The non-canonical Wnt/Ca2+ signaling cascade is less characterized than their canonical counterpart, the Wnt/β-catenin pathway. The non-canonical Wnt signaling pathways are diverse, defined as planer cell polarity pathway, Wnt-RAP1 signaling pathway, Wnt-Ror2 signaling pathway, Wnt-PKA pathway, Wnt-GSK3MT pathway, Wnt-aPKC pathway, Wnt-RYK pathway, Wnt-mTOR pathway, and Wnt/calcium signaling pathway. All these pathways exhibit a considerable degree of overlap between them. The Wnt/Ca2+ signaling pathway was deciphered as a crucial mediator in development. However, now there is substantial evidence that the signaling cascade is involved in many other molecular phenomena. Many aspects of Wnt/Ca2+ pathway are yet enigmatic. This review will give a brief overview of the fundamental and evolving concepts of the Wnt/Ca2+ signaling pathway.
The gene Wnt was first discovered in Drosophila as a mutation causing absence of wing and haltere . In Drosophila the gene is named after its mutant phenotype, and so it was named wingless. Subsequently, a conserved locus on chromosome 15 was identified in mouse, where a proviral insertion at either 5′ or 3′ end of the sequence caused malignant transformation of mammary tissues ; the sequence was named as int. The Drosophila and mouse genes were found to be homologs, and so the gene was named as Wnt by combining the two names. Since the discovery interest surged about this gene and it was found that the gene product is a lipid-modified secreted glycoprotein regulating cell polarity, morphogenetic movements, and axis development in vertebrates. The signaling cascade is a complex canonical pathway in which β-catenin is the master regulator. However, research showed that Wnt signal cascade may act independent of β-catenin, executing its effects in early development where calcium signaling is the central mediator .
While canonical Wnt signaling has been extensively dissected from the view point of molecular biology and biochemistry, non-canonical Wnt/Ca2+ signaling has been less focused on. However, non-canonical Wnt signaling pathways such as Wnt planer cell polarity pathway, Wnt-JNK signaling pathway, Wnt/Ror receptor pathway, Wnt-GSK3MT pathway, Wnt-aPKC pathway, Wnt-RYK pathway, and Wnt-mTOR pathway may or may not transduce calcium-dependent cell signaling . This review will give a brief overview of Wnt/Ca2+ signal transduction pathway with reference to development, cancer, cellular response as a consequence of inflammatory challenge, and role of Wnt/Ca2+ signaling in the nervous system with reference to neurodegeneration.
The crosstalks between the non-canonical Wnt calcium signaling and canonical Wnt β-catenin signaling pathways will also be mentioned. These pathways are not autonomous, their boundaries are not stringent and there are considerable degrees of overlapping between them .
The Non-canonical Wnt Signaling Cascade
The Wnt ligand is a secreted lipid-modified glycoprotein that binds to its cell surface receptor ligand Frizzled (Fz). The lipid modifications involve covalent attachment of palmitic acid on the first cysteine residue and palmitoleic acid on the highly conserved serine residue. The palmitoylation of Wnt is required for its binding to cognate receptor Fz, initiating a signal transduction pathway; glycosylation is required for its secretion . The lipid modifications render the proteins hydrophobic; they require detergent to remain in solution in vitro [7,8]. They are chaperoned by heparin sulphate proteoglycans in vivo to prevent clustering . The Fz receptor is a seven-transmembrane (7TM) spanning protein similar to bacteriorhodopsin. The receptor has three extracellular and three intracellular loops. The extracellular N terminus contains a cysteine-rich domain (CRD) that binds to the cognate ligand. There are sites for glycosylations on the extracellular loops. The intracellular C terminus contains the PDZ (Psd-95/dics large/ZO1)-binding domain, all direct binding proteins identified so far, which physically interact with Fz in the downstream regulatory process have PDZ domain . According to the International Union of Pharmacology and other published reports, Fz receptors are G protein-coupled receptors [11–14]. The binding of Wnt ligand to cognate Fz receptor leads to a short-lived increase in the concentration of certain intracellular signaling molecules, inositol 1,4,5-triphosphate (IP3), 1,2 diacylglycerol (DAG), and Ca2+ (Fig. 1). The elevation at the level of one or other secondary messengers leads to a rapid alteration in cellular function. IP3 and DAG are derived from membrane-bound phospholipid phosphatidyl inositol 4,5-bisphosphate by the action of phospholipase C located on the plasma membrane. Phospholipase C is activated by receptor ligand interaction. IP3 diffuses through the cytosol and interacts with the calcium channels present on the membrane of endoplasmic reticulum (ER) resulting in release of calcium ions. Calcium ions along with ubiquitously expressed eukaryotic protein calmodulin activate calcium calmodulin-dependent protein kinase II (CaMKII) . DAG produced by hydrolysis of phosphatidyl inositol 4,5-bisphosphate along with released calcium from ER activates protein kinase C (PKC) . Both CaMKII and PKC activate various regulatory proteins (NFκB and CREB), which are nuclear transcription factors. Similarly, calcium ions mobilized by IP3 from ER can activate widely expressed protein phosphatase calcinurin (Cn) that can activate cytoplasmic protein nuclear factor associated with T cells (NFAT) via dephosphorylation. Activated NFAT may boost the expression of several genes in neurons, cardiac and skeletal muscle cells, and pro-inflammatory genes in lymphocytes [17,18]. Also, Wnt/Fz receptor ligand interaction may activate phosphodiesterase 6 (PDE6) in a calcium-dependent manner, leading to a decrease in cyclic guanosine monophosphate (cGMP) .
