Lgr4 is a member of the leucine-rich, G protein-coupled receptor family of proteins, and has recently been shown to augment <em>Wnt</em>/β-catenin signaling via binding to <em>Wnt</em> agonists R-spondins. It plays an important role in skin development, but its involvement in skin tumorigenesis is unclear. Here, we report that mice deficient for Lgr4 are resistant to <em>12</em>-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced keratinocyte proliferation and papilloma formation. We show that TPA treatment activates MEK1, ERK1/2 and downstream effector AP-1 in wild-type (WT) epidermal cells and mice, but not in cells or mice where Lgr4 is depleted. <em>Wnt</em>/β-catenin signaling is also dramatically activated by TPA treatment, and this activation is abolished when Lgr4 is deleted. We provide evidences that blocking both MEK1/ERK1/2 and <em>Wnt</em>/β-catenin pathways prevents TPA-induced increase in the expression of Ccnd1 (cyclin D1), a known <em>Wnt</em>/β-catenin target gene, and that the activation of MEK1/ERK1/2 pathway lies upstream of <em>Wnt</em>/β-catenin signal pathway. Collectively, our findings identify Lgr4 as a critical positive factor for skin tumorigenesis by mediating the activation of MEK1/ERK1/2 and <em>Wnt</em>/β-catenin pathways.

Chronic rhinosinusitis (CRS) is a common disease characterized by inflammation of the nose and paranasal sinuses lasting over <em>12</em> weeks. This study aims to evaluate the effect of desmoglein 3 (DSG3) on inflammatory response and goblet cell mucin secretion in a mouse model of CRS. The CRS-related differentially expressed genes and disease genes were screened using microarray-based gene expression analysis. Subsequently, CRS mouse models were established. The levels of pro-inflammatory factors TNF-α, IL-6, and IL-8 were measured by ELISA. In addition, loss-of-function experiment was conducted using siRNAs targeting DSG3 and β-catenin. The secretion of mucins MUC5B and MUC5AC in goblet cells was detected, and the apoptosis of goblet cells was assessed. The regulatory effect of DSG3 on the <em>Wnt</em>/β-catenin signaling pathway was analyzed by determining the mRNA and protein levels of DSG3, <em>Wnt</em>, β-catenin, and GSK3β. DSG3 was identified to be an upregulated gene in CRS, which was further documented in CRS mice models. Elevated inflammation and mucin production were noted in CRS mice models. Also, it was found that DSG3 or β-catenin silencing could decrease the levels of TNF-α, IL-6, and IL-8, and the positive rates of MUC5B and MUC5AC while enhancing goblet cell apoptosis. The <em>Wnt</em>/β-catenin signaling pathway was blocked by DSG3, evidenced by downregulated <em>Wnt</em> and β-catenin as well as upregulated GSK3β mRNA and protein levels. Overall, this study provides evidence that silencing DSG3 could inhibit the activation of the <em>Wnt</em>/β-catenin signaling pathway, thus alleviating CRS.

The aim of the present study was to investigate the mechanism of metastasis in colorectal cancer (CRC) using microRNA (miRNA) and mRNA expression profiles. The mRNA and miRNA expression profiles of the GSE2509 and GSE56350 datasets were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were identified using the limma software package. The Database for Annotation, Visualization and Integrated Discovery was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs. The predicted target genes associated with the DEMs were identified using the miRWalk database and the enrichment analysis was conducted using the clusterProfiler package. The miRNA-gene molecular interaction network was visualized using the Cytoscape software platform. A total of 544 DEGs and 42 DEMs were identified. DEGs were annotated in 320 GO terms and 11 KEGG pathways. Overall, 366 miRNA-gene pairs were identified and the miRNA-gene network was visualized. Furthermore, the predicted target genes were mainly classified in <em>12</em> pathways. The results of the present study suggest that fibronectin type III domain-containing 3B, cysteine rich transmembrane BMP regulator 1 and forkhead box J2 may be potential therapeutic and prognostic targets of metastatic CRC. In addition, pathways in cancer, the <em>Wnt</em> signaling pathway and extracellular matrix-receptor interaction may play a critical role in CRC metastasis.

OBJECTIVE

The aim of this study was to investigate the possible migration of proliferating peripheral retinal pigment epithelial (RPE) cells and their association with differential gene expressions.

METHODS

The RPE layer was obtained from the inner aspect of the eyeball of dark agouti rats (<em>12</em>-13 weeks old) and was mounted on glass slides. The peripheral RPE cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry (n = 10). The cell migration was examined using the Dil tracer technique (n = 40) at the end of weeks 6, 10, 14 and 18. Affymetrix microarray analysis was used to investigate differential gene expressions in peripheral and central RPE cells, which was authenticated by RT-PCR using 4 RPE-specific genes (n = 10).

