WNT Signaling

WNT signaling pathways play essential roles in cellular proliferation, differentiation and cell migration during embryonic development. The importance of WNT signaling is indicated by conservation of its molecular components across organisms ranging from nematodes to humans. WNT pathways are classified into canonical WNT/CTNNB or non-canonical (β-catenin-independent) pathways. Canonical WNT/β- catenin signaling is the most studied, and is mediated by nuclear translocation of its central effector CTNNB and Non-canonical WNT signaling occurs independently of CTNNB–TCF/LEF and is stimulated by WNT ligands that bind to a receptor complex of FZD, ROR1/2 or RYK.WNT pathway is implicated in a variety of cancers (Ref.1 and 2).

WNT ligands signal via seven transmembrane spanning receptors of the Frizzled family (eleven members in the human genome) together with the recently identified LRP5 (Lipoprotein Receptor-related Protein-5) and LRP6 (Lipoprotein Receptor-related Protein-5) co receptors. Structurally the Frizzled receptors have an Extracellular WNT-binding domain, seven Transmembrane-spanning regions and an Intracellular C-terminal tail. Secondary structure predictions on the Frizzled sequence and involvement of proteins of the WNT signaling pathway with proteins that are characteristic for the pathways of GPCRs (G-Protein Coupled Receptors), labelled Frizzled as a GPCR family member. WNT signals are transduced through at least three distinct intracellular pathways, including the canonical WNT/ CTNN-Beta (Catenin-Beta) signaling pathway and the non-canonical WNT/Ca2+ (Calcium) pathway, and WNT/PCP (Planar Cell Polarity) pathway (Ref.1 and 3).

The WNT/ CTNN-Beta pathway is the best understood WNT signaling pathway, and is highly conserved during evolution. The WNT/ CTNN-Beta pathway is activated by WNT1, WNT3, WNT3a, WNT7a, and WNT8, and is involved in transformation. In the absence of WNT signaling, Beta-CTNN is associated with a cytoplasmic complex containing CK1a (Casein Kinase-1- Alpha), GSK3Beta (Glycogen Synthase Kinase-3-Beta), AXIN (Axis Inhibitor) and the APC (Adenomatous Polyposis Coli) protein. This promotes phosphorylation of Beta-CTNN and its interaction with Beta-TRCP (Beta-Transducin Repeat-Containing Protein), leading to the ubiquitination of Beta-CTNN and its degradation by the proteosome. In the presence of WNT signaling, WNT binds to its receptor, Frizzled which leads to activation of the DVL (Dishevelled) protein. The activated Dishevelled protein enhances the phosphorylation of GSK3B, which inhibits the ability of GSK3B leading to accumulation of free and unphosphorylated Beta-CTNN in the cytoplasm, which then translocate to the nucleus. In the nucleus prior to WNT signaling, LEF (Lymphoid-Enhancing Factor) and TCF (T-Cell Factor) homolog bind to DNA with sequence specificity in promoter/enhancer regions of target genes, and along with Groucho and CTBP (COOH-terminal Binding Protein), often function to repress gene expression. Elevation of Beta-CTNN levels by WNT signaling leads to binding of Beta-CTNN to TCF/LEF, promoting changes in the transcriptional machinery that lead to activation of several target genes. The shifting of proteins from the Cadherin-bound pool to the cytoplasmic pool can increase the amount of available free CTNN-Beta for the activation of target genes. Transcriptional activation is mediated by the interaction of CTNN-Beta with the histone acetyl transferase CBP (CREB-Binding Protein), the chromatin-remodeling SWI/SNF complex and BCL9 bound to PYGO (Pygopus) and BRG1. Chibby interacts directly with the C-terminal region of CTNN-Beta and inhibits CTNN-Beta-mediated transcriptional activation by competing with LEF1 to bind to CTNN-Beta (Ref.1, 4, 5 and 6).

