Morphological neurite changes induced by porcupine inhibition are rescued by Wnt ligands.

BACKGROUND: Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/ß-catenin-dependent or ß-catenin-independent or "non-canonical", both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluated METHODS: Cultured primary embryonic hippocampal neurons obtained from Sprague-Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, ß-catenin and GSK-3ß (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin. RESULTS: We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases ß-catenin and Wnt3a and an apparent increase in GSK-3ß (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. CONCLUSIONS: Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases. Video Abstract.

Piperine suppresses the Wnt/ß-catenin pathway and has anti-cancer effects on colorectal cancer cells.

More than 94% of colorectal cancer cases have mutations in one or more Wnt/ß-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in ß-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/ß-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of ß-catenin, ß-catenin S33A or dnTCF4 VP16, while also suppressing ß-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

Left-right asymmetry in palatal rugae is associated with genetic variants in WNT signaling pathway.

OBJECTIVE: The present study evaluated the association between genetic variants in WNT3A and WNT11, and palatal rugae phenotypes. DESIGN: Eighty-five biological unrelated orthodontic patients were included. Dental casts were assessed and data regarding the length, shape, direction and unification of rugae were recorded. The individuals were subsequently classified for each of the following rugae traits: total amount of rugae; bilateral symmetry in the amount, length and shape of the rugae; presence of secondary or fragmentary rugae; presence of unifications; predominant shape; and, direction of the rugae. Genetic variants in WNT3A (rs708111) and WNT11 (rs1533767) were genotyped by real-time PCR. Genotype and allele distributions were compared with an established alpha of 5 %. RESULTS: The wavy and curve rugae were the most common. Genotype/phenotype analyses identified that the presence of the rs708111 A allele (OR = 2.2, 95 % CI: 1.1-4.4, p = 0.01) and the rs1533767 G allele (OR = 2.3, 95 % CI: 1.0-5.3, p = 0.05) increased in more than two times the chance of having bilateral asymmetry in the amount of the rugae. In the recessive model, individuals carrying two risk alleles (AA) of WNT3A rs708111 had a higher risk of presenting this phenotype. SNP-SNP interaction analysis revealed that individuals carrying one rs708111 A allele and rs1533767 G allele showed even a higher chance of having bilateral asymmetry in the amount of rugae (OR = 5.6, 95 % CI: 1.1-28.8, p = 0.03). No associations were identified for other rugae phenotype (p > 0.05). CONCLUSION: Genetic variants in WNT3A and WNT11 were associated with the left-right asymmetry in the amount of palatal rugae.

Wnt3a ligand facilitates autophagy in hippocampal neurons by modulating a novel GSK-3ß-AMPK axis.

BACKGROUND: In the adult central nervous system (CNS), Wnt signaling regulates dendritic structure and synaptic plasticity. The Wnt signaling pathway can be divided into the canonical (ß-catenin-dependent) and non-canonical pathways. In the canonical pathway, the binding of canonical ligands such as Wnt3a to the Frizzled receptor induces inactivation of glycogen synthase kinase-3ß (GSK-3ß), which stabilizes ß-catenin and allows its translocation to the nucleus. However, to date, few studies have focused on ß-catenin-independent Wnt signaling or explained the underlying mechanisms connecting Wnt signaling to cellular energy metabolism. A recent study demonstrated negative regulation of 5' adenosine monophosphate-activated protein kinase (AMPK), a major target of GSK-3ß that regulates cellular metabolism under diverse conditions. Mainly based on these observations, we evaluated whether Wnt3a ligand modulates autophagy by regulating the GSK-3ß/AMPK axis. METHODS: Cultured primary hippocampal neurons and slices of the CA1 region of rat hippocampus were used. GSK-3ß inhibition, AMPK activation, PP2Ac expression, and LC3 processing were examined by western blotting. Autophagic compartments were studied using the CYTO-ID® fluorescent probe, and mature autophagosomes were observed via transmission electron microscopy (TEM). RESULTS: Wnt3a ligand, acting through the Frizzled receptor, promotes the rapid activation of AMPK by inactivating GSK-3ß. Biochemical analysis of downstream targets indicated that Wnt3a ligand modulates autophagy in hippocampal neurons. CONCLUSIONS: Our results revealed new aspects of Wnt signaling in neuronal metabolism. First, AMPK is an additional target downstream of the Wnt cascade, suggesting a molecular mechanism for the metabolic effects previously observed for Wnt signaling. Second, this mechanism is independent of ß-catenin, suggesting a relevant role for non-genomic activity of the Wnt pathway in cellular metabolism. Finally, these results have new implications regarding the role of Wnt signaling in the modulation of autophagy in neurons, with a possible role in the removal of accumulated intracellular proteins.

