properties of the enzyme, including: sensitivity to low pH, inhibition at high substrate concentration, and increases of the Abeta neurotoxicity. Congo Red dye stabilizes the Abeta monomer, is able to inhibit oligomerization, and inhibits the binding of AChE to Abeta. However no effect of Congo Red on the binding of AChE to the Abeta preformed fibrils was observed. These studies suggest that different interactions between Abeta soluble-AChE and Abeta fibrils-AChE take place during the association between them. Docking studies were performed to evaluate the binding of Congo Red to Abeta in order to identify putative binding sites in the Abeta monomer that might interact with AChE. The binding site involves a region between residues <em>12</em> and 16. Finally, recent studies are consistent with the idea that a attenuating beta-catenin loss of function of <em>Wnt</em> signaling components may play a role in the progression of neurodegenerative disease, such as AD, providing a connection between AChE-Abeta neurotoxicity and the <em>Wnt</em> signal transduction pathway.

OBJECTIVE

Bone marrow stem cells (BMSCs) can commit to both adipocyte and osteoblast lineages. However, the mechanism underlying how transcription factors regulate this process remains elusive. Our aims were to determine the role of runt-related transcription factor 1 (Runx1) in BMSCs lineage determination and the underlying mechanisms.

METHODS

BMSCs from mouse femur bone marrow were harvested and cultured in osteogenic medium. Runx1 was knocked down in BMSCs using lentivirus. Alkaline phosphatase (ALP), Von Kossa and Oil Red O staining were performed on the Runx1-transduced BMSCs and control cells to see the differences of osteogenic and adipogenic differentiation in these groups. Real-time quantitative PCR and Western blot were performed to analyse the expression levels of osteogenic and adipogenic factors regulated by Runx1 at gene and protein levels.

RESULTS

In BMSCs with Runx1 knockdown, the expression levels of osteogenic-related genes decreased significantly while the adipogenic genes C/EBPα, PPARγ and Fabp4 increased by <em>12</em>-fold, 10-fold, and 30-fold, respectively, compared with the control cells. ALP activity and Von kossa staining were greatly decreased in Runx1-transfected cells while the Oil Red O staining was comparable to that in the control groups. Canonical <em>Wnt</em> signaling was investigated in the Runx1-deficient BMSCs, and a 50% decrease in the expression of active β-catenin in these cells was found. Lef1 and Tcf1, which are regulated by β-catenin were also decreased in Runx1-deficient cells compared with the levels in controls. Moreover, although there was no difference in the expression of <em>Wnt</em>3a among the three groups of cells, the expression of <em>Wnt</em>10b decreased by 80% in Runx1-deficient BMSCs compared with the levels in the other two groups.

CONCLUSIONS

Our results show Runx1 promotes the capacity of osteogenesis in BMSCs while inhibits their adipogenesis through canonical Wnt/β-catenin pathway, which provides new insights into osteoblast development.