Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.
Animals ; Apoptosis ; Body Patterning ; Bone Morphogenetic Proteins/biosynthesis* ; Developmental Biology ; Ectoderm/metabolism* ; Extremities/embryology* ; Fibroblast Growth Factor 10/metabolism* ; Fibroblast Growth Factors/biosynthesis* ; Gene Expression Regulation ; Hedgehog Proteins/biosynthesis* ; Homeodomain Proteins/biosynthesis* ; Mesoderm/metabolism* ; Mice ; Signal Transduction ; Wnt Proteins/biosynthesis*

OBJECTIVE: To set up a prostate cancer cell line in which beta-catenin expression is stably suppressed and to investigate the role of Wnt/beta-catenin signaling pathway in prostate tumorgenesis. METHODS: We select 3 sites in the complete coden sequence region of beta-catenin gene as the RNAi targets, ligated the annealed double pre-DNA strands into the retroviral vectors pSUPER-retro and transfected them into the packaging cells PA317, and then collected supernatant with retrovirus to infect DU145. After selection by puromycin and culture expansion, the stable cell clones were attained. Expression of the 2 target genes of Wnt/beta-catenin signaling pathway cyclinD1 and c-myc, was detected in the beta-catenin RNAi cells by Western blot. The effect of suppressing beta-catenin by RNAi on cell proliferation was quantified by methylthiazoletetrazolium (MTT) assay. RESULTS: Western blotting and RT-PCR showed that the expression level of beta-catenin in the 2 stable cell clones apparently decreased. CyclinD1 and c-myc expression decreased in the beta-catenin RNAi cells. MTT showed that the cell number of beta-catenin expression suppression cell clones decreased significantly (P < 0. 05), suggesting the cell proliferation was prevented. CONCLUSION The beta-catenin gene stable suppression cell line was successfully established.
Cell Line, Tumor ; Genetic Vectors ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; Retroviridae ; genetics ; Signal Transduction ; Wnt Proteins ; biosynthesis ; genetics ; beta Catenin ; biosynthesis ; genetics

OBJECTIVETo observe the effect of Shenshuai Yingyang Capsule (SSYYJN) in ameliorating muscle atrophy in rats with chronic renal failure (CRF) and explore the role of Wnt7a-Akt/mTOR signal pathway in mediating this effect.

METHODSMale rats were randomly assigned to 5/6 nephrectomy group and sham-operated group, and the former group was further randomly divided into CRF model group, KA group, and SSYYJN group. The size of anterior tibia muscle was examined microscopically with HE staining. Protein synthesis in the soleus muscle was investigated by (14)C-phenylalanine experiment, and the expression of Wnt7a, frizzled-7, phospho-Akt, phospho-mTOR and GAPDH were detected with Western blotting.

RESULTSThe body weight, the wet and dry weight, cross-sectional area, and muscle protein synthesis of the anterior tibia muscles, and expressions of the proteins in the Wnt7a/Akt signaling pathway all increased significantly in SSYYJN and KA groups as compared with those in the model group.

CONCLUSIONSSYYJN can effectively improve muscle atrophy in the rat model of CRF possibly by reversing the reduction in the expressions of Wnt7a/Akt signaling pathway proteins in the skeletal muscles.

Animals ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; Kidney Failure, Chronic ; complications ; Male ; Muscle Proteins ; biosynthesis ; Muscle, Skeletal ; drug effects ; Muscular Atrophy ; drug therapy ; Nephrectomy ; Proto-Oncogene Proteins ; metabolism ; Rats ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Wnt Proteins ; metabolism

To evaluate the influence of Wnt/beta-catenin signal pathway on the porcine adipose tissue development and explore the mechanism, we detected the mRNA expression of Wnt/beta-catenin signal pathway related genes: beta-catenin, GSK3beta, Fzl and adipogenic transcription factors: PPARy, C/EBPalpha and early differentiation marker gene LPL with semi-quantitative (SQ) RT-PCR method. Immunohistochemical method (IHC) was applied to qualitatively measure the sequential expression of beta-catenin protein. The results of SQ RT-PCR showed that beta-catenin highly expressed at the first day after birth, then decreased to a low plateau after 60 days, the expression of GSK3beta and Fzl also decreased with the development process of the porcine adipose tissue development. However, the sequential expression of PPARgamma, C/EBPalpha, LPL appeared to be an opposite manner and kept at a high level after 60 days. The result of IHC showed that the expression of beta-catenin protein was strong in nucleus and cytoplasm at the first day after birth, then tended to decline with the process of adipose tissue development and could be only found in cytoplasm after 30-day old. These results suggest that beta-catenin plays an important role in the undifferentiated state maintenance of preadipocytes and the inhibition of porcine adipose tissue development, the mechanism maybe due to its regulation function on the adipogenic transcription factors PPARy, C/EBPalpha and early differentiation marker gene LPL.
Adipocytes ; metabolism ; Adipose Tissue ; growth & development ; Animals ; Gene Expression Regulation, Developmental ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; genetics ; Swine ; Transcription Factors ; Wnt Proteins ; genetics ; beta Catenin ; biosynthesis ; genetics