RESULTS

In this study, peripheral RPE cells divided and appeared in clusters, but equatorial and central RPE cells rarely divided. The peripheral RPE cells migrated to the central RPE region in a time-dependent manner up to the end of week 14, but not later. The microarray analysis showed the expression of 9,645 out of a total of 35,220 genes studied. Among the 9,645 genes, 573 were differentially expressed (438 were upregulated and 135 were downregulated) in peripheral RPE cells as compared to central RPE cells. Of these 573 genes, 56 were associated with signaling pathways related to the regulation of cell proliferation, including Pax6, TGFβ, BMP and Wnt, and 404 were associated with pathways of cell migration.

CONCLUSIONS

In this study, peripheral RPE cells divided and migrated to the central region. This process was associated with differential gene expressions in these cells.

<em>Wnt</em> signaling pathways direct key physiological decisions in development. Here, we establish a role for a pleckstrin homology domain-containing protein, PLEKHA4, as a modulator of signaling strength in <em>Wnt</em>-receiving cells. PLEKHA4 oligomerizes into clusters at PI(4,5)P₂-rich regions of the plasma membrane and recruits the Cullin-3 (CUL3) E3 ubiquitin ligase substrate adaptor Kelch-like protein <em>12</em> (KLHL<em>12</em>) to these assemblies. This recruitment decreases CUL3-KLHL<em>12</em>-mediated polyubiquitination of Dishevelled, a central intermediate in canonical and non-canonical <em>Wnt</em> signaling. Knockdown of PLEKHA4 in mammalian cells demonstrates that PLEKHA4 positively regulates canonical and non-canonical <em>Wnt</em> signaling via these effects on the Dishevelled polyubiquitination machinery. In vivo knockout of the Drosophila melanogaster PLEKHA4 homolog, kramer, selectively affects the non-canonical, planar cell polarity (PCP) signaling pathway. We propose that PLEKHA4 tunes the sensitivities of cells toward the stimulation of <em>Wnt</em> or PCP signaling by sequestering a key E3 ligase adaptor controlling Dishevelled polyubiquitination within PI(4,5)P₂-rich plasma membrane clusters.

Wnt/β-catenin signaling modulates brain development and function and its deregulation underlies pathological changes occurring in neurodegenerative and neurodevelopmental disorders. Since one of the main effects of Wnt/β-catenin signaling is the modulation of target genes, in the present work we examined global transcriptional changes induced by short-term WntWnt/β-catenin target genes such as Notum, Axin2, and Lef1, as well as novel potential candidates Fam84a, Stk32a, and Itga9. Main biological processes enriched with differentially expressed genes included neural precursor (GO:0061364, p-adjusted = 2.5 × 10-7), forebrain development (GO:0030900, p-adjusted = 7.3 × 10-7), and stem cell differentiation (GO:0048863 p-adjusted = 7.3 × 10-7). Likewise, following activation of the signaling cascade, the expression of a significant number of genes with transcription factor activity (GO:0043565, p-adjusted = 4.1 × 10-6) was induced. We also studied molecular networks enriched upon Wnt). Our results indicate that Wnt/β-catenin mediated transcription controls multiple biological processes related to neuronal structure and activity that are affected in synaptic dysfunction disorders.

BACKGROUND

Since healing of anterior cruciate ligament (ACL) grafts occurs by formation of a fibrovascular scar-tissue interface rather than by reformation of the native fibrocartilage transition zone, the purpose of our study was to examine expression of various signaling molecules and transcription factors that are known to be involved in embryologic insertion-site development following ACL reconstruction. We also aimed to characterize a murine model of ACL reconstruction to allow future study of the molecular mechanisms of healing.

METHODS

Seventy-nine mice underwent reconstruction of the ACL with autograft. Healing was assessed using histology in <em>12</em> mice and quantitative real-time polymerase chain reaction (qRT-PCR) gene-expression analysis in 3 mice at 1 week postoperatively (Group-1 mice) and by biomechanical analysis in 7, histological analysis in 7, immunohistochemical analysis in 5, microcomputed tomography analysis in 5, and qRT-PCR analyses in 8 at 2 weeks (Group-2 mice) and 4 weeks (Group-3 mice) postoperatively. Fifteen additional mice did not undergo surgery and were used for biomechanical (7 mice), qRT-PCR (3 mice), and immunohistochemical (5 mice) analyses to obtain baseline data for the native ACL.