From ‘classical’ concept it may be said that Wnt1, Wnt3a, Wnt8, and Wnt8b act in the canonical Wnt signaling pathway whereas Wnt4 and Wnt5a take part in the non-canonical Wnt signaling pathway. However, later research has shown that such classification is totally subjective and it is hard to classify Wnt ligands as ‘canonical’ or ‘non-canonical’. But several publications have shown that Wnt5a takes part in the non-canonical Wnt signaling pathway. This review will focus on Wnt5a as a ‘prototype’ of Wnt/Ca2+ signal transducer.
There are 19 Wnt genes and 12 Fz receptors in vertebrates. Both receptor and ligand are highly conserved during evolution and cross reactivity between them is very common . As for example, Wnt5a, a ‘classical’ non-canonical Wnt signal transducer, activates the calcium signaling pathway in the presence of an Fz receptor. It has been shown that Wnt5a activates the calcium signaling pathway in the presence of receptor Fz2, 3, 4, 6,  and receptor Fz 5 [21,22]; however Wnt5a may activate the β-catenin-dependent pathway leading to an expression of downstream genes, if it encounters receptor Fz4 in the presence of low density lipoprotein receptor-related protein 5 (LRP5)  or receptor Fz5 .
Also, it is worth mentioning that Fz is not the only transmembrane protein with which Wnt5a can bind. Wnt5a can bind to the membrane-bound receptor tyrosine kinase (RTK) Ror1/2, and may mediate non-canonical β-catenin-independent signal transduction . Wnt5a/Ror signaling activates the Ca2+/CaMKII pathway and plays paramount roles in axonal pathfinding in mammalian brain [26,27]. Wnt5a-mediated signal transduction promotes the growth of an axonal cone via a cleavage of cytoskeleton protein spectrin by calcium-dependent non-lysosomal cysteine protease calpain . The cleavage of cytoskeleton protein filamin by calpain is positively correlated with motility of melanoma cells [29,30]. Wnt5a signals through receptor Ror2 and Vangl to establish planer cell polarity and mediates limb development . Also, the Fz receptors for Wnt ligands activating the β-catenin pathway remains coupled to LRP5/6 co-receptors and it has already been discovered that LRP5/6 co-receptors may take part in the non-canonical Wnt signaling pathway and may inhibit or cooperate in a complex way [32,33].
Following receptor–ligand interaction, how does a cell decide whether to proceed along non-canonical calcium signaling or canonical β-catenin-dependent signaling cascade? Receptor−ligand interaction causes hyperphosphorylation and activation of cytoplasm protein Dishevelled (Dvl) . Some researchers were of the opinion that it was differential phosphorylation of Dvl by upstream proteins that might be at the crossroad of functional dichotomy . But, this hypothesis has been challenged in a recent publication . According to Grumolato et al., the outcome of Wnt signal transduction depends upon specific abilities of Wnt ligands to form a complex with shared and cognate receptors in the presence of completely unrelated co-receptors . They worked on 53S and 293T cells that endogenously express Fz4, Fz5, LRP5/6, and Ror2. They found that in the presence of Wnt3a, the cells activated the canonical pathway, whereas in the presence of Wnt5a the cells activated the non-canonical pathway, with concomitant phosphorylation of co-receptors LRP5/6 and Ror2, respectively. In both cases, phosphorylation was mediated by glycogen synthase kinase (GSK). Also, in both signaling cascades Dvl was phosphorylated to similar extent, as measured by a gel shift assay. This may tempt us to discard previous hypothesis that differential phosphorylation of Dvl is the cause of the branching off the two pathways. Further, the researchers found that in vitro expression of fusion receptor containing Ror2 extracellular domain and LRP6 intracellular domain led to the activation of the Wnt/β-catenin signaling pathway by Wnt5a. Also, a fusion ligand consisting of dickkopf2's (Dkk2's) LRP5/6 binding domain with Wnt5a led to the activation of the canonical β-catenin signaling pathway. It is worth mentioning that Dkk proteins belong to a distinct category of ligands that bind to LRP6 and are potential modulators of the canonical Wnt/β-catenin signaling cascade. There are four Dkk proteins, i.e. Dkk1, 2, 3, 4 and one Dkk3 homologous protein Soggy (Sg), present in the human genome. All these secreted proteins have two CRDs at their N terminal (Sg lacks the CRDs); of all these proteins Dkk1 and Dkk4 act as Wnt signaling antagonists . Dkk2 may antagonize or potentiate the Wnt signaling pathway by interacting with LRP6 [38,39]; similar is the case of Dkk3, it may potentiate or inhibit Wnt signaling dependent upon the cell type and the presence or absence of other receptors .
