TNF-α Activating Osteoclasts in Patients with Psoriatic Arthritis Enhances the Recruitment of Osteoclast Precursors: A Plausible Role of WNT5A-MCP-1 in Osteoclast Engagement in Psoriatic Arthritis

Psoriatic arthritis (PsA) results from joint destruction by osteoclasts. The promising efficacy of TNF-α blockage indicates its important role in osteoclastogenesis of PsA. WNT ligands actively regulate osteoclastogenesis. We investigated how WNT ligands activate osteoclasts amid the TNF-α milieu in PsA. We first profiled the expression of WNT ligands in CD14+ monocyte-derived osteoclasts (MDOC) from five PsA patients and five healthy controls (HC) and then validated the candidate WNT ligands in 32 PsA patients and 16 HC. Through RNA interference against WNT ligands in MDOC, we determined the mechanisms by which TNF-α exerts its effects on osteclastogenesis or chemotaxis. WNT5A was selectively upregulated by TNF-α in MDOC from PsA patients. The number of CD68+WNT5A+ osteoclasts increased in PsA joints. CXCL1, CXCL16, and MCP-1 was selectively increased in supernatants of MDOC from PsA patients. RNA interference against WNT5A abolished the increased MCP-1 from MDOC and THP-1-cell-derived osteoclasts. The increased migration of osteoclast precursors (OCP) induced by supernatant from PsA MDOC was abolished by the MCP-1 neutralizing antibody. WNT5A and MCP-1 expressions were decreased in MDOC from PsA patients treated by biologics against TNF-α but not IL-17. We conclude that TNF-α recruits OCP by increased MCP-1 production but does not directly activate osteoclastogenesis in PsA.

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.

Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

Colonic healing requires WNT produced by epithelium as well as Tagln+ and Acta2+ stromal cells

While Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Wnt blockage in distinct stromal cell types suggested obligatory functions of several stromal cell sources and yielded different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affected colonic Reg4+ epithelial cell differentiation, and impaired colonic epithelial regeneration after injury. Single cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin alpha 2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that Wnt blockage from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated Wnt blockage from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.

Extensive loss of Wnt genes in Tardigrada

Background Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan. Results We analyzed published genomes for two representatives of Tardigrada, Hypsibius exemplaris and Ramazzottius varieornatus. We identified single orthologs of Wnt4, Wnt5, Wnt9, Wnt11, and WntA, as well as two Wnt16 paralogs in both tardigrade genomes. We only found a Wnt2 ortholog in H. exemplaris. We could not identify orthologs of Wnt1, Wnt6, Wnt7, Wnt8, or Wnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog of arrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally, we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog of arrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes in H. exemplaris during developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes in H. exemplaris besides one of the Wnt16 paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs in H. exemplaris, rather than in broadly overlapping patterns. Conclusions Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lost Wnt1, Wnt6, Wnt7, Wnt8, and Wnt10, along with arrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

Glypicans and cytonemes unite to distribute Wnt ligands

Hu et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202009082) show that Glypican 4 participates in filopodia-mediated Wnt transport from endoderm to mesoderm in zebrafish embryos to facilitate intercellular communication between germ layers.

TNF-α activates WNT5A, which recruits osteoclast precursors by MCP-1 production, but not directly activates osteoclastogenesis, in psoriatic arthritis

Background: Psoriatic arthritis (PsA) results from joint destruction by osteoclasts. Promising efficacy of TNF-α blockage indicates its important role in osteoclastogenesis of PsA. WNT ligands actively regulate osteoclastogenesis. We investigated how WNT ligands activate osteoclasts amid the TNF-α milieu in PsA. Methods: We first profiled the expression of WNT ligands in CD14+ monocyte-derived osteoclasts (MDOC) from 3 PsA patients and 3 healthy controls (HC) and then validated the candidate WNT ligands in 32 PsA patients and 16 HC. Through RNA interference against WNT ligands in MDOC, we determined the mechanisms by which TNF-α exerts its effects on osteclastogenesis or chemotaxis. Results: The results showed numbers of CD68+WNT5A+ osteoclasts are increased in PsA joints. WNT5A was selectively upregulated by TNF-α in MDOC from PsA patients. However, direct osteoclastogenesis effect (RANK expression) by TNF-αwas not inhibited by WNT5A siRNA. Instead, CXCL1, CXCL16, and MCP-1 was selectively increased in supernatants of MDOC from PsA patients. RNA interference against WNT5A abolished the increased MCP-1 from MDOC and THP-1-cell-derived osteoclasts. The increased migration of osteoclast precursors (OCP) induced by supernatant from PsA MDOC was abolished by MCP-1 neutralizing antibody. WNT5A and MCP-1 expressions were decreased in MDOC from PsA patients treated by biologics against TNF-a but not IL-17. Conclusion: We conclude TNF-α recruits OCP by WNT5A-mediated MCP-1 production but not directly activates osteoclastogenesis in PsA.

