Primary Cilia in Hepatic Biliary Hyperplasia: Implications for Liver Diseases

Abstract

Primary cilia, hair-like projections on the surface of various cell types, play crucial roles in sensing and regulating environmental cues within the liver, particularly among cholangiocytes. These structures detect changes in bile composition, flow, and other biochemical signals, integrating this information to modulate cellular processes. Dysfunction in cholangiocyte cilia—whether due to structural abnormalities or genetic mutations—has been linked to an array of cholangiopathies and ciliopathies. These include conditions such as biliary atresia, cholangiocarcinoma, primary sclerosing cholangitis, and polycystic liver diseases, each with distinct clinical phenotypes influenced by impaired ciliary function. Given the complexity of the ciliary proteome and its role in cellular signaling, including the Hedgehog, Wnt, and TGR5 pathways, ciliary dysfunction disrupts essential signaling cascades, thus driving disease progression. While over 40 gene mutations are associated with ciliopathic features, there may be additional contributors within the expansive ciliary proteome. This study synthesizes current knowledge on cholangiocyte cilia, emphasizing their mechanistic role in liver disease, and highlights emerging therapeutic strategies aimed at restoring ciliary function. In conclusion, ciliotherapies are proposed as a promising approach for addressing cholangiopathies, with the potential to shift the current therapeutic landscape.

Lay Summary

Primary cilia are tiny, hair-like structures on the surface of certain liver cells that help them sense changes in their environment, such as bile flow and composition. These cilia play a vital role in keeping liver cells healthy and functioning properly. However, when cilia are damaged or have genetic mutations, they malfunction, leading to diseases such as biliary atresia, cholangiocarcinoma, and polycystic liver disease. This study highlights how cilia-related issues disrupt essential cell signals, contributing to the development and progression of liver diseases. Researchers are exploring new treatments, known as “ciliotherapies,” to restore proper ciliary function and create more effective treatments for liver diseases linked to ciliary dysfunction.

Graphical Abstract

Cilia are specialized cellular organelles that extend from the surface of most eukaryotic cells, serving as key structures for motility and signaling. These evolutionarily conserved projections can either facilitate movement or act as critical hubs for cellular communication.^1,2 Cilia has two primary components: (a) the basal body, which anchors the cilium at the cell surface, and (b) the axoneme, a microtubule-based extension that protrudes outward.^3–5 The basal body originates from the mother centriole and organizes the microtubules essential for ciliary function. The axoneme is the structural framework of the ciliary shaft, consisting of microtubule doublets arranged in a characteristic pattern.^3–5

Cilia are usually categorized into motile and primary cilia based on their axonemal configuration. Motile cilia typically feature nine outer doublets encircling a central pair, a structure essential for generating movement. In contrast, primary cilia lack central microtubules and function primarily as sensory organelles. Motile cilia are typically found in large numbers on epithelial surfaces, such as those lining the respiratory tract, reproductive system, and brain ventricles, where they generate coordinated movements to propel fluids or mucus.^3–5 They also play specialized roles, such as directing morphogen gradients in embryonic development. In contrast, primary cilia, which are solitary and nonmotile, were long considered evolutionary junk. However, their role as cellular “antennae,” mediating signal transduction and integrating environmental cues to regulate development, differentiation, growth, and metabolism, is now widely accepted.^3–7

The formation of primary cilia depends on the cell cycle, with growth-arrested cells being primarily ciliated during the G0 phase.² The cilium, comprising a transition zone, basal body, and axoneme, arises as the basal body differentiates from the mother centriole. Distal appendage vesicles are transported near the distal appendages of the maternal centriole during the G0 phase. These vesicles fuse to produce a sizable ciliary vesicle, which encapsulates the distal appendages. The developing cilium migrates to the plasma membrane, where it fuses with microtubules that have elongated from centriolar triplets within the ciliary vesicle. This intracellular ciliary assembly mechanism is characteristic of mesenchymal cells, whereas epithelial cells develop primary cilia via an alternative extracellular pathway on the cell surface. Following the centrosome in the G0 phase, remnants of the midbody (a microtubule structure) assist in establishing the ciliary membrane.⁸