From all these experimental observations, it may be prudent to conclude that Wnt5a may have the potential to activate the canonical β-catenin signaling pathway in the presence of a cognate receptor and LRP but may be mechanically inhibited to do so in vivo by cellular machinery. Although it is well known that Ror1/2 is an RTK, the researchers found that Wnt5a ligand led to phosphorylation of serine residue. This is in accordance with the previous observations made by Yamamoto et al.  who found that endogenously expressed Ror2 receptors in cultured cells underwent phosphorylation at serine/threonine residues in the presence of Wnt5a. Although Grumolato et al. did not investigate the role of casein kinase 1 (CK1) in co-receptor phosphorylation, its role is debated. According to MacDonald et al. , LRP6 phosphorylation at serine residue by GSK is followed by CKIɛ. However, according to Kani et al. , Ror2 undergoes priming phosphorylation by CKIɛ at C terminal serine/threonine domain followed by GSK-mediated phosphorylation at serine/threonine residues .
Can Wnt3a activate the non-canonical calcium signaling cascade? The answer is not known. But one may design a similar experiment under the same setup as done by Grumaloto et al; in vitro expression of a fusion receptor with LRP6 extracellular domain and Ror2 transmembrane and cytoplasmic domain in 53S and 293 T cells in presence of Wnt3a. Output of Wnt3a-fusion receptor interaction may be measured by Topflash luciferase reporter assay or by studying calcium dynamics. As we know that Wnt5a can not bind LRP in the cell system, it may be made to bind the fusion receptor as Dkk-Wnt5a fusion protein (as designed by Grumolato et al.), and the signaling output may be measured in the same way.
There is no thumb rule in biology and so is the Wnt signaling cascade. The signaling output is not only inherent to the Wnt protein but is also determined by a combination of factors including the receptor encountered by the ligand on the cell surface depending on the cell context.
Role of the Wnt/Ca2+ Signaling Pathway in Development
Wnt is a zygotic effect gene, which is expressed during very early embryonic development. It regulates cell fate in the blastula stage, orchestrates morphogenetic movement during gastrulation, and potentiates organogenesis. Also, it plays a role in the maintenance of neuronal and blood stem cells.
Early researchers made remarkable discoveries on Wnt5a signaling by their simple, yet elegant, experiments. It was found that only co-injection of Xenopus Wnt5a (XWnt5a) and human Fz5 (hFz5) mRNA on the ventral side of early Xenopus blastula caused dorsalization of Xenopus embryo in a dose-dependent manner; neither of them alone caused any developmental anomaly . Also, dorsal co-injection of XWnt5a and hFz5 mRNA did not have any effect. However, the dorsalization effect was inhibited by GSK. These results showed that hFz5 may be a receptor for Wnt5a and the downstream pathway may be necessary for secondary axis duplication.
Later it was discovered that Ca2+ are the second messengers in Wnt5a signaling, which arise from G protein-linked phosphatidyl inositol signaling [44,45]. Based on such findings, another research group studied any potential role of Ca2+/CaMKII during Xenopus development. They discovered dorsoventral asymmetry at the level of CaMKII in the early blastula stage of Xenopus embryo; elevated activity being present on the prospective ventral side. Also, co-injection of XWnt5a and rat Fz2 (rFz2) mRNA in the early blastula stage led to the activation of CaMKII on the ventral side with concomitant autophosphorylation of endogenous CaMKII. Further, they showed that overexpression of CaMKII on the dorsal side perturbed dorsal cell fate; whereas down-regulation of CaMKII on the ventral side promoted dorsal cell fate. The researchers concluded that the Wnt/Ca2+ signaling pathway promotes ventral cell fate during early embryogenesis . Gastrulation in animals is characterized by extensive cell movements; in Xenopus the convergent extension movement is most well known. During gastrulation the Wnt/β-catenin pathway promotes convergent extension with the TGF-β signaling pathway . We know that cadherin, one of the downstream components of the Wnt/β-catenin pathway, promotes cell adhesion by homophilic and heterophilic interaction. However, β-catenin positively regulates convergent extension because of its effect on gene expression. The downstream effectors of the Wnt/Ca2+ signaling pathway such as CaMKII and PKC blocks convergent extension by phosphorylating Dvl and Lef at critical residues, which switches off the Wnt/ β-catenin pathway.
