Cell Biology of Canonical Wnt Signaling.

Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

All the might of the osteocyte: emerging roles in chronic kidney disease.

Osteocytes are the most abundant type of bone cell and play crucial roles in bone health. Osteocytes sense mechanical stress and orchestrate osteoblasts and osteoclasts to maintain bone density and strength. Beyond this, osteocytes have also emerged as key regulators of organ crosstalk, and they function as endocrine organs via their roles in secreting factors that mediate signaling within their neighboring bone cells and in distant tissues. As such, osteocyte dysfunction has been associated with the bone abnormalities seen across a spectrum of chronic kidney disease. Specifically, dysregulated osteocyte morphology and signaling have been observed in the earliest stages of chronic kidney disease and have been suggested to contribute to kidney disease progression. More important, US Food and Drug Administration-approved inhibitors of osteocytic secreted proteins, such as fibroblast growth factor 23 and sclerostin, have been used to treat bone diseases. The present mini review highlights new research that links dysfunctional osteocytes to the pathogenesis of chronic kidney disease mineral and bone disorder.

Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na+/H+ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator ß-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Canonical Wnt is inhibited by targeting one-carbon metabolism through methotrexate or methionine deprivation.

The nutrient-sensing metabolite S-adenosylmethionine (SAM) controls one-carbon metabolism by donating methyl groups to biochemical building blocks, DNA, RNA, and protein. Our recent work uncovered a requirement for cytoplasmic arginine methylation during Wnt signaling through the activity of protein arginine methyltransferase 1 (PRMT1), which transfers one-carbon groups from SAM to many protein substrates. Here, we report that treatments that decrease levels of the universal methyl donor SAM were potent inhibitors of Wnt signaling and of Wnt-induced digestion of extracellular proteins in endolysosomes. Thus, arginine methylation provides the canonical Wnt pathway with metabolic sensing properties through SAM. The rapid accumulation of Wnt-induced endolysosomes within 30 minutes was inhibited by the depletion of methionine, an essential amino acid that serves as the direct substrate for SAM production. We also found that methionine is required for GSK3 sequestration into multivesicular bodies through microautophagy, an essential step in Wnt signaling activity. Methionine starvation greatly reduced Wnt-induced endolysosomal degradation of extracellular serum proteins. Similar results were observed by addition of nicotinamide (vitamin B3), which serves as a methyl group sink. Methotrexate, a pillar in the treatment of cancer since 1948, decreases SAM levels. We show here that methotrexate blocked Wnt-induced endocytic lysosomal activity and reduced canonical Wnt signaling. Importantly, the addition of SAM during methionine depletion or methotrexate treatment was sufficient to rescue endolysosomal function and Wnt signaling. Inhibiting the Wnt signaling pathway by decreasing one-carbon metabolism provides a platform for designing interventions in Wnt-driven disease.

Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking.

Arginine methylation has emerged as a widespread and reversible protein modification with the potential to regulate a multitude of cellular processes, but its function is poorly understood. Endolysosomes play an important role in Wnt signaling, in which glycogen synthase kinase 3 (GSK3) becomes sequestered inside multivesicular bodies (MVBs) by the process known as microautophagy, causing the stabilization of many proteins. Up to 20% of cellular proteins contain three or more consecutive putative GSK3 sites, and of these 33% also contain methylarginine (meArg) modifications. Intriguingly, a cytoskeletal protein was previously known to have meArg modifications that enhanced subsequent phosphorylations by GSK3. Here, we report the unexpected finding that protein arginine methyltransferase 1 (PRMT1) is required for canonical Wnt signaling. Treatment of cultured cells for 5-30 min with Wnt3a induced a large increase in total endocytic vesicles which were also positive for asymmetric dimethylarginine modifications. Protease protection studies, both biochemical and in situ in cultured cells, showed that many meArg-modified cytosolic proteins became rapidly translocated into MVBs together with GSK3 and Lys48-polyubiquitinated proteins by ESCRT-driven microautophagy. In the case of the transcription factor Smad4, we showed that a unique arginine methylation site was required for GSK3 phosphorylation and Wnt regulation. The enzyme PRMT1 was found to be essential for Wnt-stimulated arginine methylation, GSK3 sequestration, and canonical Wnt signaling. The results reveal a cell biological role for PRMT1 arginine methylation at the crossroads of growth factor signaling, protein phosphorylation, membrane trafficking, cytosolic proteolysis, and Wnt-regulated microautophagy.

Sclerostin and Wnt Signaling in Idiopathic Juvenile Osteoporosis Using High-Resolution Confocal Microscopy for Three-Dimensional Analyses.