RESULTS

Histological analysis demonstrated healing by formation of fibrovascular tissue at the tendon-bone interface. Immunohistochemical analysis showed a positive expression of proteins in the Indian hedgehog, Wnt, and parathyroid hormone-related protein (PTHrP) pathways. There was minimal Sox-9 expression. Gene-expression analysis showed an initial increase in markers of tissue repair and turnover, followed by a subsequent decline. Mean failure force and stiffness of the native ACL were 5.60 N and 3.44 N/mm, respectively. Mean failure force and stiffness were 1.29 N and 2.28 N/mm, respectively, in Group 2 and were 1.79 N and 2.59 N/mm, respectively, in Group 3, with <em>12</em> of 14 failures in these study groups occurring by tunnel pull-out.

CONCLUSIONS

The spatial and temporal pattern of expression of signaling molecules that direct embryologic insertion-site formation was not adequate to restore the structure and composition of the native insertion site.

CONCLUSIONS

Development of a murine model to study ACL reconstruction will allow the use of transgenic animals to investigate the cellular, molecular, and biomechanical aspects of tendon-to-bone healing following ACL reconstruction, ultimately suggesting methods to improve healing in patients.

<AbstractText>Prior studies demonstrate increased incidence of urinary incontinence (UI) in the geriatric population which affects their quality of life. Pathophysiology of UI in the geriatric population and the underlying molecular mechanisms are still unclear. To elucidate these mechanisms, we performed a pre-clinical study in a rabbit model and the objectives were to (i) determine the effect of aging as well as multiparity on urethral sphincter muscle thickness and urethral closing pressure (UCP); (ii) examine the role of fibrosis and atrophy; and (iii) elucidate the molecular pathways that mediate fibrosis and atrophy in the urethral tissue.</AbstractText><AbstractText>New Zealand White female rabbits (n = 6 each; young 6-<em>12</em> months and old over 30 months of age) were anesthetized and urethral muscle thickness and sphincter closure function were measured. Rabbits were then sacrificed and urethral tissues (bladder neck and mid-urethra) were collected to process for immunostaining as well as for molecular studies for markers for fibrosis (β-catenin which is an important mediator of <em>Wnt</em> signaling, Collagen-1, and TGF-β) and atrophy (MuRF-1).</AbstractText><AbstractText>Our studies showed a significant decrease in the urethral sphincter muscle thickness and closure function with age. Age-related increase in protein and mRNA expression levels of fibrosis, as well as atrophy markers were observed in the bladder neck and mid-urethral tissues.</AbstractText><AbstractText>Age and multiparity related increase in fibrosis and atrophy of urethral sphincter muscles may contribute to impaired urethral closure function seen in old animals.</AbstractText>

Considerable diversity exists in porcine ear size, which is an important morphological feature of pig breeds. Previously, we localized four crucial candidate genes-high mobility group AT-hook 2 (HMGA2), LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3) and <em>Wnt</em> inhibitory factor 1 (WIF1)-on Sus Scrofa chromosome 5 affecting porcine ear size, then cloned LEMD3 and MSBR3. In this study, we performed rapid amplification of cDNA ends to obtain full-length cDNA sequences of 2338-bp WIF1 and 2998-bp HMGA2. Using quantitative real-time PCR, we revealed that WIF1 expression was highest in ear cartilage of 60-day-old pigs and that this is therefore a better candidate gene for ear size than HMGA2. We further screened coding sequence variants in both genes and identified only one missense mutation (WIF1:c.1167C>G) in a conserved epidermal growth factor-like domain from the mammalian WIF1 protein. The protein-altering mutation was significantly associated with ear size across the Large White × Minzhu hybrid and Beijing Black pig populations. When WIF1:c.1167C>G was included as fixed effect in the model to re-run a genome-wide association study in the Large White × Minzhu intercross population the P-value of the peak SNP on SSC5 from re-running the genome-wide association study dropped from 2.45E-<em>12</em> to 7.33E-05. Taken together, the WIF1:c.1167C>G could be an important mutation associated with ear size. Our findings provide helpful information for further studies of the molecular mechanisms controlling porcine ear size.

OBJECTIVE

Endocrine therapies, including tamoxifen or aromatase inhibitors, are indispensable for the treatment of patients with estrogen receptor (ER)- and/or progesterone-positive breast cancer. Whereas tamoxifen displays partial ER agonistic effects in bone, aromatase inhibitors increase bone resorption and fracture risk. The Wnt inhibitors dickkopf-1 (DKK-1) and sclerostin negatively impact bone formation and are considered targets for the treatment of bone disorders. However, the effect of endocrine therapies on serum DKK-1 and sclerostin levels in patients with primary breast cancer remains elusive.

METHODS

Serum DKK-1 and sclerostin levels were measured at primary diagnosis as well as 3-5 days and 12 months after surgery in a cohort of 45 pre- and postmenopausal women with primary estrogen receptor-positive breast cancer treated with adjuvant tamoxifen or aromatase inhibitors.