Research was also focused on prospective roles of the non-canonical Wnt/Ca2+ signaling cascade during gastrulation in zebrafish . However, Wnt5a ligand is lowly expressed in zebrafish. Wnt5b is better characterized. Convergent extension movement in zebrafish requires Wnt/Ca2+ signaling but both Fz2 and receptor RYK. The researchers found that Wnt5b/RYK receptor−ligand interaction gives instructive induction to Wnt5b/Fz2 receptor−ligand expressing cells to proceed according to their prospective fate. The downstream signaling via the Wnt5b/RYK pathway was not characterized but the signaling pathway did not involve Dvl. Also, RYK knockdown blocked calcium release that perturbed convergent extension movement during gastrulation, thus leading to serious developmental defects . During gastrulation the Wnt/Ca2+ signaling cascade is positively regulated by the regulator of G protein signaling protein (RGS). This protein phenocopies the intrinsic GTPase activity of GPCR. Although RGS is necessary to suppress calcium dynamics, knocking down of either RGS or Wnt5b caused a similar somite patterning defect. Total blocking of calcium signaling induced developmental abnormalities such as gut coiling defect, edema, tumor formation in the liver, heart, and kidney, and somite during development . A recent study has demonstrated that cadmium, a potent neurotoxic, teratogenic agent induces omphalocele in chick embryo with concomitant down-regulation of Wnt11, PKC, and CaMKII transcripts . The down-regulation was observed only within 1 h of cadmium treatment with no significant change in post-1 h treatment. It is worth mentioning that omphalocele is a ventral body wall defect that causes live birth in human .
So, it may be concluded that since early vertebrate development, Wnt5a exhibits distinct signaling property. The Wnt/Ca2+ signaling pathway plays an immense role in the development of dorsoventral polarity and convergent extension movement, and various organ formations. However, the pathway needs to be expressed only at the basal level and cellular machinery has devised an intricate system to fine-tune the expression of the relevant genes since early development. Also, in order to draw any firm conclusion regarding Wnt's role during development, one must take an account of, early and late response, cell type, and development time point.
Wnt/Ca2+ Signaling Pathway in Cancer
The discovery of int gene, capable of malignant transformation of mammary tissues, prompted scientists to quickly conclude that Wnt is an oncogene. However, later it was found that some Wnts, including Wnt5a, were not capable of transforming C57MG cell lines [52,53], excluding the universal classification of Wnt gene as an oncogene. There are numerous paradoxical evidences that show functional dichotomy of Wnt5a; it may act as a proto-oncogene or a tumor suppressor gene . Why is it so? We need to recapitulate the previous statement. It depends upon cell type and receptor availability.
Wnt5a acts as a tumor suppressor gene in neuroblastoma [55,56], esophageal squamous cell carcinoma , acute myeloid lymphoma , acute lymphoblastic lymphoma , breast cancer , thyroid carcinoma , and colon carcinoma  but as proto-oncogene in prostate cancer [63,64], melanoma , breast cancer , and pancreatic cancer [67,68]. However, atypical expression of Wnt5a does not imply loss of function, gain of function, gene rearrangement, or amplification . The gene has CpG island in 5′ untranslated region, which undergoes differential methylation compared with basal level and thus executing an epigenetic regulation on gene expression [57–59,70,71]. In the following section the paradoxical role of Wnt5a as a tumor suppressor and proto-oncogene will be discussed with reference to calcium signaling.