923 Hedgehog signaling drives epithelial-to-mesenchymal transition, immune evasion, and anti-PD-1 resistance through coordinated upregulation of Wnt ligands and PGE2 synthesis

BackgroundImmunotherapy resistance has been correlated with epithelial-to-mesenchymal transition (EMT),1 2 however our understanding of tumor-intrinsic mechanisms driving this immune evasive phenotype is lacking. We have previously shown that Wnt ligands are upregulated in anti-PD-1 resistant melanomas,3 and postulated that upstream transcriptional regulation of select EMT pathways may underpin these findings. The hedgehog signaling (HH) transcription factor Gli2 promotes EMT.MethodsGli2 was constitutively activated (Gli2CA ) in a BRAFV600EPTEN-/- murine cell line via an N-terminal truncating mutation and silenced using CRISPR-Cas9. Multi-parameter flow cytometry and RNAseq was utilized to evaluate the impact of Gli2 on the tumor immune microenvironment. Anti-PD-1 resistance studies were performed in Gli2CA and control tumors. Bioinformatics studies were conducted using the melanoma TCGA and Hugo et al databases.2ResultsWe found upregulation of Gli2 targets in patients with anti-PD-1-refractory metastatic melanoma as well as in an autochthonous BRAFV600EPTEN-/- melanoma model after escape from anti-PD-1. RNAseq and Western blot studies demonstrated Gli2CA to promote EMT and Wnt ligand production in addition to upregulated COX2 in BRAFV600EPTEN-/- melanoma. This finding was reversed by genetic ablation and pharmacologic inhibition of Gli2, implicating a previously undescribed role for Gli2 in modulating COX2. These data were consistent with a notable correlation between a Gli2 signature and a prostaglandin synthesis signature in human melanoma TCGA database. Flow cytometry analysis showed exclusion of cytolytic T and NK cells, a shift from cDC1s to cDC2s, and enhanced MDSC recruitment in Gli2CA tumors. Consistent with these findings, whole tumor RNAseq of Gli2CA tumors demonstrated a decrease in Cd3e, Prf1, and Xcr1 with a concomitant increase in Cxcl1, Cxcl2, Ccl2, Ptgs2, and Arg1 relative to control tumors. RNAseq of FACS-sorted DCs from Gli2CA tumors demonstrated a loss of cDC1-associated genes including Xcr1, Wdfy4, and Clec9a compared to DCs derived from control tumors. In-line with our previous results showing that Wnt5a promotes MDSC recruitment in a Yap-dependent manner,4 we found that Yap inhibition or Wnt5a deletion in the BRAFV600EPTEN-/-Gli2CA cell line diminished MDSC-recruiting chemokines. Further consistent with these findings, Gli2CA tumors resist anti-PD-1 antibody therapy.Abstract 923 Figure 1Gli2 in tumors promotes Wnt and prostaglandin signaling, generating an immunosuppressive microenvironmentConclusionsOur data demonstrates that the HH transcription factor Gli2 drives the development of a tolerogenic tumor microenvironment unfavorable to anti-PD-1 immunotherapy by coordinating the upregulation of Wnt ligand expression and prostaglandin synthesis (figure 1). We propose that HH gene signatures are worthy of further study as a guide for selecting Wnt ligand and prostaglandin inhibitors in future immunotherapy studies.AcknowledgementsThe authors would like to acknowledge the Duke Cancer Institute Flow Cytometry Core.ReferencesBagaev A, et al. Conserved pan-cancer microenvironment subtypes predict response to immunotherapy. Cancer Cell 2021.Hugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 2016;165(1):35–44.DeVito NC, et al. Pharmacological Wnt ligand inhibition overcomes key tumor-mediated resistance pathways to anti-PD-1 immunotherapy. Cell Rep 2021;35(5):109071.Theivanthiran B, et al. A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti-PD-1 immunotherapy. J Clin Invest 2020;130(5):2570–2586.

Wnt6 plays a complex role in maintaining human limbal stem/progenitor cells

The corneal epithelium is consistently regenerated by limbal stem/progenitor cells (LSCs), a very small population of adult stem cells residing in the limbus. Several Wnt ligands, including Wnt6, are preferentially expressed in the limbus. To investigate the role of Wnt6 in regulating proliferation and maintenance of human LSCs in an in vitro LSC expansion setting, we generated NIH-3T3 feeder cells to overexpress different levels of Wnt6. Characterization of LSCs cultured on Wnt6 expressing 3T3 cells showed that high level of Wnt6 increased proliferation of LSCs. Medium and high levels of Wnt6 also increased the percentage of small cells (diameter ≤ 12 µm), a feature of the stem cell population. Additionally, the percentage of cells expressing the differentiation marker K12 was significantly reduced in the presence of medium and high Wnt6 levels. Although Wnt6 is mostly known as a canonical Wnt ligand, our data showed that canonical and non-canonical Wnt signaling pathways were activated in the Wnt6-supplemented LSC cultures, a finding suggesting that interrelationships between both pathways are required for LSC regulation.

Wnt and β-Catenin Signaling in the Bone Metastasis of Prostate Cancer

Wnt family proteins and β-catenin are critical for the regulation of many developmental and oncogenic processes. Wnts are secreted protein ligands which signal using a canonical pathway, and involve the transcriptional co-activator β-catenin or non-canonical pathways that are independent of β-catenin. Bone metastasis is unfortunately a common occurrence in prostate cancer and can be conceptualized as a series of related steps or processes, most of which are regulated by Wnt ligands and/or β-catenin. At the primary tumor site, cancer cells often take on mesenchymal properties, termed epithelial mesenchymal transition (EMT), which are regulated in part by the Wnt receptor FZD4. Then, Wnt signaling, especially Wnt5A, is of importance as the cells circulate in the blood stream. Upon arriving in the bones, cancer cells migrate and take on stem-like or tumorigenic properties, as aided through Wnt or β-catenin signaling involving CHD11, CD24, and Wnt5A. Additionally, cancer cells can become dormant and evade therapy, in part due to regulation by Wnt5A. In the bones, E-selectin can aid in the reversal of EMT, a process termed mesenchymal epithelial transition (MET), as a part of metastatic tumorigenesis. Once bone tumors are established, Wnt/β-catenin signaling is involved in the suppression of osteoblast function largely through DKK1.