In both pathways, distal appendage vesicles support early membrane formation and coordinate the recruitment of intraflagellar transport protein (IFT) essential for ciliary elongation (►Fig. 1). Ciliary protein synthesis occurs primarily in the cytoplasm and is transported into the cilium via the IFT complex, as ribosomes are not present in primary cilia. The anterograde transport (IFT-B complex) is propelled by the KIF3A/KIF3B/KAP kinesin-2 motor, whereas the retrograde transport (IFT-A complex) is driven by dynein. IFT-A and IFT-B can participate in both retrograde and anterograde transport.^7,9 The axoneme, the main structural component of primary cilia, serves as a core support for ciliary function. The transition zone regulates the entry and separation of cargo into the ciliary compartment, while the axoneme plays a crucial role in cilium dynamics and stability (►Fig. 1). Posttranslational modifications, such as tubulin acetylation, further contribute to its regulation.^6,8 Ciliary genetic disorders, characterized by clinical manifestations such as intellectual disability, skeletal defects, retinal degeneration, kidney cysts, liver cysts, and polydactyly, are frequently caused by mutations in genes involved in primary cilium formation and trafficking. Cilium resorption occurs when the cell cycle restarts in response to growth factor or hormone stimulation.

Fig. 1.

Ciliogenesis. During extracellular ciliogenesis, the cilium emerges directly at the cell membrane. Key proteins, including ARL13B and IFT-A/B (intraflagellar transport proteins), participate in the transport and regulation of components along the cilium. The ciliary membrane covers the axoneme, which extends from the basal body, connecting the cilium to the cell surface. The ciliary pocket, near the base of the cilium, is an incision of the plasma membrane that facilitates membrane trafficking. Ciliogenesis begins when the mother centriole, targeted to become the basal body, links with a small vesicle to form a ciliary vesicle precursor. The early phase is assisted by MYO5A, a motor protein. EHD1 subsequently promotes the growth of the ciliary vesicle, which combines with the basal body. As the cilium begins to develop, so does its associated ciliary sheath. The proteins ARL13B and IFT-A/B are recruited to promote the elongation of the axoneme. The transition zone (TZ), comprising MKS/NPHP proteins, regulates access to the cilium, consequently maintaining appropriate protein composition. As the cilium elongates, it ultimately contacts the plasma membrane, enabling the axoneme to protrude from the cell surface. Insert: Cross-sectional view of the cilium. Primary cilia exhibit a 9 + 0 microtubule structure, while motile cilia feature a 9 + 2 arrangement and possess dynein arms and radial spokes for movement. (Created with BioRender).

This study highlights primary cilia biology in biliary diseases, including new diagnostic and therapeutic research directions.

Cholangiocyte Primary Cilia: Normal Physiology

Primary cilia on cholangiocytes detect changes in bile flow, composition, and tonicity.⁴ (►Fig. 2). Ciliary proteins detect environmental ligands and transmit signals, enabling multisensory functions. The ciliary-related proteins polycystin-1 (PC-1), a cell surface receptor, and polycystin-2 (PC-2), a calcium channel, enable the mechanosensory function of primary cilia. By causing the main cilia of cholangiocytes to bend, luminal flow increases intracellular ionic calcium, which depends on PC-1 and PC-2 receptors.^10–12 Transient receptor potential vanilloid 4, activated by extracellular hypotonicity and inhibited by extracellular hypertonicity, mediates osmosensory activity by increasing intracellular calcium, which depends on external calcium sources.¹³ Important purinergic receptors, such as P2Y12 and P2Y11, are activated by nucleotides and influence intracellular cAMP levels, playing a key role in chemosensory functions^14,15 (►Fig. 3). In addition, the AKAP (A-kinase anchoring protein) signaling complex, including AKAP150, protein kinase A, and one isoform of Exchange Protein Directly Activated by CAMP 1 (EPAC), is linked to chemosensory function of primary cilia and signal transduction via cAMP.^15,16 Unlike Gαs, which is localized at the apical plasma membrane, the bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) is abundant in the primary cilia of cholangiocytes and is coupled to Gi.^17,18 This chemosensory function may modify cAMP signaling since all these proteins are engaged in the cAMP signaling cascade.¹⁹

Fig. 2.

Cholangiocytes express primary cilia. An enlarged cross-section of the liver shows its functional microscopic organization into lobules, where the portal triad (the hepatic artery, portal vein, and bile duct) regulates important liver functions. The primary cilia of cholangiocytes, which line the bile ducts, are sensors for bile flow, osmosis, and chemical signals. The cilia regulate essential signaling pathways like EGFR, FGFR, Wnt, and TGFßR, which impact cell proliferation and survival. Calcium and cAMP signaling are at the core of the multisensory functions of primary cilia and regulate the functions of cholangiocytes. (Created with BioRender).

