Functional role of Vangl2 in bile duct injury

Abstract

The Liver is comprised of hepatocytes, the parenchymal cells, and non-parenchymal cells which includes stellate cells, portal fibroblasts, inflammatory cells and cholangiocytes.

Cholangiocytes are the epithelial cells of the bile ducts, which carry bile from the liver to the duodenum. Bile ducts and specifically cholangiocytes are susceptible to disease; these diseases are classified as cholangiopathies. Primary Sclerosing Cholangitis (PSC) is a disease of both the extra- and intrahepatic bile ducts involving inflammation and obliterative fibrosis. The cause of PSC is, as yet unclear, but is thought to be autoimmune related.

Inflammation of tissue surrounding the ducts leads to stricture and sclerosing of the duct and results in scar formation, thereby, leading to obstruction of bile flow.

Chronic impairment of the duct accompanied by dysfunction of bile transport results in biliary fibrosis, progressive biliary cirrhosis and liver failure. PSC is increasing in prevalence, untreatable and, ultimately, requires transplantation. Given donor liver numbers have remained static over the last 30 years, modulating fibrosis is an attractive alternative to transplantation. Tissue fibrosis is a complex process involving the formation of an excess of fibrous tissue. Fibrosis is an important part of the regenerative process during injury. It requires balanced expression of fibrotic promotors (e.g. Transforming Growth Factor β (TGFβ) and Matrix Metalloproteinases (MMPs)) and inhibitors (e.g. Tissue Inhibitor of Metalloproteinases (TIMPs)) to maintain an environment conducive to tissue repair and continued organ function.

The non-canonical Wnt signaling pathway has been, traditionally, shown to determine cellular polarity and cell migration during embryogenesis, however, its role in adult tissue homeostasis and regeneration is poorly understood. Examples of non-canonical Wnt pathway activation have been described during regeneration, for example, in skeletal muscle, however the role of non-canonical Wnt signaling in the liver has not been elucidated. Briefly, the non-canonical Wnt pathway does not involve β-catenin, unlike the canonical pathway or calcium, as in the calcium mediated Wnt Pathway. Wnt ligand binds to a Frizzled (Fzd) receptor or a single pass receptor tyrosine-kinase (such as Ror2 or Ptk7) which can then interact with the co-receptor Van Gogh-like 2 (Vangl2).

Activation of this receptor recruits Disheveled (Dsh) to the cell membrane. Downstream of Dsh the pathway can utilise a number of signaling cascades involving activation of DAAM1, Rho, Rac, ROCK and Jnk that ultimately leads to actin cytoskeletal modifications and/or transcription via AP- 1. Inhibition of these key downstream components has been shown to phenocopy knockout of the cell surface receptor elements demonstrating the specific nature of the pathway to its desired biological effect. Here we use a mouse model of PSC to show up-regulation of the non-canonical Wnt pathway receptor-ligand complex and downstream activation of the Vangl2 mediated non-canonical Wnt pathway. Particularly, we show that Vangl2 and Wnt5a are up regulated at both the transcript and protein level and also demonstrate that these proteins are expressed in the regenerating ducts following PSC-modelled injury. To functionally evaluate the role of Vangl2 in bile duct fibrosis we used Vangl2 mouse mutants, which contain either a C-terminus point mutation in Vangl2 or conditional loss of the transmembrane domain of Vangl2 to knockout Vangl2 function.

Here we show that loss of Vangl2 results in a significant reduction of fibrosis following bile duct injury when compared to mice with intact Vangl2. The loss of Vangl2 demonstrates that it has a central and hitherto unidentified role in liver fibrosis. In mice where Vangl2 is abrogated we have defined that Vangl2 regulates a number of gene families associated with inflammation and have, furthermore, identified candidate pathways through which Vangl2 can potentially act. These data together highlight the role of the non-canonical Wnt signaling pathway in the maintenance of the fibrotic niche.

Moreover, with the use of therapeutic Wnt inhibitors we are able to modulate bile duct fibrogenesis and, for the first time, identify a targetable pathway for the treatment of PSC.