Protein kinase CK2 is required for Wntless internalization and Wnt secretion

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization.     

Downregulating the Canonical Wnt/β-catenin Signaling Pathway Attenuates the Susceptibility to Autism-like Phenotypes by Decreasing Oxidative Stress

Although a growing body of evidence supports the importance of the Wnt/β-catenin signaling pathway and oxidative stress in the pathogenesis of autism, it is unclear whether a relationship exists between the Wnt/β-catenin pathway and oxidative homeostasis. The present study examines the effects of sulindac, a small molecule inhibitor of the Wnt/β-catenin signaling pathway, on the oxidative status of rats that are prenatally exposed to valproic acid (VPA), which is used in an animal model of autism. Our data show that sulindac treatment downregulated the canonical Wnt/β-catenin signaling pathway by enhancing the expression of Glycogen Synthase Kinase 3β and attenuating the expression of β-catenin in comparison to levels in VPA-treated rats. Concomitantly, a marker of lipid peroxidation, 4-hydroxynonenal, was reduced as well. Sulindac treatment ameliorated the pain threshold, repetitive/stereotypic activity, learning and memory abilities and behavioral abnormalities of rats in our autism model. Our working model suggests that the upregulation of the Wnt/β-catenin signaling pathway induced by VPA administration during early pregnancy produces an imbalance of oxidative homeostasis that facilitates susceptibility to autism. This information may be instrumental in designing appropriate therapeutic regimens with small molecule inhibitors of the Wnt/β-catenin pathway for the treatment of autism-like behavioral phenotypes.     

Flavonoids: potential Wnt/beta-catenin signaling modulators in cancer

Flavonoids are polyphenolic compounds found throughout the plant kingdom. They occur in every organ but are usually concentrated in leaves and flowers. During the last two decades, in vitro and in vivo studies demonstrated that flavonoids have inhibitory effects on human diseases through targeting of multiple cellular signaling components. Wnt/β-catenin signaling regulates proliferation, differentiation and fate specification in developmental stages and controls tissue homeostasis in adult life. For these reasons, this pathway has received great attention in the last years as potential pathway involved in distinct Human pathologies. In this review we discuss the emerging potential mechanisms for flavonoids on Wnt/β-catenin signaling in cancer and possible investigation strategies to understand flavonoids mode of action on this signaling pathway.     

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.     

ATP-binding cassette transporter G1 and lipid homeostasis

PURPOSE OF REVIEW: This review briefly discusses the ATP-binding cassette transporter G (ABCG) family members and emphasizes recent studies that identify ABCG1 as a key regulator of cellular lipid homeostasis. RECENT FINDINGS: The in-vivo importance of ABCG1 has recently been demonstrated with both loss-of-function and gain-of-function studies in mice. Administration of a diet high in both fat and cholesterol to ABCG1 mice results in massive cholesterol accumulation in both the liver and lungs. In contrast, lipid accumulation is greatly attenuated in transgenic mice that express both the murine and human ABCG1 genes. Despite the observed tissue lipid accumulation, plasma lipid levels and lipoprotein cholesterol distribution are not significantly different between wild-type, ABCG1, and hABCG1 transgenic mice. Other studies show that ABCG1 expression is induced following activation of the nuclear receptor LXR and that over expression of ABCG1 results in increased efflux of cellular cholesterol to HDL or phospholipid vesicles. SUMMARY: The ABCG1 transporter plays a key role in regulating cellular cholesterol and lipid homeostasis. Elucidation of the molecular mechanism by which ABCG1 controls sterol flux should provide critical information that may link ABCG1 to the reverse cholesterol transport pathway or diseases such as atherosclerosis.     

D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol alters cellular cholesterol homeostasis by modulating the endosome lipid domains

D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is a frequently used inhibitor of glycosphingolipid biosynthesis. However, some interesting characteristics of D-PDMP cannot be explained by the inhibition of glycolipid synthesis alone. In the present study, we showed that d-PDMP inhibits the activation of lysosomal acid lipase by late endosome/lysosome specific lipid, bis(monoacylglycero)phosphate (also called as lysobisphosphatidic acid), through alteration of membrane structure of the lipid. When added to cultured fibroblasts, D-PDMP inhibits the degradation of low-density lipoprotein (LDL) and thus accumulates both cholesterol ester and free cholesterol in late endosomes/lysosomes. This accumulation results in the inhibition of LDL-derived cholesterol esterification and the decrease of cell surface cholesterol. We showed that D-PDMP alters cellular cholesterol homeostasis in a glycosphingolipid-independent manner using L-PDMP, a stereoisomer of D-PDMP, which does not inhibit glycosphingolipid synthesis, and mutant melanoma cell which is defective in glycolipid synthesis. Altering cholesterol homeostasis by D-PDMP explains the unique characteristics of sensitizing multidrug resistant cells by this drug.     