BACKGROUND: Idiopathic juvenile osteoporosis (IJO) is a rare condition characterized by low bone mass that can increase the risk of fractures in children. Treatment options for these patients are limited as the molecular mechanisms of disease initiation and progression are incompletely understood. Sclerostin inhibits canonical Wnt signaling, which is important for the bone formation activity of osteoblasts, and elevated sclerostin has been implicated in adult osteoporosis. OBJECTIVE: To evaluate the role of sclerostin in IJO, high-resolution confocal microscopy analyses were performed on bone biopsies collected from 13 pediatric patients. METHODS: Bone biopsies were stained with sclerostin, and ß-catenin antibodies showed elevated expression across osteocytes and increased sclerostin-positive osteocytes in 8 of the 13 total IJO patients (62%). RESULTS: Skeletal sclerostin was associated with static and dynamic histomorphometric parameters. Further, colocalization analyses showed that bone sclerostin colocalized with phosphorylated ß-catenin, a hallmark of Wnt signaling that indicates Wnt inhibition. In contrast, sclerostin-positive osteocytes were not colocalized with an "active" unphosphorylated form of ß-catenin. CONCLUSIONS: These results support a model that altered levels of sclerostin and Wnt signaling activity occur in IJO patients.

Hitting the Sweet Spot: How Glucose Metabolism Is Orchestrated in Space and Time by Phosphofructokinase-1.

Glycolysis is the central metabolic pathway across all kingdoms of life. Intensive research efforts have been devoted to understanding the tightly orchestrated processes of converting glucose into energy in health and disease. Our review highlights the advances in knowledge of how metabolic and gene networks are integrated through the precise spatiotemporal compartmentalization of rate-limiting enzymes. We provide an overview of technically innovative approaches that have been applied to study phosphofructokinase-1 (PFK1), which represents the fate-determining step of oxidative glucose metabolism. Specifically, we discuss fast-acting chemical biology and optogenetic tools that have delineated new links between metabolite fluxes and transcriptional reprogramming, which operate together to enact tissue-specific processes. Finally, we discuss how recent paradigm-shifting insights into the fundamental basis of glycolytic regulatory control have shed light on the mechanisms of tumorigenesis and could provide insight into new therapeutic vulnerabilities in cancer.

Sclerostin, Osteocytes, and Wnt Signaling in Pediatric Renal Osteodystrophy.

The pathophysiology of chronic kidney disease-mineral and bone disorder (CKD-MBD) is not well understood. Specific factors secreted by osteocytes are elevated in the serum of adults and pediatric patients with CKD-MBD, including FGF-23 and sclerostin, a known inhibitor of the Wnt signaling pathway. The molecular mechanisms that promote bone disease during the progression of CKD are incompletely understood. In this study, we performed a cross-sectional analysis of 87 pediatric patients with pre-dialysis CKD and post-dialysis (CKD 5D). We assessed the associations between serum and bone sclerostin levels and biomarkers of bone turnover and bone histomorphometry. We report that serum sclerostin levels were elevated in both early and late CKD. Higher circulating and bone sclerostin levels were associated with histomorphometric parameters of bone turnover and mineralization. Immunofluorescence analyses of bone biopsies evaluated osteocyte staining of antibodies towards the canonical Wnt target, ß-catenin, in the phosphorylated (inhibited) or unphosphorylated (active) forms. Bone sclerostin was found to be colocalized with phosphorylated ß-catenin, which suggests that Wnt signaling was inhibited. In patients with low serum sclerostin levels, increased unphosphorylated "active" ß-catenin staining was observed in osteocytes. These data provide new mechanistic insight into the pathogenesis of CKD-MBD and suggest that sclerostin may offer a potential biomarker or therapeutic target in pediatric renal osteodystrophy.

Vitamin B6 is governed by the local compartmentalization of metabolic enzymes during growth.

Vitamin B6 is a vital micronutrient across cell types and tissues, and dysregulated B6 levels contribute to human disease. Despite its importance, how B6 vitamer levels are regulated is not well understood. Here, we provide evidence that B6 dynamics are rapidly tuned by precise compartmentation of pyridoxal kinase (PDXK), the rate-limiting B6 enzyme. We show that canonical Wnt rapidly led to the accumulation of inactive B6 by shunting cytosolic PDXK into lysosomes. PDXK was modified with methyl-arginine Degron (MrDegron), a protein tag for lysosomes, which enabled delivery via microautophagy. Hyperactive lysosomes resulted in the continuous degradation of PDXK and B6 deficiency that promoted proliferation in Wnt-driven colorectal cancer (CRC) cells. Pharmacological or genetic disruption of the coordinated MrDegron proteolytic pathway was sufficient to reduce CRC survival in cells and organoid models. In sum, this work contributes to the repertoire of micronutrient-regulated processes that enable cancer cell growth and provides insight into the functional impact of B6 deficiencies for survival.

Metabolism and Endocrine Disorders: What Wnt Wrong?