RESULTS

Mean baseline levels ±SD for DKK-1 and sclerostin were 29.7 ± 14.6 and 27.1 ± 16.2 pmol/l, respectively. A significant negative correlation of DKK-1 levels and age was observed (r = -0.32; p < 0.05), but not for sclerostin. Of note, DKK-1 levels were significantly lower in peri- and postmenopausal women compared to premenopausal patients (-47%; p < 0.05). In tamoxifen-treated patients, DKK-1 levels were reduced by 35% (p < 0.01) one year after surgery but remained unaltered in patients treated with aromatase inhibitors. No significant changes were observed for sclerostin.

CONCLUSIONS

DKK-1 serum levels were reduced in breast cancer patients receiving an adjuvant therapy with tamoxifen, possibly contributing to its bone-protective properties.

In Caenorhabditiselegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9-11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1 degrees , 2 degrees , or 3 degrees ), while eight anterior cells, P(1-8).p, fuse with the hyp7 epidermal syncytium. Here we show that activation of the canonical BAR-1 beta-catenin pathway of <em>Wnt</em> signaling alters the competence of P(3-8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1-8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-<em>12</em> or EGF signaling. While increased <em>Wnt</em> signaling induces predominantly 1 degrees HCG fates, increased LIN-<em>12</em> or EGF signaling in combination with MAB-5 overexpression promotes 2 degrees HCG fates in anterior Pn.p cells, suggesting distinctive functions of <em>Wnt</em>, LIN-<em>12</em>, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9-11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development.

Dipeptidyl peptidase-4 inhibitors, such as saxagliptin, have been reported to have beneficial effects on β-cell function, but the specific underlying mechanism remains unclear. Stromal cell-derived factor-1α (SDF-1α), a chemokine produced in multiple organs, has been considered as a crucial regulator in promoting β-cell survival. Here, we speculate that SDF-1α might mediate the effect of saxagliptin on improving β-cell function. After <em>12</em>-week saxagliptin treatment in high-fat diet/streptozotocin-induced diabetic rats, significant improvement in pancreas insulin secretion capacity evaluated by hyperglycemia clamp and increased β-cell to α-cell areas ratio were observed. Saxagliptin significantly induced β-cell proliferation and upregulated the expression of proliferation-related factors including c-myc and cyclind D1 determined with western blotting from the isolated islets. The expression/activity of DPP-4 was significantly reduced and paralleled with the restoration of SDF-1α levels in the saxagliptin-treated diabetic rats, subsequently the key <em>WNT</em>-signaling regulators, β-catenin, and AKT were activated. However, the effect of saxagliptin inducing β-cell proliferation was attenuated when we silenced the SDF-1α receptor (CXCR4) with RNAi in INS cell lines. Collectively, our data indicate that SDF-1α mediates the protective effect of saxagliptin on β-cell proliferation, suggesting that DPP-4 inhibitors have the potential role on delaying β-cell failure and SDF-1α could be a therapeutic target of β-cell regeneration.

Since our first description of the microcystic stromal tumor (MST) of the ovary, a rare and distinctive neoplasm with a definitional, usually striking microcystic pattern and a CD10+/vimentin+/inhibin-/calretinin- immunophenotype, 3 examples with β-catenin nuclear localization, and CTNNB1 mutation have been reported. We undertook a detailed immunohistochemical study and molecular analysis of CTNNB1 and FOXL2 of 15 cases of MST to further characterize this neoplasm and establish its histogenesis. Diffuse nuclear staining for FOXL2, WT-1, cyclin D1, and β-catenin was present in all tumors tested, and <em>12</em>/15 were positive for steroidogenic factor-1 (SF-1). Heterozygous missense point mutations in exon 3 of CTNNB1 were detected in 8 of 14 cases, resulting in amino acid changes at codons 32, 34, 35, and 37. There was no correlation between CTNNB1 exon 3 mutation status and tumor immunophenotype. All 14 cases tested showed wild-type FOXL2. Our study establishes that MST of the ovary exhibits a characteristic FOXL2/SF-1/WT-1/cyclin D1/nuclear β-catenin-positive immunohistochemical profile, which may be useful in diagnosis and in the exclusion of histologic mimics. The presence of diffuse nuclear FOXL2 and WT-1 immunostaining in all cases and SF-1 in most supports the classification of MST within the sex cord-stromal category. Aberrant nuclear β-catenin expression, detected in all MSTs, appears to be the result of stabilizing CTNNB1 mutations in 57% of cases, providing further evidence that dysregulation of the <em>Wnt</em>/B-catenin pathway is involved in the tumorigenesis of MST and may involve activation of β-catenin with upregulation of cyclin D1.

