Genome-wide identification and expression profiling analysis of Wnt family genes affecting adipocyte differentiation in cattle

The Wnt family features conserved glycoproteins that play roles in tissue regeneration, animal development and cell proliferation and differentiation. For its functional diversity and importance, this family has been studied in several species, but not in the Bovinae. Herein we identified 19 Wnt genes in cattle, and seven other species of Bovinae, and described their corresponding protein properties. Phylogenetic analysis clustered the 149 Wnt proteins in Bovinae, and 38 Wnt proteins from the human and mouse into 12 major clades. Wnt genes from the same subfamilies shared similar protein motif compositions and exon–intron patterns. Chromosomal distribution and collinearity analysis revealed that they were conservative in cattle and five species of Bovinae. RNA-seq data analysis indicated that Wnt genes exhibited tissue-specific expression in cattle. qPCR analysis revealed a unique expression pattern of each gene during bovine adipocytes differentiation. Finally, the comprehensive analysis indicated that Wnt2B may regulate adipose differentiation by activating FZD5, which is worthy of further study. Our study presents the first genome-wide study of the Wnt gene family in Bovinae, and lays the foundation for further functional characterization of this family in bovine adipocytes differentiation.

The scaffolding protein Flot2 regulates cytoneme-based transport of Wnt3 in gastric cancer

The Wnt/β-catenin signalling pathway regulates multiple cellular processes during development and many diseases, including cell proliferation, migration, and differentiation. Despite their hydrophobic nature, Wnt proteins exert their function over long distances to induce paracrine signalling. Recent studies have identified several factors involved in Wnt secretion, however, our understanding of how Wnt ligands are transported between cells to interact with their cognate receptors is still debated. Here, we demonstrate that gastric cancer cells utilise cytonemes to transport Wnt3 intercellularly to promote proliferation. Furthermore, we identify the membrane-bound scaffolding protein Flotillin-2 (Flot2), frequently overexpressed in gastric cancer, as a regulator of these cytonemes. Together with the Wnt co-receptor and cytoneme initiator Ror2, Flot2 determines the number and length of Wnt3 cytonemes in gastric cancer. Finally, we show that Flot2 is necessary for Wnt8a cytonemes during zebrafish embryogenesis, suggesting a conserved mechanism for Flot2-mediated Wnt transport on cytonemes in development and disease.

Connective Tissue Growth Factor (CTGF)/CCN2 Knockout: A New Murine Model of Craniofacial Development

Animal models represent an invaluable tool, which must be judiciously used to provide the greatest benefit to human kind, due to their cost and time effectiveness. The CCN2 null mouse model described in this paper represents a new murine model of craniofacial development. This model is notable for its remarkably consistent phenotype and ease of colony care and propagation. The interaction of CCN2 with the TGF-β, BMP, FGF, EGF, Integrin, and WNT proteins is currently under investigated and signifies a plethora of research opportunities that may help elucidate novel therapeutic options for future patients. This paper presents a descriptive overview of the known craniofacial developmental abnormalities of this model as well as the known molecular signaling aberrances that provide clues to direct future investigative endeavors.

QSAR Docking on Azoles as inhibitors of Notum carboxylesterase

Background: Carboxylesterase Notum is a negative regulator of Wnt signaling. Notum carboxylesterase is a carboxylic ester hydrolase enzyme that functions as a negative feedback regulator of Wnt proteins by depalmitoylation reaction. It is of great importance to understand the pathway of Wnt regulation because, conversely, misregulation of Wnt signaling is a telltale sign of cancer and other degenerative diseases. The Wnt inhibition is important in the control of colorectal cancer. Objective: In the present study, we carried out a QSAR analysis of a series of reported compounds with carboxylesterase Notum inhibitory activity using multiple regression analysis. A series of 83 compound datasets of pyrrole derivatives with carboxy Notum inhibitory values were taken from the reported literature. Methods: The study was performed by conducting multiple linear regression analysis followed by validation of the model. The multiple linear regression (MLR) models with the highest coefficients of correlation (R2) and explained variance in leave-one-out (Q2 LOO) prediction and leave-many-out (Q2 LMO) were selected for the whole dataset. The developed models were subjected to internal and external validation. The reliability of the predicted model was checked by plotting the Williams plot. The docking methodology was performed using Autodock 4 for the designed compounds to study the interaction between the ligand and the receptor. Results: The best model generated exhibited an r2 value of 0.7413, Q2LOO =0.6379, Q2LMO =0.6368. Novel compounds of phenyl pyrrolidine were designed based on generated QSAR equations. The carboxylesterase Notum inhibitory activity was predicted using the QSAR equations. The docking studies were carried out for designed compounds using Autodock against Carboxylesterase Notum esterase. Conclusion: From the results, the designed compounds were found to inhibit Notum Carboxylase. Thus, the study led to the development of a novel lead compound for Carboxylesterase Notum.

