Deciphering the Cell-Specific Role of Wnts in the Liver: New Tools for a Difficult Task

Wnt Signing in the Liver

Wnt signaling pathway plays a critical role in a number of pathophysiological processes, including stem cell biology, embryonic development, cell differentiation, and several pathologies, including cancer.¹ In the liver, Wnt signaling in general and Wnt/β-catenin pathway in particular is central to various aspects of hepatic physiology, such as embryonic liver development, postnatal liver growth, hepatic differentiation, and liver regeneration.^{2, 3} Dysregulation of Wnt signaling is reported in pathogenesis of several hepatic disorders, including alcoholic and nonalcoholic liver steatohepatitis, cholestasis and biliary disorders, and in hepatic malignancies.³ Wnt pathway remains a popular therapeutic target in the liver pathobiology.

The downstream regulator of the canonical Wnt pathway is the transcriptional coactivator, β-catenin. In the “off” state, β-catenin is targeted for degradation in the cytoplasm by multiple phosphorylations induced in a multiprotein complex containing glycogen synthase kinase-3 beta, casein kinase, Axin, and adenomatous polyposis coli (APC). The pathway is turned “on” when the extracellular ligands of the pathway, the Wnt lipoproteins, bind to cell-surface receptors called Frizzled. Several subsequent steps in signal transduction, including recruitment of coreceptor low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6), activation of Dishevelled, and inactivation of the destruction complex, culminate in prevention of β-catenin phosphorylation. The unphosphorylated (or dephosphorylated) β-catenin enters the nucleus where it can bind to multiple transcription factors, including transcription factor 4, hypothia-inducible factor, and others to initiate signaling. Studies have shown that activation of this signaling is absolutely dependent on presence of Wnts.¹

Mammals, including humans, have a total of 19 Wnt genes further divided into 12 subfamilies. Wnts have a unique lipoprotein structure containing a 40-KD cysteine-rich protein backbone ornamented with palmitoleic acid. The lipoprotein chemistry of the Wnts makes them relatively hydrophobic and thus they function more in paracrine fashion than endocrine fashion, that is, they are secreted locally and act locally rather than widely circulating by blood. A major molecule that is indispensible for Wnt secretion outside the cell where it is made is a sorting receptor called Wntless (Wls)/Evenness interrupted. Because of the multiplicity of Wnt gene products and redundancy in their function, tissue- and/or cell-specific deletion of Wls has become the primary mechanism to study Wnt function using the loss-of-function approach. Deletion of Wls results in inhibition of Wnt secretion from cells, and thus rather than deleting individual Wnt genes, which could be tedious, deletion of Wls has been a standard practice.

Wnt Signaling and Liver Zonation

Initial studies on the role of Wnt/β-catenin pathway in the liver were focused mainly on the role of β-catenin. These studies, performed using hepatocyte-specific deletion of β-catenin, revealed its role in embryonic and postnatal liver growth, regeneration, and cancer.² However, apart from its well-known role in cancer pathogenesis, Wnt/β-catenin signaling has now been recognized as a major regulator of liver zonation.^{4, 5} Studies showed that β-catenin is active mainly in the centrilobular area and changing the expression of APC, a regulator of β-catenin, can change the zonal expression of β-catenin, resulting in marked alteration in hepatic zonation.⁶ Further studies indicated that β-catenin involved in liver zonation is primarily driven by paracrine Wnt signaling. These studies used Lrp5/6-floxed and Wls-floxed mice bred with mice expressing various cell-specific cre recombinases to delete Wls in epithelial cells (albumin cre-hepatocytes and biliary cells), macrophages (LyzM cre-Kupffer cells), and in endothelial cells (Tie2 cre).⁷ Deletion of Lrp5/6, the coreceptors for Wnts, resulted in loss of hepatic zonation attributed to inhibition of Wnt signaling. The next set of experiments indicated that hepatocytes, cholangiocytes, or Kupffer cells are not the major sources of Wnts involved in liver zonation given that deletion of Wls in these cells had no effect of liver zonation (although there were changes in liver regeneration after partial hepatectomy). Interestingly, these studies also reported that deletion of Wls in endothelial cells using a Tie2-cre resulted in embryonic lethality. Taken together, these studies show that liver zonation is regulated by Wnt/β-catenin pathway. Paracrine secretion of Wnts in the liver turns on β-catenin in hepatocytes, which, in turn, regulate zone-specific gene expression. However, the cellular source of these Wnts and an effective methodology to study them had been a challenge in the field.