In vitro studies have demonstrated that the Wnt/β-catenin signaling pathway is up-regulated in many cancers. Often (but not always) prognosis of cancer in these cases is associated with simultaneous down-regulation of Wnt/Ca2+ signaling. Wnt5a acts as a tumor suppressor gene in these cell populations. Blanc et al. reported that Wnt5a signaling via PKC was down-regulated in neuroblastoma and cultured metastatic neuroblasts . Their research showed that β-catenin was indeed expressed in those cells but the interaction between Wnt/β-catenin and Wnt calcium signaling pathway was not elucidated. Several other reports are more explicit to suggest that Wnt5a suppresses cell growth by phosphorylating β-catenin, thus acting as a tumor suppressor. This happens in thyroid and colon carcinoma [61,62]. Wnt5a executes its tumor suppressor effect in thyroid carcinoma by down-regulating c-myc, a well-known proto-oncogene activated via the Wnt/β-catenin pathway . Transfection of Wnt5a mRNA in thyroid carcinoma cell line reduced motility and invasiveness with activation of the Ca2+/CaMKII pathway. Result showed that CaMKII phosphorylated β-catenin independently and downstream of GSK3β making the protein susceptible to degradation. Colon carcinoma may be caused by loss of function in adenosis polyposis coli (APC) gene in intestinal cells that leads to stabilization of β-catenin and subsequent expression of downstream genes that may promote cancer. MacLeod et al. reported that constitutively active β-catenin protein in colon cancer cell lines can be degraded by exposing colon cancer cell lines to extracellular Ca2+ . They found that extracellular Ca2+ activated calcium-sensitive receptors (CaSRs) in intestinal epithelial cells, which in turn led to transcription and translation of Wnt5a. Once secreted, Wnt5a exhibited autocrine signaling. It activated the Wnt5a/Ror2 signaling pathway, which subsequently led to a degradation of β-catenin via ubiquitin ligase Siah2. These findings have immense therapeutic importance elucidating the role of calcium therapy to prevent prognosis of cancer, where the β-catenin pathway is active. It is well known that the intestinal and colon epithelial cells rest on the basement membrane underlined by myofibroblasts, exhibiting a mesenchyme-like property. These cells have CaSRs on their plasma membrane. In another study, Pacheco and MacLeod deciphered that extracellular Ca2+ led to the activation of CaSR, culminating in the secretion of Wnt5a protein. CaSR activation also led to an increased expression of Ror2 receptors in intestinal and colonic epithelial cells. Secreted Wnt5a protein exhibited paracrine (and autocrine) signaling to active Wnt5a/Ror singling leading to transcription and translation of genes responsible for maintenance of intestinal and colonic stem cells . The therapeutic stronghold of calcium therapy was also deciphered in the work of Gwak et al. . In a cell-based screen of compounds to identify the modulators of the β-catenin pathway, the researchers found calcium ionophore A23187, which activated PKC, a downstream component of the Wnt/Ca2+ pathway. PKC was able to phosphorylate β-catenin independent of GSK3β, thus promoting its degradation. However, the most intriguing evidence of Wnt5a's role as a tumor suppressor comes from a study of the murine model of basal cell carcinoma (BCC). In a recent study, researchers created a patched gene knock-out mutant mouse exhibiting BCC, one of the most common skin cancers in humans . It should be mentioned that the patched gene knock-out mouse had a persistently active hedgehog signaling pathway. Expression of Wnt5a in late-stage tumors, which were subsequently undergoing regression, was observed in some patients suffering from BCC. Wnt5a was signaling via the CaMKII pathway. This suggests that activation of the Wnt5a signaling cascade may be important for tumor regression and increased expression of Wnt5a in late-stage tumors served to inhibit hedgehog signaling and reversed cancer growth.
In some cells, Wnt5a is a proto-oncogene whose expression is required since early embryonic development . A knock-out mouse without the function of Wnt5a gene exhibits numerous developmental abnormalities. Proto-oncogene Wnt5a not only transforms cell lines in vitro, but is also required for cell growth and viability. It has been reported that adenovirus-mediated expression of Wnt5a in endothelial cells kept them viable under serum-deprived stressful condition . Under similar condition Wnt5a played an anti-apoptotic role in primary dermal fibroblasts . This was maintained by PKA-mediated phosphorylation of GSK3β and cyclic AMP response element binding protein (CREB). Inactivation of GSK3β by phosphorylation promoted nuclear translocation of β-catenin, which along with CREB transcribed the cell survival promoting genes. As an oncogene Wnt5a can metastasize several cells lines. For example, it has been reported that Wnt5a was capable of inducing invasiveness of breast cancer cell lines, acting in parallel with the Wnt/β-catenin pathway. The role of calcium signaling was not characterized but the expression of TNF-α and matrix metalloproteases-7 (MMP-7) regulated invasiveness . Wnt5a signaling was up-regulated in melanoma cells that directly increased cell motility, invasiveness, and cell morphology via reorganization actin cytoskeleton. All these properties were mediated by PKC showing direct involvement of calcium signaling [66,78,79]. Another research group has shown that in prostate cancer cell line the Wnt5a signaling cascade is up-regulated due to hypomethylation of the Wnt5a promoter region . The up-regulated Wnt expression made the prostate cancer cell lines highly motile, invasive; the cells exhibited different morphology from the wild-type ones. All these properties were reported to be regulated in a Ca2+/CaMKII-dependent manner . In all the above examples Wnt5a exhibited its hallmark manifestation as an oncogene.
Role of the Wnt/Ca2+ Signaling Pathway in Arousing Inflammatory Response
The fact that Wnt signaling molecules can mediate inflammatory response was first discovered in Drosophila when it was deciphered that WntD, a Drosophila homolog of Wnt protein, can evoke inflammatory response by binding with cognate receptor Toll . The receptor−ligand interaction activated Dorsal, the Drosophila homolog of NFκB, which in turn activated cytokine genes. Subsequently, Wnt5a signaling mediated by cognate Fz receptor 5 has been implicated in many inflammatory conditions such as rheumatoid arthritis , psoriasis vulgaris , sepsis , and endothelial function [83,84]. It should be borne in mind that endothelial dysfunction characterized by macrophage anomalies is a hallmark of atherosclerosis and is also manifested in patients suffering from diabetes mellitus, hypercholesteromia, hypertension, and coronary artery diseases.
