Roles of Non-Canonical Wnt Signalling Pathways in Bone Biology

Abstract

The Wnt signalling pathway is one of the central signalling pathways in bone development, homeostasis and regulation of bone mineral density. It consists of numerous Wnt ligands, receptors and co-receptors, which ensure tight spatiotemporal regulation of Wnt signalling pathway activity and thus tight regulation of bone tissue homeostasis. This enables maintenance of optimal mineral density, tissue healing and adaptation to changes in bone loading. While the role of the canonical/β-catenin Wnt signalling pathway in bone homeostasis is relatively well researched, Wnt ligands can also activate several non-canonical, β-catenin independent signalling pathways with important effects on bone tissue. In this review, we will provide a thorough overview of the current knowledge on different non-canonical Wnt signalling pathways involved in bone biology, focusing especially on the pathways that affect bone cell differentiation, maturation and function, processes involved in bone tissue structure regulation. We will describe the role of the two most known non-canonical pathways (Wnt/planar cell polarity pathways and Wnt/Ca²⁺ pathway), as well as other signalling pathways with a strong role in bone biology that communicate with the Wnt signalling pathway through non-canonical Wnt signalling. Our goal is to bring additional attention to these still not well researched but important pathways in the regulation of bone biology in the hope of prompting additional research in the area of non-canonical Wnt signalling pathways.

Keywords: bone mineral density; non-canonical Wnt signalling pathway; osteogenesis; signalling crosstalk.

Scheme 1

Schematic representation of (a) the inactive Wnt signalling pathway, (b) activated canonical Wnt signalling pathway and (c) non-canonical Wnt/Ca²⁺ signalling pathway. (a) In the absence of a Wnt ligand, the canonical Wnt signalling pathway is inactive, and β-catenin is degraded by a multiprotein complex termed axin degradosome. Degradosome is composed of scaffold proteins adenomatous polyposis coli (APC) and axin and kinases glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). The two kinases phosphorylate β-catenin and release it into the cytosol, where it is ubiquitinated and degraded by a proteasome complex. (b) Canonical Wnt signal transduction is initiated by the binding of Wnt ligand (WNT) to membrane frizzled (Fzd) receptor and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptor. Fzd and LRP5/6 undergo dimerization and conformational changes, which recruit dishevelled (DVL) and degradosome to the membrane and trigger the assembly of a multiprotein complex termed signalosome. Inhibition of kinases releases β-catenin from the complex and enables its accumulation in the cytosol and translocation to the nucleus. In the nucleus, β-catenin incorporates into a multiprotein complex termed enhanceosome, composed of Pygopus (PYGO), ChiLs complex, adaptor protein B-cell lymphoma 9 (BCL9) and BAF (Brg/Brahma-associated factors) chromatin-remodelling complex. The main proteins of enhanceosome are transcriptional co-repressors TLE (transducin-like enhancer of split) and T cell factor (TCF) family of DNA-bound transcription factors, which mediate transcription of β-catenin dependent genes through changes in histone acetylation and chromatin condensation. (c) Non-canonical Wnt signalling is activated by binding a non-canonical Wnt ligand to Fzd receptor and Receptor tyrosine kinase-like orphan receptor 2 (ROR2). Receptor dimerization recruits DVL to the membrane and activates heterotrimeric G-proteins, which in turn activate phospholipase C (PLC). The PLC cleaves the membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and 1,2 diacylglycerol (DAG). IP3 induces the release of Ca²⁺ from intracellular calcium stores and activation of calcium-sensitive enzymes, such as protein kinase C (PKC), calmodulin-dependent protein kinase II (CaMKII) and calcineurin. These activated proteins, in turn, activate several transcription factors, such as nuclear factor κB (NF-κB), cAMP-responsive element-binding protein (CREB) and nuclear factor associated with T cells (NFAT).

Scheme 2

Schematic representation of Wnt/planar cell polarity (PCP) establishment, core components and signal transduction pathway. (a) Cell polarity is established in an epithelium based on the gradient of certain signalling molecules, such as Wnt5a ligands. It is mediated by two protein complexes that form on the opposite cell sites, and inter- and intracellular communication between these complexes provides the cells with a planar orientation axis pattern that can extend through the whole tissue. (b) The first complex is formed by a transmembrane frizzled receptor (Fzd) and cadherin EGF LAG seven-pass G-Type receptor (CELSR), which enable intercellular communication and cell–cell connection, and intracellular disheveled (DVL) and ankyrin repeat domain 6 (ANKRD6) proteins, which mediate intracellular signals. Similarly, the second complex is composed of transmembrane CELSR and Vangl planar cell polarity protein (VANGL) and the intracellular Prickle. The intracellular components are essential for negative feedback loops within each cell (they antagonise each other) and for activating signal transduction pathways. (c) Following Wnt ligand binding to Fzd receptor and ROR2 co-receptor, DVL is recruited to the membrane, which activates downstream signalling transductions. The activation signal is then transmitted to the adaptor protein dishevelled associated activator of morphogenesis (DAAM1-2) and small G proteins, such as Rac1, RhoA and Cdc42 as well as Rho-associated coiled kinase (ROCK1/2). The activation of GTPases triggers cytoskeleton rearrangements and activates transcriptions factors, such as c-jun NH2-terminal kinase (JNK) and activator protein1 (AP-1).

Scheme 3

Schematic representation of cross-communication of non-canonical Wnt signalling pathways observed in bone cells. (a) Non-canonical Wnt signalling inhibits the RANK-RANKL signalling pathway through the inhibition of the formation of the TRAF6-TAB2-TAK1 complex by sequestering TAK1 to the Wnt-induced TAB2-TAK1-NLK complex. This complex activates NLK kinase, which inhibits the TCF/LEF protein family through phosphorylation. (b) The non-canonical Wnt signalling pathway interacts with mTOR in osteoblasts through two mechanisms. mTORC1 complex is activated through LRP6/Fzd-PI3K-AKT pathway and results in increased activity of S6 kinase 1 (S6K1) and increased protein synthesis. On the other hand, mTORC2 activation is mediated through Rho-family small GTPase RAC1 and leads to the activation of AKT and other signal transduction pathways, which results in changes in glucose and lipid energy metabolism, cell survival and cytoskeleton reorganisation. Both processes are required for adequate cell growth and proliferation. (c) The Hippo signalling pathway is activated through cell–cell attachment and cell adhesion. When active, it activates MST1/2 kinases and LATS1/2 kinases, which inhibit YAP/TAZ activity by inducing its proteosomal degradation. Non-canonical Wnt signalling through ROR2/Gα_12/13 G protein/RhoA kinase inhibits YAP/TAZ inhibitor LATS1/2. This releases YAP/TAZ from degradation and enables it to translocate to the nucleus, where it forms complexes with different transcription factors, such as TEAD or β-catenin, to regulate transcription of downstream genes. YAP/TAZ also interacts with the canonical Wnt signalling pathway by blocking the degradosome function and promoting β-catenin cytoplasmic release. (d) MAPK signalling pathways involve the activation of small GTPases RAS and a cascade of sequentially activated kinases that lead to the activation of ERK1/2, JNK and p38 MAPKs. Activation of canonical Wnt signalling and inhibition of degradosome not only leads to the release of β-catenin but also stabilises RAS and thus activates the MAPK cascade. At the same time, p38 promotes Wnt/β-catenin signalling through the inhibition of the destruction complex. Not enough information is known to reliably construct the interactions of the non-canonical Wnt with MAPK signalling pathway in bone cells.