A fundamental question in cell biology underlies how nutrients are regenerated to maintain and renew tissues. Physiologically, the canonical Wnt signaling is a vital pathway for cell growth, tissue remodeling, and organ formation; pathologically, Wnt signaling contributes to the development of myriad human diseases such as cancer. Despite being the focus of intense research, how Wnt intersects with the metabolic networks to promote tissue growth and remodeling has remained mysterious. Our understanding of metabolism has been revolutionized by technological advances in the fields of chemical biology, metabolomics, and live microscopy that have now made it possible to visualize and manipulate metabolism in living cells and tissues. The application of these toolsets to innovative model systems have propelled the Wnt field into new realms at the forefront answering the most pressing paradigms of cell metabolism in health and disease states. Elucidating the basis of Wnt signaling and metabolism in a cell-type and tissue-specific manner will provide a powerful base of knowledge for both basic biomedical fields and clinician scientists, and has the promise to generate new, transformative therapies in disease and even processes of aging.

Simplifying the B Complex: How Vitamins B6 and B9 Modulate One Carbon Metabolism in Cancer and Beyond.

Vitamin B micronutrients are essential regulators of one carbon metabolism that ensures human health. Vitamin B9, or folate, lies at the heart of the folate cycle and converges with the methionine cycle to complete the one carbon pathway. Additionally, vitamin B6 contributes by orchestrating the flux of one carbon cycling. Dysregulation of vitamin B contributes to altered biochemical signaling that manifests in a spectrum of human diseases. This review presents an analysis of the past, present, and future work, highlighting the interplay between folate and vitamin B6 in one carbon metabolism. Emerging insights include advances in metabolomic-based mass spectrometry and the use of live-cell metabolic labeling. Cancer is used as a focal point to dissect vitamin crosstalk and highlight new insights into the roles of folate and vitamin B6 in metabolic control. This collection of vitamin-based research detailing the trends of one carbon metabolism in human disease exemplifies how the future of personalized medicine could unfold using this new base of knowledge and ultimately provide next-generation therapeutics.

Characterization of Primary Cilia in Osteoblasts Isolated From Patients With ADPKD and CKD.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of chronic kidney disease (CKD) and leads to a specific type of bone disease. The primary cilium is a major cellular organelle implicated in the pathophysiology of ADPKD caused by mutations in polycystin-1 (PKD1) and polycystin-2 (PKD2). In this study, for the first time, cilia were characterized in primary preosteoblasts isolated from patients with ADPKD. All patients with ADPKD had low bone turnover and primary osteoblasts were also obtained from patients with non-ADPKD CKD with low bone turnover. Image-based immunofluorescence assays analyzed cilia using standard markers, pericentrin, and acetylated-α-tubulin, where cilia induction and elongation were chosen as relevant endpoints for these initial investigations. Osteoblastic activity was examined by measuring alkaline phosphatase levels and mineralized matrix deposition rates. It was found that primary cilia can be visualized in patient-derived osteoblasts and respond to elongation treatments. Compared with control cells, ADPKD osteoblasts displayed abnormal cilia elongation that was significantly more responsive in cells with PKD2 nontruncating mutations and PKD1 mutations. In contrast, non-ADPKD CKD osteoblasts were unresponsive and had shorter cilia. Finally, ADPKD osteoblasts showed increased rates of mineralized matrix deposition compared with non-ADPKD CKD. This work represents the first study of cilia in primary human-derived osteoblasts from patients with CKD and patients with ADPKD who have normal kidney function, offering new insights as bone disease phenotypes are not well recapitulated in animal models. These data support a model whereby altered cilia occurs in PKD-mutated osteoblasts, and that ADPKD-related defects in bone cell activity and mineralization are distinct from adynamic bone disease from patients with non-ADPKD CKD. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Wnt-inducible Lrp6-APEX2 interacting proteins identify ESCRT machinery and Trk-fused gene as components of the Wnt signaling pathway.

The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including ß-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of ß-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/ß-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.

GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the ß-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, ß-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

GSK3- and PRMT-1-dependent modifications of desmoplakin control desmoplakin-cytoskeleton dynamics.

Intermediate filament (IF) attachment to intercellular junctions is required for skin and heart integrity, but how the strength and dynamics of this attachment are modulated during normal and pathological remodeling is poorly understood. We show that glycogen synthase kinase 3 (GSK3) and protein arginine methyltransferase 1 (PRMT-1) cooperate to orchestrate a series of posttranslational modifications on the IF-anchoring protein desmoplakin (DP) that play an essential role in coordinating cytoskeletal dynamics and cellular adhesion. Front-end electron transfer dissociation mass spectrometry analyses of DP revealed six novel serine phosphorylation sites dependent on GSK3 signaling and four novel arginine methylation sites including R2834, the mutation of which has been associated with arrhythmogenic cardiomyopathy (AC). Inhibition of GSK3 or PRMT-1 or overexpression of the AC-associated mutant R2834H enhanced DP-IF associations and delayed junction assembly. R2834H blocked the GSK3 phosphorylation cascade and reduced DP-GSK3 interactions in cultured keratinocytes and in the hearts of transgenic R2834H DP mice. Interference with this regulatory machinery may contribute to skin and heart diseases.