Sen et al. showed that the Wnt5a/Fz5 signaling pathway was persistently active in the fibroblast-like synoviocytes (FLS) of rheumatoid patients even in the absence of inflammatory stimulus, when the cells are grown in culture in the absence of inflammatory challenge . The downstream signal mediators were not deciphered; however, the cells exhibited a high level of pro-inflammatory like interleukin 6 (IL6), IL15, and receptor activator for NFκB (RANKL). It was found that blockage of Wnt5a/Fz5 signaling by cell transfection with Wnt5a antisense RNA or dominant negative Wnt5a expression vector down-regulated IL6 and IL15 at both mRNA and protein levels. Similar effects were observed when RANKL was down-regulated or the Fz receptor was blocked with an antagonist. This showed that the Wnt5a/Fz5 cascade was mediating inflammatory response by secreting IL6 and IL15 with an implicating role of NFκB. No direct role of calcium signaling was implicated. In another work, Blumenthal et al. demonstrated that Wnt5a signaling was at the hub of inflammatory response making a bridge between innate and adaptive immunity . They showed that Wnt5a has both autocrine and paracrine signaling properties. Researchers demonstrated that in response to pathogenic challenge, macrophages expressed Wnt5a and cytokines via the toll like receptor (TLR) signaling pathway. Also, the macrophages and T lymphocytes up-regulated the expression of Fz5. Further, IL12 expressed in macrophages induced T cells to produce interferon (IFN)-γ; with an implicating role of Wnt5a at the epicenter of inflammation. Pereira et al. studied the gene expression profile of cultured macrophages from patients suffering from chronic sepsis, an inflammatory disease . They showed that both Wnt/Fz5 signaling pathway mediated by CaMKII and TLR signaling pathway were indeed operating in the macrophages of patients suffering from sepsis, but the further downstream mediators were not deciphered. They found that inflammatory response was exacerbated by pathogenic challenge of lipopolysaccharide (LPS) and IFN-γ; but down-regulated by anti-inflammatory cytokine IL10 and APC. A high level of Wnt5a was found in both sera and bone marrow of patients. Similarly, Cheng et al. deciphered that growth, migration, and proliferation of human endothelial cells were regulated by Wnt5a in a CaMKII-dependent way . The researchers were able to show that endothelial cells may be made to abrogate the above properties by blocking the CaMKII pathway. Also, a challenge with inflammatory cytokines, such as IL1 and IL8, is to up-regulate the Wnt5a mRNA expression. As a further extrapolation of the above study Kim et al. studied the physiological response of human endothelial cells in the presence of Wnt5a protein . They observed a robust up-regulation in the expression of COX2 gene with concomitant rise at the level of other inflammatory cytokines such as IL3, IL5, IL6, and IL8. The signal was transmitted via the Ca2+/PKC route, as calcium ionophores boosted endothelial inflammatory response, whereas PKC inhibitors or calcium chelators blocked the output. The signal was NFκB-mediated expression, as they observed nuclear binding of Rel A by immunofluorescence microscopy in the presence of inflammatory challenge. Further, their results showed that another signaling pathway mediated by TNF-α was simultaneously active in the endothelial cells with limited degree of overlap with the Wnt5a/Ca2+ signaling pathway.
In light of the above findings it may be reasonable to conclude that undoubtedly the non-canonical Wnt signaling pathway mediates inflammatory response. To summarize, it may be suggested that inflammatory response in macrophages is mediated by a dual-signaling complex cascade consisting of Wnt calcium signaling and TLR signaling pathways (Fig. 2). It may be prudent to conclude that TLR signaling pathway that leads to the expression of Wnt and cytokine genes is the primary immune response, but such conclusion needs further investigation. Once Wnt is expressed, it acts as a positive feedback loop, boosting its expression by the Wnt/Ca2+ signaling pathway via CaMKII and PKC, which culminates the expression of downstream cytokine genes via transcription factor NFκB. It is known that pro-inflammatory cytokine, such as IL12, is produced by macrophage-stimulated T cells , which also express inflammatory cytokine IFN-γ via the Wnt/Fz signaling pathway, thus further promoting inflammation. The TLR signaling pathway and Wnt5a/Fz5-mediated downstream signaling cascade operating in macrophages have been dissected; however, their counterparts present on T cells are yet to be thoroughly deciphered. Also from recent research, it may be reasonably concluded that Wnt/Ca2+ signaling pathway-mediated inflammatory response via NFκB indeed operates in the endothelial cells, but whether the TLR signaling pathway is active or not needs further investigation.