Wnt/β-catenin pathway proteins in end-stage renal disease

Aim: To delineate the association of end-stage renal disease (ESRD) and Wnt-proteins including the agonist R-spondin-1, the transducer β-catenin and the antagonists DKK1 and sclerostin. Materials & methods: Serum Wnt-pathway proteins levels were measured by ELISA in 60 ESRD patients and 30 normal controls. Results: DKK1 and sclerostin were significantly higher in ESRD than in controls, and β-catenin and the catenin + R-spondin-1/DKK1 + sclerostin ratio, reflecting the ratio of agonist and transducer on antagonists (AT/ANTA), were significantly lower in ESRD. Estimated glomerular filtration rate was significantly associated with DKK1 and sclerostin (inversely), β-catenin (positively) and the AT/ANTA ratio (r = 0.468, p < 0.001). Conclusion: Wnt/β-catenin pathway proteins show significant alterations in ESRD, indicating significantly increased levels of antagonists.

Inhibition of Wnt/β-Catenin Signaling Sensitizes Esophageal Cancer Cells to Chemoradiotherapy

The standard treatment of locally advanced esophageal cancer comprises multimodal treatment concepts including preoperative chemoradiotherapy (CRT) followed by radical surgical resection. However, despite intensified treatment approaches, 5-year survival rates are still low. Therefore, new strategies are required to overcome treatment resistance, and to improve patients’ outcome. In this study, we investigated the impact of Wnt/β-catenin signaling on CRT resistance in esophageal cancer cells. Experiments were conducted in adenocarcinoma and squamous cell carcinoma cell lines with varying expression levels of Wnt proteins and Wnt/β-catenin signaling activities. To investigate the effect of Wnt/β-catenin signaling on CRT responsiveness, we genetically or pharmacologically inhibited Wnt/β-catenin signaling. Our experiments revealed that inhibition of Wnt/β-catenin signaling sensitizes cell lines with robust pathway activity to CRT. In conclusion, Wnt/β-catenin activity may guide precision therapies in esophageal carcinoma patients.

Abstract 121: Interleukin 11 Mediates Wnt/β-catenin-dependent Fibrotic Response Of Human Cardiac Fibroblasts