Fig. 3.

Primary cilia sense the extracellular environment. Primary cilia detect external signals via receptors on their surface, leading to the activation or inhibition of distinct signaling pathways. EGFR, epidermal growth factor receptor; LKB1, liver kinase B1; P2YR, purinergic receptors; PC-1, polycystin-1; PC-2, polycystin-2; PDGFRα, platelet-derived growth factor receptor α; PTCH, patched receptor; SMO, smoothened receptor; TGR5, G-protein-coupled bile acid receptor 1; TRPV4, transient receptor potential vanilloid 4. (Created with BioRender).

Cholangiocytes and hepatocytes release exosomes,²⁰ which primarily mediate intercellular communication.²¹ Exosomes interact with primary cilia on cholangiocytes, stimulating proliferation, microRNA synthesis, and intracellular signaling. The cilia detect biliary exosomes, known as ectosomes, and convert these extracellular signals into intracellular responses, including changes in cAMP levels.^20,22 Exposure to the biliary exosome significantly reduces extracellular signal-regulated kinase signaling and inhibits cholangiocyte proliferation.^20,22 However, these effects decrease significantly after cilia are removed, indicating that primary cilia are essential for ciliary exosome-dependent regulatory mechanisms controlling cholangiocyte growth.^20,22 The cholangiocyte-exosome-primary cilia relationship highlights the complexity of cellular communication in the biliary system and suggests potential therapeutic interventions for cholangiopathies, when disruption of these pathways may occur.

Primary cilia in cholangiocytes appear to have a physiologic role in the fine-tuning regulation of bile secretion by feedback loops, in which changes in bile flow and/or composition are sensed by the cilia, which further transmit signals to the cells to modulate their activities. For instance, an increase in bile flow due to the hormone secretin causes the primary cilia to bend, signaling the cessation of bicarbonate production.⁶ On the other hand, primary cilia produce a signal to promote bicarbonate secretion when changes in osmolarity or nucleotides are recognized.⁶

Molecular Mechanisms Affected by Primary Cilia

Primary cilia act as signaling hubs, regulating pathways that control proliferation, differentiation, and homeostasis. Ciliary integrity in cholangiocytes and other cells is crucial for regulating pathways like Hedgehog, Wnt, Notch, Hippo, mTOR, TGR5, and receptor tyrosine kinases. Through its unique structural composition, the cilium mediates the localization and activity of receptors and downstream signaling molecules (►Table 1).

Table 1.

Cilia-mediated regulation of liver signaling pathways and associated liver diseases

Signaling pathway	Role in cellular function	Mechanism of regulation by primary cilia	Associated liver diseases
Hedgehog (Hh) signaling	Regulates liver development, regeneration, and fibrosis	Facilitates Hh signaling by modulating Gli transcription factors	Cholangiocarcinoma25
Wnt signaling (canonical and noncanonical)	Controls hepatocyte proliferation, biliary differentiation, and tumorigenesis	Suppresses β-catenin-dependent Wnt signaling while enhancing noncanonical Wnt/PCP signaling	Cholangiocarcinoma95 and biliary atresia96
TGF-β/Smad signaling	Modulates liver fibrosis, regeneration, and hepatocyte differentiation	Mediates TGF-β receptor trafficking and Smad-dependent signaling	Primary sclerosing cholangitis97
Epidermal growth factor receptor signaling	Promotes hepatocyte proliferation, survival, and liver cancer progression	Sequestering receptors and modulating their activation	Cholangiocarcinoma and polycystic liver disease46
Mammalian target of rapamycin (mTOR) signaling	Controls hepatic metabolism, autophagy, and liver cancer growth	Regulates mTOR activity via Lkb1 and AMPK-dependent mechanisms	Cholangiocarcinoma98
Calcium signaling	Controls bile duct homeostasis and hepatic metabolism	Regulates intracellular calcium ion levels via polycystin-1 and polycystin-2	Polycystic liver disease99
cAMP/PKA signaling	Influences bile duct function and hepatic metabolism	Regulates cAMP levels via adenylyl cyclase and phosphodiesterase	Polycystic liver disease99 and cholangiocarcinoma15

Hedgehog Signaling Pathway

The Hedgehog signaling pathway is important to the control of cellular processes such as development, tissue homeostasis, and organogenesis. Cholangiocytes in the liver produce key Hedgehog ligands, particularly Sonic Hedgehog and Indian Hedgehog, which affect neighboring cells via paracrine signaling and act on the cholangiocytes themselves through autocrine signaling.²³ Both ligands are secreted and bind to and activate the patched receptor, which releases inhibition of the smoothened receptor inhibition. The production of genes targeted by this pathway is facilitated by the transformation of the glioblastoma protein transcription factor from transcriptional repressors to transcriptional activators via activation of the smoothened receptor.