Biliary tract cancer (BTC) represents a heterogeneous disease with dismal outcome. Herein, we examined genotype and angiogenesis features in BTC. We applied genotyping (Sanger, qPCR, 101-gene panel NGS), mRNA relative quantification methods, and β-catenin immunohistochemistry in 84 FFPE BTC (55 gallbladder [GBC], 14 intrahepatic [ICC], 15 extrahepatic [ECC] carcinomas). We identified 541 mutations in 68 (81%) tumors. Top mutated genes were CTNNB1 (36%); PTEN (33%); TP53 (31%); PIK3R1 (29%); PIK3CA (13%); BRCA2 and KRAS (<em>12</em>%); BRCA1 (11%). Six GBCs were hypermutated [hm] displaying a distinct mutational pattern. Mutations in TP53 and PI3K, <em>Wnt</em> and RAS components were prevalent among non-hypermutated tumors. All hmGBCs carried mutations in BRCA2 and other homologous recombination repair (HRR) genes, in PD1, but not in CTNNB1 and KRAS. None of the pathogenic BRCA2 p.D2723G and BRCA1 p.Q563* and c.5266dupC was present at frequencies expected for germline mutations. We observed copy gains (>6 copies) in EGFR (9% of informative tumors), PRKAR1A (7%), PIK3CA (6%), ERBB2 (5%) and MET (4%). TP53 mutations were prevalent in GBC (P<0.001) and PRKAR1A copy gains in ICC (P=0.007). PTEN was frequently co-mutated with CTNNB1 (P<0.001). Unrelated to CTNNB1 mutations, nuclear β-catenin was detected in 45% of tumors, among them in 5/6 hmGBC. We observed strong mRNA expression correlation of the two neuropilins (NRP1 and NRP2) with each other (Spearman's rho 0.59) and with the endothelin receptor (NRP2 rho 0.66; NRP2 rho 0.51), and between VEGFA and its receptors (FLT1 rho 0.49; KDR rho 0.45). All PIK3CA mutated tumors expressed endothelin 1 mRNA (P=0.010). Most tumors expressing nuclear β-catenin were negative for VEGFC (P=0.009) and FLT4 (P=0.002) mRNA expression. In conclusion, we confirmed the presence of known genomic aberrations in BTC and different genotypes between BTC subsets. Novel findings are the coexistence of PI3K and <em>WNT</em> pathway gene alterations in BTC, their association with angiogenesis, and the hypermutated GBCs with HRR gene mutations, all of which may be considered for new treatment options in this difficult to treat disease.

The protein β-catenin exhibits a dual function in cells, by acting as a major structural component of cell-cell adherens junctions and as a central signaling molecule in the <em>Wnt</em> signaling pathway. However, how the regulation of β-catenin expression during tumor metastasis in non-small cell lung cancer (NSCLC) varies according to histological type remains unclear. To investigate the regulatory mechanism of β-catenin on tumor metastasis, the present study compared the expression of <em>Wnt</em>1, β-catenin and E-cadherin in 41 primary NSCLC tumors and their corresponding metastatic lesions by immunohistochemistry. Altered expression of β-catenin was more frequent in the metastatic tumors (34/41, 82.9%) than in the corresponding primary tumors (24/41, 58.5%; P<0.05). There were <em>12</em> cases [nine of adenocarcinoma (ADC) and three of squamous cell carcinoma (SqCC)] that revealed discordant β-catenin expression between the primary tumors and the corresponding metastatic lesions. Of these, 11 cases (11/<em>12</em>, 91.7%; nine ADCs and two SqCCs) demonstrated acquired β-catenin alterations in the metastatic lesions. Subgroup analysis of these nine ADCs revealed that six cases (6/9, 66.7%) were accompanied by E-cadherin loss but no <em>Wnt</em>1 overexpression. Subgroup analysis of the three SqCCs revealed discordant β-catenin expression. Two cases (2/3, 66.7%) demonstrated acquired β-catenin expression during metastatic progression with <em>Wnt</em>1 overexpression but no change in E-cadherin expression. One case of SqCC revealed normal β-catenin expression in the metastasis although the expression was aberrant in the primary tumor. The results of the present study revealed that the changes in β-catenin expression occurred during tumor metastasis by different mechanisms, depending on histological type. The alterations in β-catenin expression may be regulated by a cadherin-catenin system in ADCs with reduced membranous expression of E-cadherin, but mediated by <em>Wnt</em>1 overexpression in SqCCs with cytoplasmic or nuclear transition types.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC<em>12</em> cells. Cells were cultured to 70%-80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC<em>12</em> genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: <em>12</em> at 1 h, 26 at 3 h; <em>Wnt</em> pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.