Wnt proteins family represents secreted glycoproteins implicated in the number of fibrotic cardiac pathologies. The transcriptional activity of Wnts is broad and involves β-catenin-dependent or β-catenin-independent responses. In this study, we examined the effect of exogenous Wnt3a (β-catenin-dependent) and Wnt5a (β-catenin-independent) in TGF-β-activated human cardiac fibroblasts. Furthermore, we assessed the hypothesis that Wnt3a could regulate IL-11 production and analyzed its contribution to profibrotic response in cardiac fibroblasts.By employing a full genome transcriptomics, we analyzed transformation of human cardiac fibroblasts induced by TGF-β in the presence of Wnt3a or Wnt5a produced by cell culture supernatant of L-Wnt3a, L-Wnt5a or control L-cells. Stimulation with Wnt3a of TGF-β-activated fibroblasts resulted in induction of 66 genes, specifically involved in myofibroblast differentiation including ACTA2 (encoding alpha smooth muscle actin; αSMA) ACTG2 (encoding gamma smooth muscle actin; γSMA) and VCL (encoding vinculin). In contrast to Wnt3a, treatment with Wnt5a upregulated expression of only 2 genes in TGF-β-activated cells. Additionally, in the presence of TGF-β, Wnt3a enhanced phosphorylation of TAK1 and production and secretion of IL-11. Importantly, in the absence of TGF-β, Wnt3a did not promote fibroblast-to-myofibroblast transition, TAK1 phosphorylation and IL-11 production. To determine, if Wnt3a-dependent production of IL-11 could contribute to profibrotic response we blocked IL-11 activity with anti-IL-11 neutralizing antibody in cardiac fibroblasts activated with TGF-β and Wnt3a. We found that neutralizing anti-IL11 antibody effectively suppressed production of αSMA, γSMA, fibronectin and pro-collagen I alpha 1, both on mRNA and protein levels. In line with these findings, blockade of IL-11 suppressed contractile properties of TGF-β/Wnt3a-activated cardiac fibroblasts. In conclusion, Wnt3a and Wnt5a differentially regulate gene expression of TGF-β-activated cardiac. Activation of the Wnt/β-catenin pathway promotes fibroblast-to-myofibroblast transition by enhancing production of profibrotic IL-11. It seems that identifying the profibrotic Wnt/β-catenin-IL11 mechanism in cardiac fibroblasts might represent a promising strategy in development of new therapies against cardiac fibrosis.

Cryo-EM structure of human Wntless in complex with Wnt3a

Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A β-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.

EVI/WLS function is regulated by ubiquitination and linked to ER-associated degradation by ERLIN2

WNT signalling is important for development in all metazoan animals and is associated with various human diseases. The Ubiquitin-Proteasome System (UPS) and regulatory ER-associated degradation (ERAD) have been implicated in the production of WNT proteins. Here, we investigated how the WNT secretory factor EVI/WLS is ubiquitinated, recognised by ERAD components and subsequently removed from the secretory pathway. We performed a focused, immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitination machinery. Interestingly, we found in addition that EVI/WLS is ubiquitinated and degraded in cells irrespective of their level of WNT production. This K11, K48, and K63-linked ubiquitination is mediated by the E2 ubiquitin conjugating enzymes UBE2J2, UBE2K, and UBE2N, but independent of the E3 ligases HRD1/SYVN or GP78/AMFR. Taken together, our study identified factors that link the UPS to the WNT secretory pathway and provides mechanistic details on the fate of an endogenous substrate of regulatory ERAD in mammalian cells.

Genome-Wide Identification and Expression Profiling Analysis of Wnt Family Genes Affecting Adipocyte Differentiation in Cattle

Wnt is a family of conserved glycoproteins that functions in a variety of crucial biological processes including tissue regeneration, animal development, and cell proliferation and differentiation. For its functional diversity and importance, Wnt gene family has gained considerable research interest in a variety of species. However, comprehensive identification and analysis of Wnt genes in Bovinae is lacking. In this study, we identified the repertoire of Wnt genes in cattle and seven other species of Bovinae and obtained 19 Wnt genes. Protein properties of these Wnt genes were also described. Phylogenetic analysis showed that the 149 Wnt proteins in Bovinae, together with 38 Wnt proteins from human and mouse, were clustered into 12 major clades. The Wnt genes belonging to the same subfamilies shared similar protein motif compositions and exon-intron patterns. Chromosomal distribution and collinearity analysis of Wnt genes among cattle and five species of Bovinae revealed that this gene family was conservative in evolution. RNA-seq data analysis indicated that Wnt genes exhibited tissue-specific expression patterns in cattle. qPCR analysis of Wnt gene family showed that each gene had a unique expression pattern during bovine adipocytes differentiation. And the comprehensive analysis indicated that Wnt2B may regulate adipose differentiation through activation of FZD5, which is worthy of further study. Our study presents the first genome-wide study of Wnt gene family in Bovinae, and lay the foundation for further functional characterization of the Wnt family in bovine adipocytes differentiation.