The New Stab2-cre Model

The studies published by Leibing etal. in this issue provide further evidence supporting the role of Wnt signaling in liver zonation.⁸ Additionally, these studies also highlight a mouse model that can transform studying liver sinusoidal endothelial cell (LSEC) biology. These studies show that LSECs are the primary source of Wnts in the liver and Stabilin2 promoter-driven cre recombinase mice can be used to study gene expression in LSECs. Stablins are scavenger receptors expressed on endothelial cells. Stabilin2 (Stab2) is expressed primarily on LSECs and also on endothelial cells in the spleen and lymph nodes.⁹ Therole of Stab2 in scavenging extracellular matrix proteins, such as hyaluronic acid, chondroitin sulfate, and heparin, is well recognized. Deletion of Stab1 and Stab2 both resulted in moderate perisinusoidal fibrosis and significant glomerular fibrosis and macroalbuminuria.¹⁰ The next set of experiments using the newlygenerated Stab2-cre demonstrated that GATA4 regulates embryonic LSEC generation.

In the current studies, the investigators utilize these Stab2-cre mice to obtain LSEC-specific deletion of Wls, which has been elusive so far because of lack of an LSEC-specific cre driver. The primary finding of these studies is that deletion of Wls in LSECs using Stab2 cre resulted in disruption of liver zonation. This was accompanied by lack of characteristic β-catenin activation in perivenous hepatocytes and decrease in expression of centrilobular zone-specific canonical Wnt signaling target genes, including glutamine synthase and cytochrome P450 2E1. Interestingly, deletion of Wls, which presumably results in inhibition of Wnt secretion from LSECs, did not result in any notable pathology in the liver, but induced a moderate change in cholesterol metabolism. Taken together, these studies provide conclusive evidence that LSECs are the major source of Wnts in the liver and are responsible for activation of Wnt/β-catenin signaling that drives liver zonation.

Opportunities and Challenges

These studies have advanced our knowledge of the molecular mechanisms behind liver zonation and also provided a very useful reagent to the scientific community. Stab2 cre will be instrumental in studying physiology and pathobiology of LSECs, cells that are integral to maintenance of liver homeostasis and are on the forefront of disease progression in the liver. The advent of Stab2 cre will enable gene deletion in LSECsand even targeted killing of LSECs to further study their role in hepatobiology. Similarly, the role of LSEC-derived Wnts in homeostatic and injury-induced liver regeneration and in various liver diseases can now be studied with much more precision. However, many unanswered questions remain: for example, which specific Wnt(s) are involved in the liver zonation? Whereas previous studies have indicated a role of Wnt2 and Wnt9, this can now be confirmed with more precise experiments. What is the role of canonical versus noncanonical Wnts in liver biology in general and LSEC biology specifically? When LSECs in centrilobular region are injured, such as after CCl4-induced liver injury, will LSECs from other zones of the liver take over the function of secreting Wnts? Answering these questions is necessary to not only further our understanding of LSEC biology or improve our understanding of the role of Wnts in the liver biology, but also critical in determining therapeutic targets for various liver diseases.

Abbreviations

LSECs

liver sinusoidal endothelial cells

LRP5/6

low-density lipoprotein receptor-related proteins 5 and 6

Stab2

stabilin2

Wls

Wntless