BACKGROUND

Beta-catenin (CTNNB1), as a key transcriptional regulator in the WNT signal transduction cascade, plays a pivotal role in multiple biological functions such as embryonic development and homeostasis in adults. Although it has been suggested that CTNNB1 is required for gonad development and maintenance of ovarian function in mice, little is known about the expression and functional role of CTNNB1 in gonadal development and differentiation in the chicken reproductive system.

METHODS

To examine sex-specific, cell-specific and temporal expression of CTNNB1 mRNA and protein during gonadal development to maturation of reproductive organs, we collected left and right gonads apart from mesonephric kidney of chicken embryos on embryonic day (E) 6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens and performed quantitative PCR, in situ hybridization, and immunohistochemical analyses. In addition, localization of Sertoli cell markers such as anti-Müllerian hormone (AMH), estrogen receptor alpha (ESR1), cyclin D1 (CCND1) and N-cadherin (CDH2) during testicular development was evaluated.

RESULTS

Results of the present study showed that CTNNB1 mRNA and protein are expressed predominantly in the seminiferous cords on E6 to E14 in the male embryonic gonad, and are mainly localized to the medullary region of female embryonic gonads from E6 to E9. In addition, CTNNB1 mRNA and protein are abundant in the Sertoli cells in the testes and expressed predominantly in luminal epithelial cells of the oviduct, but not in the ovaries from 12-week-old and adult chickens. Concomitant with CTNNB1, AMH, ESR1, CCND1 and CDH2 were detected predominantly in the seminiferous cord of the medullary region of male gonads at E9 (after sex determination) and then maintained or decreased until hatching. Interestingly, AMH, ESR1, CCND1 and CDH2 were located in seminiferous tubules of the testes from 12-weeks-old chickens and ESR1, CCND1 and CDH2 were expressed predominantly in the Sertoli cells within seminiferous tubules of adult testes.

CONCLUSIONS

Collectively, these results revealed that CTNNB1 is present in gonads of both sexes during embryonic development and it may play essential roles in differentiation of Sertoli cells during formation of seminiferous tubules during development of the testes.

OBJECTIVE

To investigate whether the 5/6 nephrectomized (5/6Nx) rats' <em>12</em>-week serum could lead to tubular epithelial-to-mesenchymal transition (EMT) and its molecular mechanism, so as to probe the potential stimulation from circulation in chronic progressive kidney disease.

METHODS

A total of 24 Sprague Dawley (SD) rats were randomly divided into two groups: sham operation group (sham group) and 5/6Nx group. Rats were killed <em>12</em> weeks after surgery to obtain 5/6Nx rats' <em>12</em>-week serum. Then we detected the expression of E-cadherin in renal tubular epithelial cells of the remaining kidney and we investigated whether the <em>12</em>th week serum of 5/6Nx rats could cause HK-2 (human kidney proximal tubular cell line) cells to transdifferentiate into fibroblasts.

RESULTS

Our data confirmed that E-cadherin expression decreased significantly in the remaining kidney at <em>12</em> weeks, and the 5/6Nx rats' <em>12</em>-week serum could suppress E-cadherin protein and mRNA expression (p < 0.05). We also found that the 5/6Nx rats' <em>12</em>-week serum could upregulate ZEB1, β-catenin, and wnt3 protein expression (p < 0.05).

CONCLUSIONS

Our results demonstrated that the 5/6Nx rats' <em>12</em>-week serum could suppress the expression of E-cadherin in HK-2 cells. It was partially through modulating the increase of ZEB1. The loss of E-cadherin could lead β-catenin to localize to the cytoplasm and nucleus, and feed into the Wnt signaling pathway. It means that the pathogenic serum in chronic kidney disease (CKD) plays an important role in the loss of renal function and turns to be a new avenue of research with potential clinical implications.

The G protein-coupled bradykinin B2 receptor (Bdkrb2) plays an important role in regulation of blood pressure under conditions of excess salt intake. Our previous work has shown that Bdkrb2 also plays a developmental role since Bdkrb2(-/-) embryos, but not their wild-type or heterozygous littermates, are prone to renal dysgenesis in response to gestational high salt intake. Although impaired terminal differentiation and apoptosis are consistent findings in the Bdkrb2(-/-) mutant kidneys, the developmental pathways downstream of gene-environment interactions leading to the renal phenotype remain unknown. Here, we performed genome-wide transcriptional profiling on embryonic kidneys from salt-stressed Bdkrb2(+/+) and Bdkrb2(-/-) embryos. The results reveal significant alterations in key pathways regulating <em>Wnt</em> signaling, apoptosis, embryonic development, and cell-matrix interactions. In silico analysis reveal that nearly <em>12</em>% of differentially regulated genes harbor one or more Pax2 DNA-binding sites in their promoter region. Further analysis shows that metanephric kidneys of salt-stressed Bdkrb2(-/-) have a significant downregulation of Pax2 gene expression. This was corroborated in Bdkrb2(-/-);Pax2(GFP+/tg) mice, demonstrating that Pax2 transcriptional activity is significantly repressed by gestational salt-Bdkrb2 interactions. We conclude that gestational gene (Bdkrb2) and environment (salt) interactions cooperate to impact gene expression programs in the developing kidney. Suppression of Pax2 likely contributes to the defects in epithelial survival, growth, and differentiation in salt-stressed BdkrB2(-/-) mice.