Beta-catenin plays a central role in Wnt signaling pathway. The aberrant localization of beta-catenin in nucleus causes the transcription of down-stream target genes, which is the pathogenesy of some solid tumours. As the expression of adheren junction on hemopoietic cells is very low, there are a few studies on beta-catenin expression in leukaemia. This study was aimed to investigate beta-catenin localization and beta-catenin mRNA expression levels in 4 leukemia cell lines so as to explore a new oncogenic mechanism and to find out a new therapeutic target. The beta-catenin localization in leukemia cell lines was detected by immunocytochemistry, the beta-catenin mRNA expression level was assayed by real-time quantitative RT-PCR. The results showed that there was aberrant localization of beta-catenin in Jurkat and Thp-1, and beta-catenin mRNA expression level was not increased in these two cell lines, however, the mRNA expression levels of Jurkat and Thp-1 were lower than those of Daudi and K562. The beta-catenin mRNA expression level was not correlated with beta-catenin aberrant localization in these 4 cell lines. It is concluded that the aberrant localization of beta-catenin may play a role in the development of some leukemia, and the mechanism resulting in beta-catenin aberrant localization not take place at transcription level.
Burkitt Lymphoma ; metabolism ; pathology ; Cell Line, Tumor ; Hematologic Neoplasms ; pathology ; Humans ; Jurkat Cells ; K562 Cells ; Leukemia, Monocytic, Acute ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Wnt Proteins ; metabolism ; beta Catenin ; analysis ; biosynthesis ; genetics

The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.
Cadherins/biosynthesis ; Carcinoma, Non-Small-Cell Lung/*genetics/mortality ; DNA Methylation/*genetics ; Female ; Humans ; Lung Neoplasms/*genetics/mortality ; Male ; Middle Aged ; Neoplasm Metastasis/genetics ; Neoplasm Staging ; Promoter Regions, Genetic/*genetics ; Republic of Korea ; Tumor Markers, Biological/genetics ; Wnt Proteins/*genetics

The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.
Cadherins/biosynthesis ; Carcinoma, Non-Small-Cell Lung/*genetics/mortality ; DNA Methylation/*genetics ; Female ; Humans ; Lung Neoplasms/*genetics/mortality ; Male ; Middle Aged ; Neoplasm Metastasis/genetics ; Neoplasm Staging ; Promoter Regions, Genetic/*genetics ; Republic of Korea ; Tumor Markers, Biological/genetics ; Wnt Proteins/*genetics

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.
Activating Transcription Factor 2/metabolism ; Animals ; Cell Separation ; Chemokines/*biosynthesis ; Chemotaxis/*drug effects ; Culture Media, Conditioned/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; GTP-Binding Proteins/metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Mice ; NF-kappa B/metabolism ; Neutrophils/*cytology/drug effects/enzymology/*metabolism ; Pertussis Toxin/pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Receptors, Wnt/metabolism ; Type C Phospholipases/metabolism ; Wnt Proteins/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo investigate the effects of hydrocamptothecin on the expression of Wnt signaling pathway inhibitor DKK-1 in tumor cells.

METHODSHuman HepG2, Hep3B, LoVo and U251 cells were treated with the antitumor drug Hydrocamptothecin. DKK-1 mRNA expression in the cells was detected with RT-PCR, and beta-catenin expression was measured by fluorescence-activated cell sorting (FACS).

RESULTSDKK mRNA in Hep3B, HepG2, LoVo and U251 cells was significantly increased after hydrocamptothecin treatment for 24 h, and the percentage of beta-catenin-positive cells and fluorescence intensity for beta-catenin expression was lowered in the cells after the treatment.

CONCLUSIONHydrocamptothecin promotes mRNA expression of Wnt signaling pathway inhibitor DKK-1 in Hep3B, HepG2, LoVo and U251 cells.

Antineoplastic Agents, Phytogenic ; pharmacology ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Flow Cytometry ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Liver Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Wnt Proteins ; metabolism

OBJECTIVETo re-analyze the data published in order to explore plausible biological pathways that can be used to explain the anti-aging effect of curcumin.

METHODSMicroarray data generated from other study aiming to investigate effect of curcumin on extending lifespan of Drosophila melanogaster were further used for pathway prediction analysis. The differentially expressed genes were identified by using GeneSpring GX with a criterion of 3.0-fold change. Two Cytoscape plugins including BisoGenet and molecular complex detection (MCODE) were used to establish the protein-protein interaction (PPI) network based upon differential genes in order to detect highly connected regions. The function annotation clustering tool of Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for pathway analysis.

RESULTSA total of 87 genes expressed differentially in D. melanogaster melanogaster treated with curcumin were identified, among which 50 were up-regulated significantly and 37 were remarkably down-regulated in D. melanogaster melanogaster treated with curcumin. Based upon these differential genes, PPI network was constructed with 1,082 nodes and 2,412 edges. Five highly connected regions in PPI networks were detected by MCODE algorithm, suggesting anti-aging effect of curcumin may be underlined through five different pathways including Notch signaling pathway, basal transcription factors, cell cycle regulation, ribosome, Wnt signaling pathway, and p53 pathway.

CONCLUSIONGenes and their associated pathways in D. melanogaster melanogaster treated with anti-aging agent curcumin were identified using PPI network and MCODE algorithm, suggesting that curcumin may be developed as an alternative therapeutic medicine for treating aging-associated diseases.

Aging ; drug effects ; genetics ; Animals ; Cell Cycle ; drug effects ; genetics ; Curcumin ; pharmacology ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; drug effects ; genetics ; Gene Expression Regulation ; drug effects ; Gene Regulatory Networks ; drug effects ; Genes, Insect ; Protein Biosynthesis ; drug effects ; genetics ; Protein Interaction Maps ; drug effects ; genetics ; Receptors, Notch ; genetics ; metabolism ; Ribosomes ; drug effects ; metabolism ; Signal Transduction ; drug effects ; genetics ; Tumor Suppressor Protein p53 ; metabolism ; Wnt Signaling Pathway ; drug effects ; genetics