Wnt family member 4 (WNT4) and WNT3A activate cell-autonomous Wnt signaling independent of porcupine O-acyltransferase or Wnt secretion.

Porcupine O-acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned media from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion (WLS) mediator. We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling.

Embryonic Stem Cells Derived Kidney Organoids as Faithful Models to Target Programmed Nephrogenesis.

The kidney is a complex organ that is comprised of thousands of nephrons developing through reciprocal inductive interactions between metanephric mesenchyme (MM) and ureteric bud (UB). The MM undergoes mesenchymal to epithelial transition (MET) in response to the signaling from the UB. The secreted protein Wnt4, one of the Wnt family members, is critical for nephrogenesis as mouse Wnt4-/- mutants fail to form pretubular aggregates (PTA) and therefore lack functional nephrons. Here, we generated mouse embryonic stem cell (mESC) line lacking Wnt4 by applying the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems 9 (Cas9). We describe here, differentiation of the wild type and Wnt4 knockout mESCs into kidney progenitors, and such cells induced to undergo nephrogenesis by the mouse E11.5 UB mediated induction. The wild type three-dimensional (3D) self-organized organoids depict appropriately segmented nephron structures, while the Wnt4-deficient organoids fail to undergo the MET, as is the case in the phenotype of the Wnt4 knockout mouse model in vivo. In summary, we have established a platform that combine CRISPR/Cas9 and kidney organoid technologies to model kidney development in vitro and confirmed that mutant organoids are able to present similar actions as in the in vivo studies.

Zhen-wu-tang ameliorates adenine-induced chronic renal failure in rats: regulation of the canonical Wnt4/beta-catenin signaling in the kidneys.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhen-wu-tang (ZWT), composed of Radix Aconiti lateralis, Rhizoma Atractylodis macrocephalae, Poria, Radix Paeoniae alba and ginger, is a classic Chinese herbal formula for the treatment of chronic kidney diseases that may cause chronic renal failure (CRF). AIM OF THE STUDY: To better understand its clinical use, this study investigated the effects and underlying mechanisms of action of ZWT on CRF. MATERIALS AND METHODS: CRF was induced by adenine. ZWT was given via an oral gavage method. The serum biochemical parameters were measured enzymatically or by ELISA. The kidneys were examined pathohistologically. The gene expression was analyzed by real time PCR and Western blot. RESULTS: Similar to the positive control losartan, ZWT extract inhibited adenine-induced increase in serum concentrations of creatinine, BUN and advanced oxidation protein products in rats. These effects were accompanied by attenuation of proteinuria and renal pathological changes and suppression of renal mRNA and protein overexpression of Collagen IV and fibronectin, two of the key components of fibrosis. Mechanistically, renal mRNA and protein expression of Wnt4, a Wnt signaling ligand, was increased in the adenine-treated group, compared to the vehicle-treated control. Consistently, Wnt4 downstream genes beta-catenin and Axin were also overexpressed. Treatment with ZWT extract and losartan suppressed adenine-stimulated overexpression of these mRNAs and proteins. CONCLUSIONS: The present results demonstrate that ZWT extract ameliorates adenine-induced CRF in rats by regulation of the canonical Wnt4/beta-catenin signaling in the kidneys. Our findings provide new insight into the underlying renoprotective mechanisms of the ancient formula.

Structural dynamics and inhibitor searching for Wnt-4 protein using comparative computational studies.

Wnt-4 (wingless mouse mammary tumor virus integration site-4) protein is involved in many crucial embryonic pathways regulating essential processes. Aberrant Wnt-4 activity causes various anomalies leading to gastric, colon, or breast cancer. Wnt-4 is a conserved protein in structure and sequence. All Wnt proteins contain an unusual fold comprising of a thumb (or N-terminal domain) and index finger (or C-terminal domain) bifurcated by a palm domain. The aim of this study was to identify the best inhibitors of Wnt-4 that not only interact with Wnt-4 protein but also with the covalently bound acyl group to inhibit aberrant Wnt-4 activity. A systematic computational approach was used to analyze inhibition of Wnt-4. Palmitoleic acid was docked into Wnt-4 protein, followed by ligand-based virtual screening of nearly 209,847 compounds; conformer generation of 271 compounds resulted from extensive virtual screening and comparative docking of 10,531 conformers of 271 unique compounds through GOLD (Genetic Optimization for Ligand Docking), AutoDock-Vina, and FRED (Fast Rigid Exhaustive Docking) was subsequently performed. Linux scripts was used to handle the libraries of compounds. The best compounds were selected on the basis of having maximum interactions to protein with bound palmitoleic acid. These represented lead inhibitors in further experiments. Palmitoleic acid is important for efficient Wnt activity, but aberrant Wnt-4 expression can be inhibited by designing inhibitors interacting with both protein and palmitoleic acid.