The primary cilium is necessary for Hedgehog signaling in mammalian cells. Without a ligand, the patched receptor at the ciliary basal body blocks the smoothened receptor from entering the axoneme. The patched receptor dissociates from the cilia upon ligand interaction, allowing the smoothened receptor to move to the ciliary membranes^23,24 and promote the full-length forms of glioblastoma proteins, preventing their cleavage into repressor forms. These proteins then migrate to the nucleus and activate target genes. Without activation via the smoothened receptor, glioblastoma proteins are processed into their repressor forms or bound to the inhibitor SuFu, which facilitates their proteasomal degradation.

Increased Hedgehog signaling promotes epithelial-to-mesenchymal transition, aiding cancer invasion and metastasis.^21,25 Loss of primary cilia dysregulates Sonic Hedgehog signaling, promoting chemokine production and epithelial-to-mesenchymal transition in cholangiocytes.²⁵ Blocking Hedgehog signaling significantly inhibits cholangiocarcinoma and polycystic liver disease, suggesting that targeting this route may represent a viable treatment strategy.^25–27 Additionally, inhibition of Rab23, a negative regulator of the Sonic Hedgehog pathway, results in aberrant ciliogenesis and enhanced basal Sonic Hedgehog signaling, consequently promoting proliferation in medulloblastoma cells. However, it renders the cells unresponsive to stimulation by Hedgehog agonists.²⁸

Wnt Signaling

Wnt are secreted proteins that, via the canonical route, bind to the Frizzled receptor and induce Dishevelled to be recruited to the plasma membrane. This results in the stabilization of β-catenin in the cytoplasm, followed by its translocation to the nucleus where it functions as a transcription factor in conjunction with TCF/LEF. Glycogen Synthase Kinase 3 Beta (GSK-3) phosphorylates β-catenin, leading to its degradation via the proteasome in the absence of Wnt ligand. Dishevelled also regulates small GTPases via downstream protein targets in a noncanonical pathway.²⁹ Canonical Wnt signaling is active in human cholangiocarcinoma due to overexpression of Wnt ligands and activation of an increased number of β-catenin targets.^27,29 Pharmacologic blocking of canonical Wnt signaling decreased the formation of cholangiocarcinoma in a rat model by inducing apoptosis and reducing cell cycle entrance.³⁰

Wnt signaling is crucial for the survival and proliferation of cholangiocarcinoma cells. Primarycilia control thebalance between canonical and noncanonical Wnt signaling pathways. In medulloblastomas characterized by abnormally active Wnt signaling, primary cilia are prevalent. However, the absence of primary cilia suppresses tumor growth, reduces the rate at which cells re-enter the cell cycle, and increases the rate at which they exit. In contrast, for the G3 subtype of medulloblastoma, characterized by MYC amplification, loss of primary cilia leads to accelerated tumor development, and the absence of cilia is associated with a shorter cell cycle duration.³¹

Notch and Hippo Signaling

The transmembrane receptor known as Notch is bound to and activated by ligands of the Delta family of Jagged proteins. When Notch is activated, proteolytic enzymes cleave the Notch receptor’s intracellular domain fragment. These enzymes then translocate to the nucleus to stimulate transcription-targeting genes linked to suppression of differentiation and proliferation.³¹

Cilia and Notch are both involved in a complex feedback mechanism, although it is unknown how cilia regulate Notch.³² In polycystic liver disease, increased nuclear expression of Notch1, Notch2, Notch3, and the Notch effector Hes1 in biliary epithelial cells, and elevated levels of the ligand Delta-like 1, are associated with heightened cell proliferation, suggesting that aberrant activation of the Notch-Hes1 pathway drives biliary cystogenesis.³³ Notch signaling is crucial for cholangiocyte differentiation, as inhibition of the Notch pathway prevents cholestatic liver fibrosis by reducing the differentiation of hepatic progenitor cells into cholangiocytes.³⁴ Furthermore, the Notch pathway is a major driver of cholangiocarcinogenesis, with its components (receptors, ligands, and downstream signaling molecules) playing key roles.³⁵