From all these observations, it is may be concluded that Wnt5a may be regarded as an inflammatory marker, which is expressed above the basal level in pro-inflammatory conditions .
However, in a recent study, in contrast to the above pro-inflammatory role of the non-canonical Wnt signaling cascade, Kelly et al. have demonstrated a novel anti-inflammatory pathway mediated by Wnt5a-mediated signal transduction . As I have already mentioned before, the intestinal and colonic epithelial cells rest on the basement membrane underlined by myofibroblasts, surrounded by macrophages. They also demonstrated that in response to inflammatory challenges, the macrophages and myofibroblasts of intestinal subepithelial produced inflammatory cytokine TNF-α via the activation of TLR4 signaling pathway. However, extracellular high concentration of calcium ion titrated the inflammatory effect. Extracellular calcium ions activated the CaSR signaling cascade in the subepithelial region leading to the production of Wnt5a; once expressed, Wnt5a not only reduced the expression of TNF-α via inhibition of the transcription factor NFκB but also acted as a paracrine signal transducer. It activated the Wnt5a/Ror2 signaling cascade in the intestinal epithelia, thus down-regulating the expression of membrane-bound receptor TNFR1, thereby reducing inflammation of the transepithelial region. These observations further reinforce the benefit of high dietary calcium as a chemoprotective agent for colon cancer. However, cell- and tissue-specific anti-inflammatory signaling cascade mediated by Wnt5a has so far been unreported; its molecular network needs further characterization.
Role of the Wnt/Ca2+ Signaling Cascade in the Nervous System with Reference to Neurodegeneration
Neuronal system is a dynamic system. Neurons are born, eliminated, attracted, and repelled during the course of development. Neurons are not static, for example, they undergo changes in shape, the dendrites undergo morphogenesis, the length of the axons increases or decreases, and their tips are fine-tuned during the course of development. There was no doubt that all the above phenomena were guided by chemical cues, but their identity and molecular mechanism of action remained reclusive for a long time. Now it is well known that along with other morphogens, Wnt does mediate neuronal circuit formation often exhibiting antagonism between them [88–90]. Calcium ions, the predominant second messenger in the non-canonical signaling pathway is required for axon pathfinding, and the required calcium ion concentration is achieved not only by released calcium ions from ER in response to the upstream signal but also by the entry of calcium ions from extracellular space.
Early reports on Xenopus embryonic spinal cord neuron showed that the growth of neurite was dependent on calcium-mediated expression of CaMKII in response to the chemotropic gradient . Loss of any of the above signaling molecules, such as destruction of chemotropic gradient or intracellular calcium concentration, blocking of CaMKII, retarded nerve growth. Also, IP3 channels were concentrated at the tips of growing neurons; blocking IP3 channels by any means caused retraction growth cones .
Another research group deciphered that the growth of mammalian corticospinal nerve in the anteroposterior direction was mediated by the Wnt4 ligand and the cognate Fz receptor . Although none of the mentioned examples showed any firm evidence of operating Wnt calcium signaling cascade in early neurogenesis, there were some more experimental evidences where the researchers discovered that various components of the Wnt/Ca2+ signaling pathway were operating, but upstream or downstream the signaling cascade often remained elusive [94–98]. However, from experimental evidence it may be justified to conclude that Wnt/Ca2+ signaling does operate in the nervous system in which both PKC and CaMKII take part . Besides archetypal Wnt–Fz interaction, abundant data are available emphasizing the role of RYK in axon repulsion [100–103]. A recent study has demonstrated that Wnt5a commands over both growth and repulsion of neuritis to their ultimate pruning . However, growth and repulsion are regulated via different receptor−ligand interactions with some degree of overlap. While growth was regulated by Wnt5a and RYK interaction, it was Wnt5a/Fz–RYK interaction that caused axon repulsion . The required calcium ion concentration for axon growth was attained by orchestered mobilization of calcium from ER and transient receptor potential (TRP) channel-mediated extracellular entry, and neurite retraction was mediated only by TRP channel calcium entry.
In addition to neuronal pathfinding, Wnt/Ca2+ and Wnt/β-catenin pathways partake in pre- and post-synaptic receptor localization, mediating a balance between excitatory and inhibitory neurotransmission. Canonical ligand Wnt7a induces localization of α7 nicotinic acetylcholine receptor along with dissociated APC from β-catenin destruction complex on the pre-synaptic terminus ; Wnt5a regulates assemblage and recycling of inhibitory GABA receptors via a CaMKII-dependent mechanism . Some research groups have focused on the roles of Wnt/Ca2+ signaling pathway in the nervous system in the light of neurodegeneration. Alzheimer's disease (AD) is a severe neurodegenerative disease bearing a hallmark expression of extracellular amyloid β neurotoxic plaques and intracellular hyperphosphorylated Tau proteins present as neurofibrillary tangles. It is known that the Wnt/β-catenin pathway is perpetually switched off in AD. This is because toxic amyloid β protein binds to Fz receptor protein and blocks the downstream signaling cascade . Several research groups have deciphered that activation of various downstream proteins of the non-canonical Wnt signaling cascade may mitigate AD.