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/β-catenin pathway. Here, we show that RNF43 suppresses both Wnt/β-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/β-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/β-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/β-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/β-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.     

Pdro,a Protein Associated with Late Endosomes and Lysosomes and Implicated in Cellular Cholesterol Homeostasis

Background

Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content.

Methodology/Principal Findings

Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY.

Conclusions/Significance

LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.     

The lipoprotein receptor-related protein-1 (LRP) adapter protein GULP mediates trafficking of the LRP ligand prosaposin, leading to sphingolipid and free cholesterol accumulation in late endosomes and impaired efflux

One of the conserved functional pathways linked to engulfment of apoptotic corpses involves two membrane proteins low density lipoprotein receptor-related protein-1 (LRP) and ABCA1 and the LRP adapter protein GULP. Because LRP and ABCA1 play roles in cellular lipid trafficking and efflux, here we addressed whether the third member, the LRP adapter protein GULP, also affects cellular lipid transport. Several lines of evidence show that overexpression of GULP causes glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment that is accompanied by down-regulation of ABCA1 and decreased efflux. Conversely, knockdown of endogenous GULP expression promoted cholesterol flux through the late endosomes and up-regulation of ABCA1, even in the context of a disease state such as Niemann-Pick Type C disease. Mechanistically, we were able to show that trafficking of the LRP ligands alpha2-macroglobulin and prosaposin, a protein cofactor necessary for glycosphingolipid degradation, are impaired in cells expressing full-length GULP protein, resulting in glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment. On the other hand, knockdown of endogenous GULP results in enhanced targeting of prosaposin and enhanced clearance of glycosphingolipids and cholesterol from the late endosomes. Taken together, these data reveal that GULP/LRP/ABCA1 represents a triad of molecules involved in engulfment and cellular lipid homeostasis.     

STARD3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

StAR‐related lipid transfer domain‐3 (STARD3) is a sterol‐binding protein that creates endoplasmic reticulum (ER)–endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill‐defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3‐mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER–endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER‐anchored partner, Vesicle‐associated membrane protein‐associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts. Thus, STARD3 is a cholesterol transporter scaffolding ER–endosome contacts and modulating cellular cholesterol repartition by delivering cholesterol to endosomes.     

Lipid modifications of Sonic hedgehog ligand dictate cellular reception and signal response

Background

Sonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. However, the influences of the lipid moieties on cellular reception and signal response are not well understood.

Methodology/Principal Findings

We analyzed fully lipidated Shh and mutant forms to eliminate one or both adducts in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We demonstrate that lipid modification is required for cell reception, that either adduct is sufficient to confer cellular association, that the cholesterol adduct anchors ligand to the plasma membrane and that the palmitate adduct augments ligand internalization. We further show that signaling potency correlates directly with cellular concentration of Shh ligand.

Conclusions/Significance

The findings of this study demonstrate that lipid modification of Shh determines cell concentration and potency, revealing complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and augmenting reception of Shh protein in target tissues.     

Wnts and wing: Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis

In the past twenty years, secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development from hydra to human. In the developing vertebrate limb, Wnt signaling is required for limb bud initiation, early limb patterning (which is governed by several well-characterized signaling centers), and, finally, late limb morphogenesis events. Wnt ligands are unique, in that they can activate several different receptor-mediated signal transduction pathways. The most extensively studied Wnt pathway is the canonical Wnt pathway, which controls gene expression by stabilizing beta-catenin in regulating a diverse array of biological processes. Recently, more attention has been given to the noncanonical Wnt pathway, which is beta-catenin-independent. The noncanonical Wnt pathway signals through activating Ca(2+) flux, JNK activation, and both small and heterotrimeric G proteins, to induce changes in gene expression, cell adhesion, migration, and polarity. Abnormal Wnt signaling leads to developmental defects and human diseases affecting either tissue development or homeostasis. Further understanding of the biological function and signaling mechanism of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. This review provides a critical perspective on how Wnt signaling regulates different developmental processes. As Wnt signaling in tumor formation has been reviewed extensively elsewhere, this part is not included in the review of the clinical significance of Wnt signaling.     

Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.     

Apc tumor suppressor gene is the "zonation-keeper" of mouse liver

The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.     

Differential molecular assemblies underlie the dual function of Axin in modulating the WNT and JNK pathways.

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3 beta (GSK-3 beta) and kinase-dead GSK-3 beta-Y216F (capable of binding to Axin), but not GSK-3 beta-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I epsilon also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, beta-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free beta-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.