Several genes have now been identified as the target of CTNN-Beta/TCF transcriptional regulation. These include MMP7 (Matrix Metalloproteinase-7), UPAR (Urokinase-type Plasminogen Activator Receptor), CD44, c-Myc, c-Jun, FRA1 (Fos-Related Antigen-1), CCND1 (Cyclin-D1), PPARD (Peroxisome Proliferative Activated Receptor-Delta), TCF1 (Transcription Factor-1), Fibronectin, Gastrin and COX2 (Cyclooxygenase-2). In addition, the complexes of TCF/LEF and CTNN-Beta may cooperate with factors activated by other signaling pathways to alter cellular remodeling processes. Another target of CTNN-Beta is Vimentin, a protein involved in cell migration. WNT signaling can prevent apoptosis by up-regulating anti-apoptotic proteins, such as the Caspase inhibitor, Survivin, and stimulate Angiogenesis via up-regulation of VEGF (Vascular Endothelial Growth Factor). Several proteases capable of degrading extracellular matrix, such as Matrilysin/MMP7 and MMP26, as well as cell adhesion molecules such as CD44 and NRCAM (Neuronal Cell Adhesion Molecule) are WNT targets that could aid the tumour cells in invasion and metastasis.CLDN1 (Claudin-1) is also involved in the CTNN-Beta -TCF/LEF signaling pathway, and increased expression of CLDN1 may have some role in colorectal tumorigenesis. The increased expression of Beta-TRCP and the Ubiquitin Conjugating enzyme, both involved in degradation of CTNN-Beta, could act as a negative feedback loop in WNT signaling. Recent studies have identified four families of inhibitors of the WNT signaling pathway: FRP (Frizzled-Related Protein), CER (Cerberus), WIF1 (WNT-Inhibitory Factor-1), and DKK1 (Dickkopf-1) (Ref.4, 6, 7, 8, 9, 10, 11 and 12).

The non-canonical WNT signaling pathway, also termed the atypical WNT-Frizzled signaling pathway, has two intracellular signaling cascades that consist of the WNT/ Ca2+ pathway and the WNT/PCP pathway. In the WNT/ Ca2+ pathway, WNT protein consisting primarily of WNT1, WNT5a, and WNT11, binds to Frizzled transmembrane receptors on the cell surface resulting in several cellular processes that involve stimulation of Heterotrimeric G-proteins, which further activates PLC (Phospholipase-C). PLC lead to increased intracellular Ca2+ release, decreased cGMP (cyclic Guanosine Monophosphate) levels, and activation of the two kinases CAMKII (Ca2+- Calmodulin-dependent Protein Kinase-II) or CALN (Calcineurin) and PKC (Protein Kinase-C). These processes can stimulate nuclear factor NFAT and other transcription factors like CREB (cAMP Response Element-Binding Protein-1). Thus, the WNT/Ca2+pathway is most likely a G-protein dependent signaling pathway. In the WNT/PCP pathway, WNT proteins bind to Frizzled transmembrane receptors on the cell surface followed by activating RHO/RAC small GTPase and JNK (Jun N-terminal Kinase) via DVL to assist in the subsequent regulation of cytoskeletal organization and gene expression. DVL is connected via DAAM1 to downstream effector RHO and ROCK (Rho-Associated Kinase). RAC is directly activated by DVL, which further activates JNK by activating MAP3Ks (Mitogen-Activated Protein Kinase Kinase Kinase) and MAP2Ks (Mitogen-Activated Protein Kinase Kinase) respectively. The product of the WNT target gene NKD (Naked) was recently identified as an antagonist for WNT signaling that binds to DVL and blocks CTNN-Beta but stimulates the JNK pathway. WNT5 also signals via the FZD2 receptor and FYN activates STAT3 transcription leading to EMT (epithelial-mesenchymal transition) in cancer cells (Ref.1, 5 and 6).

The WNT-Frizzled signaling pathway plays an important role in the biology of the development of the nervous system. In the nervous system, WNT proteins are required during development for early patterning by acting as posteriorising signals, for neural crest cell induction, neural precursor, cell proliferation, and neurogenesis. Dysfunction of the WNT-Frizzled pathway can lead to neurodegenerative disorders, such as Alzheimer’s disease and heart failure. Through further identification and targeting of the critical elements that shape and control the WNT-Frizzled signaling pathway, a greater understanding of the biological potential of WNT-Frizzled signaling pathway can emerge for the development of new therapeutic options against neurodegenerative and vascular diseases (Ref.1, 13 and 14).