Regulation of Wnt4 in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is associated with persistent inflammation and oxidative stress in susceptible individuals. Using microarray analysis of bronchial biopsy samples from patients with COPD and controls, we identified Wnt4 as being up-regulated in COPD. Analysis of bronchial biopsy samples showed a very strong correlation between Wnt4 and IL8 gene expression, suggesting that Wnt4 plays a role in chronic lung inflammation. In vitro, Wnt4 induced proliferation and inflammation in human epithelial cells (BEAS-2B) and normal primary human bronchial epithelial cells in a concentration-dependent manner. This effect was enhanced in the presence of interleukin-1ß (IL-1ß) as a result of activation of the p38 and c-Jun NH2-terminal kinase mitogen-activated protein kinase pathways. Hydrogen peroxide, but not proinflammatory stimuli, up-regulated Wnt4 expression in epithelial cells. In monocytic THP-1 and primary airway smooth muscle cells, Wnt4 induced inflammation and enhanced the inflammatory response to lipopolysaccharide and IL-1ß but did not induce proliferation. In addition, these other cell types did not have enhanced Wnt4 expression in response to hydrogen peroxide. Our results indicate that airway epithelial activation, due to oxidative stress, may lead to Wnt4 induction. Wnt4, in turn, acts through the noncanonical pathway to activate epithelial cell remodeling and IL8 gene expression, leading to neutrophil infiltration and inflammation.

Exendin-4 upregulates the expression of Wnt-4, a novel regulator of pancreatic ß-cell proliferation.

The Wnt-signaling pathway regulates ß-cell functions. It is not known how the expression of endogenous Wnt-signaling molecules is regulated in ß-cells. Therefore, we investigated the effect of antidiabetic drugs and glucose on the expression of Wnt-signaling molecules in ß-cells. Primary islets were isolated and cultured. The expression of Wnt-signaling molecules (Wnt-4, Wnt-10b, Frizzled-4, LRP5, TCF7L2) and TNFα was analyzed by semiquantitative PCR and Western blotting. Transient transfections were carried out and proliferation assays of INS-1 ß-cells performed using [(3)H]thymidine uptake and BrdU ELISA. Insulin secretion was quantified. A knockdown (siRNA) of Wnt-4 in ß-cells was carried out. Exendin-4 significantly increased the expression of Wnt-4 in ß-cells on the mRNA level (2.8-fold) and the protein level (3-fold) (P < 0.001). The effect was dose dependent, with strongest stimulation at 10 nM, and it was maintained after long-term stimulation over 4 wk. Addition of exd-(9-39), a GLP-1 receptor antagonist, abolished the effect of exendin-4. Treatment with glucose, insulin, or other antidiabetic drugs had no effect on the expression of any of the examined Wnt-signaling molecules. Functionally, Wnt-4 antagonized the activation of canonical Wnt-signaling in ß-cells. Wnt-4 had no effect on glucose-stimulated insulin secretion or insulin gene expression. Knocking down Wnt-4 decreased ß-cell proliferation to 45% of controls (P < 0.05). In addition, Wnt-4 and exendin-4 treatment decreased the expression of TNFaα mRNA in primary ß-cells. These data demonstrate that stimulation with exendin-4 increases the expression of Wnt-4 in ß-cells. Wnt-4 modulates canonical Wnt signaling and acts as regulator of ß-cell proliferation and inflammatory cytokine release. This suggests a novel mechanism through which GLP-1 can regulate ß-cell proliferation.