Evidences have shown that pulsed electromagnetic fields (PEMFs) can partially prevent bone loss in streptozotocin (STZ)-induced diabetic rats. However, the precise mechanisms accounting for these favorable effects are unclear. This study aimed to investigate the effects of PEMFs on bone mass and receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) and <em>Wnt</em>/β-catenin signaling pathway in STZ rats. Thirty 3-month-old Sprague Dawley rats were randomly divided into the following three groups (n = 10): control group (injection of saline vehicle), DM group (injection of STZ), and PEMFs group (injection of STZ + PEMFs exposure). One week following injection of STZ, rats in the PEMFs group were subject to PEMFs stimulus for 40 min/day, 5 days/week, and lasted for <em>12</em> weeks. After <em>12</em> week intervention, the results showed that PEMFs increased serum bone-specific alkaline phosphatase level and bone mineral density, and inhibited deterioration of bone microarchitecture and strength in STZ rats. Furthermore, PEMFs up-regulated the mRNA expressions of low-density lipoprotein receptor-related protein 5, β-catenin and runt-related gene 2 (Runx2), and down-regulated dickkopf1 in STZ rats. However, mRNA expressions of RANKL and OPG were not affected by PEMFs. PEMFs can prevent the diabetes-induced bone loss and reverse the deterioration of bone microarchitecture and strength by restoring Runx2 expression through regulation of <em>Wnt</em>/β-catenin signaling, regardless of its no glucose lowering effect.

The <em>Wnt</em>/Frizzled signaling pathway participates in many inflammation-linked diseases. However, the inflammatory response mediated by the <em>Wnt</em>/Frizzled signaling pathway in experimental subarachnoid hemorrhage has not been thoroughly investigated. Consequently, in this study, we examined the potential role of the <em>Wnt</em>/Frizzled signaling pathway in early brain injury in rat models of subarachnoid hemorrhage. Simultaneously, possible neuroprotective mechanisms were also investigated. Experimental subarachnoid hemorrhage rat models were induced by injecting autologous blood into the prechiasmatic cistern. Experiment 1 was designed to examine expression of the <em>Wnt</em>/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. In total, 42 adult rats were divided into sham (injection of equivalent volume of saline), 6-, <em>12</em>-, 24-, 48-, 72-hour, and 1-week subarachnoid hemorrhage groups. Experiment 2 was designed to examine neuroprotective mechanisms of the <em>Wnt</em>/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. Rats were treated with recombinant human <em>Wnt</em>1 (rhwnt1), small interfering <em>Wnt</em>1 (siwnt1) RNA, and monoclonal antibody of Frizzled1 (anti-Frizzled1) at 48 hours after subarachnoid hemorrhage. Expression levels of <em>Wnt</em>1, Frizzled1, β-catenin, peroxisome proliferator-activated receptor-γ, CD36, and active nuclear factor-κB were examined by western blot assay and immunofluorescence staining. Microglia type conversion and inflammatory cytokine levels in brain tissue were examined by immunofluorescence staining and enzyme-linked immunosorbent assay. Our results show that compared with the sham group, expression levels of <em>Wnt</em>1, Frizzled1, and β-catenin were low and reduced to a minimum at 48 hours, gradually returning to baseline at 1 week after subarachnoid hemorrhage. rhwnt1 treatment markedly increased <em>Wnt</em>1 expression and alleviated subarachnoid hemorrhage-induced early brain injury (within 72 hours), including cortical cell apoptosis, brain edema, and neurobehavioral deficits, accompanied by increasing protein levels of β-catenin, CD36, and peroxisome proliferator-activated receptor-γ and decreasing protein levels of nuclear factor-κB. Of note, rhwnt1 promoted M2-type microglia conversion and inhibited release of inflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α). In contrast, siwnt1 RNA and anti-Frizzled1 treatment both resulted in an opposite effect. In conclusion, the <em>Wnt</em>/Frizzled1 signaling pathway may participate in subarachnoid hemorrhage-induced early brain injury via inhibiting the inflammatory response, including regulating microglia type conversion and decreasing inflammatory cytokine release. The study was approved by the Animal Ethics Committee of Anhui Medical University and First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China (approval No. LLSC-20180202) in May 2017.

