The cholangiocyte primary cilium in health and disease

Abstract

Cholangiocytes, like most cells, express primary cilia extending from their membranes. These organelles function as antennae which detect stimuli from bile and transmit the information into cells regulating several signaling pathways involved in secretion, proliferation and apoptosis. The ability of primary cilia to detect different signals is provided by ciliary associated proteins which are expressed in its membrane. Defects in the structure and/or function of these organelles lead to cholangiociliopathies that result in cholangiocyte hyperproliferation, altered fluid secretion and absorption. Since primary cilia dysfunction has been observed in several epithelial tumors, including cholangiocarcinoma (CCA), primary cilia have been proposed as tumor suppressor organelles. In addition, the loss of cilia is associated with dysregulation of several molecular pathways resulting in CCA development and progression. Thus, restoration of the primary cilia may be a potential therapeutic approach for several ciliopathies and CCA.

Figure 1. Primary cilium

Confocal immunofluorescence showing a cross-section of a bile duct, where the nuclei of cholangiocytes lining the duct are stained in blue, and primary cilium extending into the ductal lumen in red.

Figure 2. Cholangiocyte primary cilium regulates several signaling pathways

The ability of primary cilia to detect different signals is a consequence of the expression of specific ciliary proteins and ciliary associated receptors. The activation of these receptors results in activation or inhibition of different signaling pathways. TGR5: G-protein-coupled bile acid receptor 1; PC-1: polycystin-1; PC-2: polycystin-2; TRPV4: Transient Receptor Potential Vanilloid 4; PTCH: patched receptor; SMO: Smoothened receptor; PDGFRα: Platelet-derived growth factor receptor α; P2Y12: Purinergic receptor 12; Inv: inversin; AMPK: AMP-activated protein kinase; LKB1: liver kinase B1.

Figure 3. Primary cilia function as mechano-, chemo- and osmosensors

PC-1 and PC-2 allows primary cilia to function as a mechanosensor. The bending of cholangiocytes primary cilia by luminal flow induces an increase in the intracellular ionic calcium through activation of PC-1 and PC-2, leading to inhibition of AC6, which is inhibited by calcium, resulting in a decrease in levels of cAMP and inhibition of PKA. TRPV4 participates in the osmosensory activity. This receptor is activated by extracellular hypotonicity and inhibited by extracellular hypertonicity, leading to an increase in intracellular calcium that results in PKA inhibition. The cilia chemosensory response is a consequence of purinergic receptors (P2Y12) and bile acid receptor (TGR5), which are activated causing changes in intracellular cAMP levels, possibly via AC6, that induces PKA activation or inhibition. PC-1: polycystin-1; PC-2: polycystin-2; AC6: adenylyl cyclase 6.

Figure 4. PDGRF signaling pathway

Quiescent cells express PDGFRα in primary cilia. Its activation induces ERK1/2 phosphorylation by induction of MEK1/2 and activation of AKT through PI3K resulting in cell cycle entry.

Figure 5. Hedgehog signaling pathways

Canonical Hedgehog signaling in mammals is ligand dependent. In the absence of Hh ligands, PTCH accumulates in the primary cilium and inhibits the function of Smo. PTCH facilitates the activation of several kinases (CK1, PKA, and GSK3) at the base of the primary cilium, which differentially phosphorylates the Gli protein. This can lead to complete degradation of the Gli protein by the proteasome, as well as partial cleavage of Gli2 and Gli3. Partially cleaved Gli3 (Gli3R) is translocated to the nucleus and functions as a transcriptional repressor for Hh target genes. In the presence of Hh ligands, Shh, Ihh, or Dhh binds to PTCH, which induces its lysosomal degradation. This relieves its inhibitory effect on Smo, which accumulates in the primary cilium and prevents degradation of Gli proteins. Activated Gli protein is translocated to the nucleus and functions as a transcriptional activator for Hh target genes.

Figure 6. Wnt signalling pathways

In the canonical pathway, when the Frizzled receptor is not activated, the destruction complex (Apc and Axin) binds β-catenin inducing its phosphorylation as mediated by GSK-3β and CKI, which results in β-catenin degradation. When the Frizzled receptor is activated, the destruction complex is inactivated resulting in the stabilization of β-catenin that translocates to the nucleus and induces target gene expression in coordination with TCF/LEF. In the non-canonical pathway, the activation of Frizzled receptors lead to activation of the small GTPases Rho and Rac. Inversin, localized in the basal body, functions as a switch between both pathways. DVL: Dishevelled; APC: Adenomatous polyposis coli, Inv: inversin.

Figure 7. Primary cilia regulate mTOR

Changes in the bile flow would induce activation of LKB1, which is localized along the entire length of the cilia. Once activated LKB1 phosphorylates and activates AMPK, which is recruited in the basal body. TSC is phosphorylated by AMPK leading to inhibition of Rheb that normally activates mTOR. As result, mTOR and its downstream proteins are inhibited. Thus, primary cilia participate in the regulation of cell size, cell metabolism, cell growth, proliferation and survival.

Figure 8

Cholangiocyte primary cilia regulate PKA activation trough TGR5. In the apical membrane TGR5 is coupled to Gαs, while in primary cilia TGR5 is coupled to Gαi. The activation of apical TGR5 leads to adenylyl cyclase activation and increase in the level of cAMP resulting in the activation of PKA and increase in ERK1/2 signaling, while the activation of ciliary TGR5 has an opposite effect.

Figure 9. Ciliotherapy

Primary cilia function as tumor suppressor organelles by controlling several signaling pathways involved in metabolic process, cell growth, proliferation and survival. In CCA, the loss of primary cilia, which is associated with HDAC6 overexpression, generates malignant cholangiocytes unable to properly regulate these signaling pathways. Therefore, ciliotherapies, which are based on ciliary restoration, might have a potential value for cancer prevention and treatment.