Dishevelled stability is positively regulated by PKCζ-mediated phosphorylation induced by Wnt agonists.

Dishevelled (Dvl) proteins are central mediators of both canonical and non-canonical Wnt signaling. It is well known that, upon Wnt stimulation, Dvl becomes phosphorylated. However, how Wnt-induced phosphorylation of Dvl is regulated and its consequences are poorly understood. Here we found that Dvl proteins are overexpressed in colon cancer cells. In addition, we found that Wnt3a treatment rapidly induces hyperphosphorylation and stabilization of Dvl2 and Dvl3. The latter can be blocked by inhibition of Protein Kinase C (PKC)α, PKCδ, and PKCζ isoforms. We also found that Wnt3a-induced phosphorylation of Dvl3 by PKCζ is required to avoid Dvl3 degradation via proteasome. This demonstrated, to our knowledge for the first time, that hyperphosphorylation of Dvl by PKCζ results in Dvl stabilization. This is clear contrast with the consequences reported to date of CK1δ/ε-mediated Dvl phosphorylation upon Wnt treatment. Mapping the interaction domain between PKCζ and Dvl3 indicated that, although the Dvl-DIX domain is required to stabilize PKCζ-phosphorylated Dvl, it is not the region phosphorylated by this kinase. Our data show that the Dvl-DEP domain, required for specific interaction with PKCζ, is the site phosphorylated by this kinase, and also probably the Dvl-C terminus. Our findings suggest a model of positive regulation of PKCζ-mediated Dvl signaling activity, to produce a strong and sustained response to Wnt3a treatment by stabilizing Dvl protein levels.

WNT3A gene polymorphisms are associated with bone mineral density variation in postmenopausal mestizo women of an urban Mexican population: findings of a pathway-based high-density single nucleotide screening.

Osteoporosis (OP) is a common skeletal disorder characterized by low bone mineral density (BMD) and is a common health problem in Mexico. To date, few genes affecting BMD variation in the Mexican population have been identified. The aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) located in genes of the Wnt pathway with BMD variation at various skeletal sites in a cohort of postmenopausal Mexican women. A total of 121 SNPs in or near 15 Wnt signaling pathway genes and 96 ancestry informative markers were genotyped in 425 postmenopausal women using the Illumina GoldenGate microarray SNP genotyping method. BMD was measured by dual-energy X-ray absorptiometry in total hip, femoral neck, Ward's triangle, and lumbar spine. Associations were tested by linear regression for quantitative traits adjusting for possible confounding factors. SNP rs752107 in WNT3A was strongly associated with decreased total hip BMD showing the highest significance under the recessive model (P = 0.00012). This SNP is predicted to disrupt a binding site for microRNA-149. In addition, a polymorphism of the Wnt antagonist DKK2 was associated with BMD in femoral neck under a recessive model (P = 0.009). Several LRP4, LRP5, and LRP6 gene variants showed site-specific associations with BMD. In conclusion, this is the first report associating Wnt pathway gene variants with BMD in the Mexican population.

Effect of the Jianpi Bushen Prescription on expressions of the Wnt3a and Cyclin D1 genes in radiation-damaged mice.

The effects of the traditional Chinese drug Jianpi Bushen Prescription (JBP) were investigated on expressions of Wnt3a and Cyclin D1 genes in radiation-damaged mice. The radiation damage model was induced in Kumming mice by single total body irradiation treatment for 9 days. Mice were divided into the radiation group, low-dose (100%) JBP group, high-dose (200%) JBP group, or batyl alcohol group (positive control), which were administered twice a day for 9 days. mRNA and protein expressions of Wnt3a were detected in bone marrow mononuclear cells by real-time polymerase chain reaction and Western blot, whereas Cyclin D1 mRNA was detected by in situ hybridization. Wnt3a expressions were significantly downregulated in the radiation damage model group compared with all other groups (P < 0.05). The positive cell rate of Cyclin D1 mRNA expression and the number of granulocyte macrophage colonies were significantly decreased in the radiation damage model group relative to all other groups (P < 0.05). Furthermore, mRNA and protein expressions of Wnt3a, the positive cell rate of Cyclin D1 mRNA expression in bone marrow cells, and the number of granulocyte macrophage colonies were all significantly higher in the low-dose JBP group than in the high-dose JBP group (P < 0.05). In summary, JBP plays a protective role on radiation-induced bone marrow through the activation of the Wnt3a signaling pathway, and promotes the transcription and expression of Cyclin D1.

Protein kinase C delta negatively modulates canonical Wnt pathway and cell proliferation in colon tumor cell lines.

The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional ß-catenin destruction complex, did not affect ß-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal ß-catenin destruction complex, negatively affected ß-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of ß-catenin degradation, because PKCδ inhibition induces ß-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.