OBJECTIVE

Glycogen synthase kinase-3 beta (GSK-3β) is a serine/threonine kinase involved in glycogen metabolism, cell cycle progression, differentiation, embryogenesis, migration, metabolism, survival and cellular senescence. Its main biological function is to inhibit β-catenin by sequestration and promotion of its proteasomal degradation in the Wnt canonical pathway; however, GSK-3β interacts with multiple signalling pathways, and aberrant expression of the enzyme was reported in many solid neoplasms. This study aimed to investigate the biological relevance of GSK-3β in classical Hodgkin lymphomas (cHL).

RESULTS

We analysed the functional status of GSK-3β enzyme in cHL by using antibodies raised against fixation-resistant epitopes of phospho Y216 GSK-3β (active form), phospho S9 GSK-3β (inactive form) and β-catenin protein. We first detected the pY216 GSK-3β active form of the enzyme in 100 of 100 (100%) of the cases, and in line with the latter expression profile, the β-catenin protein was found in only 12 of 100 (12%) of the samples. As reported previously in bladder cancer, pancreatic adenocarcinoma and chronic lymphocytic leukaemia, we showed an aberrant nuclear localization in the neoplastic clone of active pY216 GSK-3β in 78 of 100 (78%) of cHL cases.

CONCLUSIONS

We demonstrated the activation of GSK-3β in cHL resulting in inhibition of the Wnt/β-catenin signal cascade and the aberrant accumulation of its activated form in nuclei of Hodgkin Reed-Sternberg and Hodgkin cells. These findings may be relevant for future clinical studies, identifying GSK-3β as a potential therapeutic target for cHL.

This work aimed to investigate the therapeutic effect of curcumin on sublesional bone loss induced by spinal cord injury (SCI) in rats. SCI model in this work was generated in rats by surgical transaction of the cord at the T10-<em>12</em> level. After the surgery, animals were treated with curcumin (110 mg/kg body mass/day, via oral gavages) for 2 weeks. Treatment of SCI rats with curcumin prevented the reduction of bone mass in tibiae and femurs, preserved bone microstructure including trabecular bone volume fraction, trabecular number, and trabecular thickness in proximal tibiae, and preserved mechanical properties of femoral midshaft. Treatment of SCI rats with curcumin increased osteoblast surface and reduced osteoclast surface in proximal tibiae. Treatment of SCI rats with curcumin increased osteocalcin mRNA expression and reduced mRNA levels of tartrate-resistant acid phosphatase and mRNA ratio of receptor activator of NF-κB ligand/osteoprotegerin in distal femurs. Treatment of SCI rats with curcumin reduced serum and femoral levels of thiobarbituric acid reactive substances. Treatment of SCI rats with curcumin had no significant effect on serum 25(OH)D, but enhanced mRNA and protein expression of vitamin D receptor (VDR) in distal femurs. Treatment of SCI rats with curcumin enhanced mRNA levels of <em>Wnt</em>3a, Lrp5, and ctnnb1 and upregulated protein expression of β-catenin in distal femurs. In conclusions, treatment with curcumin abated oxidative stress, activated VDR, and enhanced <em>Wnt</em>/β-catenin pathway, which might explain its beneficial effect against sublesional bone loss following SCI in rats, at least in part.

Melanocyte stem cells (McSCs) undergo cyclical activation and quiescence together with hair follicle stem cells (HFSCs). This process is strictly controlled by the autonomous and extrinsic signaling environment. However, the modulation of factors important for the activation of McSCs for hair pigmentation remains unclear. <em>12</em>-O-tetradecanoylphorbol-13-acetate (TPA) mimics vital signaling pathways involved in melanocyte growth and melanogenesis in vitro. To investigate whether TPA regulates quiescent McSCs for hair pigmentation, we topically smeared TPA on 7-week-old mouse dorsal skin and found that TPA stimulated hair growth and hair matrix pigmentation. These changes were associated with a significant increase in the number of hair bulb melanocytes. Moreover, in the TPA-treated group, hair bulge McSCs and hair bulb melanoblasts actively proliferated. At the molecular level, nuclear β-catenin, a key factor of <em>Wnt</em>/β-catenin signaling, was highly synthesized in melanocytes and keratinocytes in TPA-induced hair bulbs. Inhibition of <em>Wnt</em>/β-catenin signaling by injecting Dickkopf1 plasmids into TPA-treated skin decreased hair matrix pigmentation and inhibited the proliferation and differentiation of McSCs. Our findings suggest that the topical application of TPA stimulates the proliferation and differentiation of McSCs and their progeny for hair matrix pigmentation by activating <em>Wnt</em>/β-catenin signaling. This might provide a useful experimental model for the study of signals controlling the activation of McSCs.