Hyperphosphorylation of Tau is mediated by GSK3β. Therapeutic intervention by Wnt calcium signaling via PKC-mediated inactivation of GSK3β has been postulated, as it has been already documented that Ser9 phosphorylation of GSK3β can activate the Wnt/β-catenin pathway, subsequently activating target genes that have neuroprotective roles . Further, β-amyloid plaques are neurotoxic, which can block excitatory synaptic transmission . Research has shown that expression of Wnt5a can mitigate the cytotoxicity of amyloid proteins. Another research group has shown that PKC may promote α-secretase activity that causes non-amyloidogenic deposition of amyloid precursor protein (APP) in the extracellular region . Similar effects have been deciphered by overexpression of Dvl .
So, for the clarity of understanding let us summarize the role of Wnt/Ca2+ signaling pathway in AD. (i) Wnt5a can ameliorate the cytotoxic effects of Aβ protein which blocks synaptic transmission. (ii) Overexpression of Dvl or PKC may ensure neuroprotection at various levels. They cause inactivation of GSK3β. Inactivated GSK3β reduces phosphorylation of tau, and leads to the expression of β-catenin regulated neuroprotective genes. (iii) Both Dvl and PKC reduce deposition of Aβ by promoting α-secretase activity.
Understanding the intricate molecular mechanism of action of the Wnt signaling cascade is of immense therapeutic interest in amelioration of various diseases such as cancer, neurodegeneration, and inflammatory disorders. There is no doubt that research in the past two decades has made considerable progresses on these aspects of cell signaling; however, the following aspects of non-canonical Wnt/calcium cascade need further attention and investigation. It may be concluded that focused research concentrated on the above aspects will give us a better understanding of Wnt cell signaling. Some scientists are of the opinion that targeting Wnt signaling pathway for gene therapy, however, any such approach should be taken with very much caution and utmost precaution. Wnt signaling cascades (both canonical and non-canonical) are expressed very early in development, which maintain close links with other signaling cascades like hedgehog, Notch, and TGF-β. Any kind of molecular or genetic misadventure with Wnt cascades may screw up other signaling networks.
Wnt signaling pathway is needed to be studied in the context of receptor expression on the cell types. It can not be studied in isolation. Unfortunately, many research articles about Wnt signaling cascade mention no details about receptor expression. But there is always a strong possibility that ‘misexpression’ of Wnt receptor is causing up- or down-regulation of downstream proteins compared with the basal level, leading to disease.
Do the pro-inflammatory cytokines such as TNF-α, IL1, and IL6 mediate inflammatory response via any positive feedback loop once they have been expressed in inflamed cells? Is it via the TLR signaling pathway?
We need a thorough understanding about the molecular basis of regulation of GSK3β. GSK3β is a paradoxical protein with pro- and anti-apoptotic roles. It is a facilitator of mitochondrial extrinsic apoptotic pathway and an inhibitor of TNF-α-mediated intrinsic pathway . It is phosphorylated by upstream regulators of β-catenin pathway  that may promote cancer. Similarly, it may be phosphorylated by PKC (at serine 9 residue) leading to down-regulation of Tau protein and ameliorates β amyloid induced neurotoxicity [114,115]. The mechanism of neuroprotection ensured by GSK3β is not well characterized but it can always be via the expression of neuroprotective genes . Also, chemotherapeutic drug lithium has been shown to exhibit neuroprotection via phosphorylation of GSK3β at Ser9/Ser21 residue, though its clinical implication is an open question . How does inactivation of GSK3β cause cancer and ensure neuroprotection? Is it solely via tissue-specific phosphorylation or do some other mechanisms exist?
Another seemingly intriguing aspect is crosstalk among canonical and non-canonical signaling pathway-mediated PKC, GSK3β, and β-catenin. As I have stated above, GSK3β may be phosphorylated by PKC culminating in the expression of neuroprotective genes (via stabilization and nuclear translocation of β-catenin). Also, PKCα phosphorylates Ser33, Ser37/Ser45 residues of β catenin via calcium signaling that may promote degradation of β-catenin . We know that PKC has several different isoforms . Do they exhibit cell-specific action?
Professor S. Raha is acknowledged for constructive criticism and critical reading of the manuscript. All lab members are acknowledged for stimulating discussions.
This work was supported by postdoctoral research fellowship awarded to the author by Saha Institute of Nuclear Physics, Government of India.