Loss ofAnks6leads to YAP deficiency and liver abnormalities

Abstract

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations (DPM) are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6, and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. Loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights to the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Mitigation of portal fibrosis and cholestatic liver disease in ANKS6 ‐deficient livers by macrophage depletion

“…Our analysis above showed that ANKS6 deficiency causes perinatal liver fibrosis (Figures 1 and 3). 1,7 To characterize the onset and progression of portal fibrosis in detail, we stained Anks6 KO livers with Sirius red. We observed a steady increase in Sirius red‐positive area in mutant livers as they aged compared to control livers (Figure 5A).…”

“…CHF is a heterogeneous cholangiopathy that frequently manifests as part of cilia‐related syndromic disorders, such as autosomal recessive polycystic kidney disease (OMIM #263200), Meckel‐Gruber syndrome (OMIM #249000), Joubert syndrome (OMIM #213300), Bardet‐Biedl syndrome (OMIM #209900), nephronophthisis (NPHP) (OMIM #256100), or oral‐facial‐digital syndrome (OMIM #311200), indicating that ciliary dysfunction which underpins the ductal plate remodeling abnormalities in these disorders can secondarily lead to progressive fibrogenesis in the portal tract 10,33,34 . Indeed, work by several groups, including ours, have demonstrated that defective bile duct morphogenesis gives rise to damaged and immature cholangiocytes, which secrete different profibrotic cytokines (Tgfb1) and growth factors (Ctgf) that promote cell proliferation and fibrogenesis in the surrounding tissue 7,35,36 . Immature cholangiocytes also secrete pro‐inflammatory cytokines (Il1β, Il6, Tnf) and chemokines (Ccl2, Cxcl10), which leads to the accumulation of activated liver resident macrophages (Kupffer cells) and further propagation of inflammatory signaling in the portal tract and recruitment of inflammatory (Ly6C + ) bone marrow‐derived monocytes to the sites of injury 19,20 .…”

“…Depending on the severity of the mutations, phenotypes can include kidney cysts, cardiovascular abnormalities, laterality defects, and congenital hepatic fibrosis (CHF) 1–3 . While the precise function of ANKS6 remains to be determined, biochemical and functional studies of Anks6 mutations have demonstrated that it is required for the formation and function of the cilium localized signaling compartment, known as inversin (Inv)‐compartment (IC) 4–7 . Indeed, mutations in genes that encode IC components—NPHP3, NPHP2/INV, NEK8, ANKS3, and ANKS6, result in phenotypically overlapping developmental abnormalities in affected humans 1,8 and orthologous animal models, 4,8 underlining the critical role of each protein in the function of IC.…”

Compensatory hepatic adaptation accompanies permanent absence of intrahepatic biliary network due to YAP1 loss in liver progenitors

“…First, YAP1 may be involved in the establishment and maintenance of cell polarization in ductal plate cells, as well as adjacent HBs. A recent study showed that Anks6, a protein localized to the primary cilium of developing ductal plate cells, regulates YAP1 transcriptional activity, and loss of Anks6 results in defects of bile duct morphogenesis ( Airik et al, 2020 ). Although this suggests that YAP1 activity in the ductal plate depends on appropriate polarization, our data suggest that YAP1 loss impairs primary cilium formation and polarization, pointing to a positive feedback loop that may be required for maintenance of bile duct cell polarity.…”

Congenital hepatic fibrosis and its mimics: a clinicopathologic study of 19 cases at a single institution

“…Fortunately, despite the wide variations in clinical presentations, the liver biopsy in CHF patients often shows classic histomorphology for which we coined the term “triads” to describe the abnormal bile duct profile, hypoplastic portal vein, and progressive fibrosis that are almost invariably presented in every case [ 3 ]. The molecular mechanisms of this “vein-duct” interaction was nicely illustrated in a Anks6 knock-out mouse study demonstrating the involvement of Hippo-YAP/TAZ angiogenesis pathway during bile duct embryogenesis [ 26 ], suggesting a finely tuned interaction among the portal veins, ductal plates, and the intervening mesenchyme during ductal plate remodeling [ 24 ]. DPM is also a known risk factor for hepatobiliary neoplasms, more frequently cholangiocarcinoma than hepatocellular carcinoma [ 27 – 29 ].…”