The Hippo pathway regulates cellular processes such as proliferation, survival, differentiation, and homeostasis, and its dysregulation is linked to cancer. It controls the transcriptional activators Yes-associated protein and transcriptional coactivator with PDZ-binding motif (YAP/TAZ) by inhibiting their nuclear roles in promoting cell proliferation and preventing apoptosis. This pathway both regulates and is influenced by cilia.³⁶ Nephronophthisis proteins can stimulate TAZ activity,³⁷ while the upstream Hippo components MST and Salvador Family WW Domain Containing Protein 1 prevent ciliary disassembly by phosphorylating Aurora-A.³⁸ In polycystic liver disease, the Wnt and Hippo signaling pathways intersect through the PKD1–TAZ–Wnt–β-catenin–c-MYC axis. In PKD1-deficient cells, TAZ drives cyst formation by interacting with AXIN1 and increasing β-catenin activity. This activation leads to upregulated c-MYC expression and cystogenesis.³⁹ Hippo signaling, via YAP and TAZ, is critical in cholangiocarcinoma; its dysregulation promotes aggressive growth and poor prognosis. Activating YAP/TAZ, particularly with oncogenic signals like AKT, drives tumorigenesis in cholangiocarcinoma.⁴⁰

mTOR

Primary cilia control mTOR activity via the tumor suppressor LKB1.⁴¹ The multiprotein complexes mTORC1 and mTORC2, whose catalytic core is the serine/threonine kinase mTOR, are crucial for numerous cellular processes, including cell size, metabolism, growth, proliferation, and survival. mTORC1 regulates several anabolic processes involved in cell and tissue growth, such as protein synthesis, ribosome formation, lipogenesis, and nucleotide synthesis.⁴¹ Additionally, it prevents the formation of lysosomes, which are necessary for autophagy. Cell proliferation is regulated by the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 and S6 kinase β−1 when mTORC1 is activated.⁴¹ Conversely, mTORC2 controls several proteins, including AKT, which is involved in the anabolic pathwayand is controlled by protein kinase C to regulate the actin cytoskeleton and cell motility.⁴² The precise function of mTORC2 is not fully understood, but it may be associated with the PI3K pathway.^41,42 The PI3K/Akt/mTOR signaling pathways, which are upregulated in several cholangiopathies including primary sclerosing cholangitis, cholangiocarcinoma, and polycystic liver disease; govern cell survival, apoptosis, metabolism, movement, and angiogenesis.⁴¹ mTORC1 activity appears to be altered by the loss of primary cilia. Although mechanical stimulation of primary cilia triggers mTOR inhibition through activation of AMPK in an LKB1-dependent pathway, loss of cilia results in dysregulation of mTOR activity, which is associated with the development of cholangiocarcinoma and polycystic liver disease.^41,42 This aligns with our earlier finding that experimental deciliation of cholangiocytes and cholangiocarcinoma cells results in poor AMPK activation and reduced LKB1 activity. Specifically, LKB1 activation through cilia-dependent chemosensation inhibits migration and invasion in normal cholangiocytes, whereas the loss of cilia promotes these processes. This suggests that primary cilia have a tumor-suppressive mechanism by regulating LKB1 activity in response to extracellular signals.¹⁴

TGR5 Signaling

Bile acids are derived from cholesterol and play an important role in dietary lipid absorption and cholesterol excretion. After food ingestion, the gallbladder contracts, releasing bile acids into the small intestine to facilitate fat breakdown and absorption. Most conjugated bile acids are absorbed in the terminal ileum and recycled to the liver.⁴³ Bile acids play a crucial role in enterohepatic communication, integrating microbiota-derived signals into this pathway. One of the key discoveries in bile acid signaling was the identification of natural receptors for bile acids, such as the farnesoid X receptor and the G protein-coupled bile acid receptor 1, also known as Takeda G-protein-coupled receptor 5 (TGR5).⁴³ TGR5 is expressed in cholangiocytes, intestinal cells, the basolateral surface of smooth muscle cells, neural cells, brown adipose tissue, and immune cells, including dendritic cells and macrophages. It is most potently activated by bile acids and is a key receptor mediating their effects.⁴⁴ TGR5 is important in regulating the bile acid pool, bile composition, hepatic inflammation, insulin sensitivity, and glucose metabolism. It is activated by unconjugated and conjugated bile acids in the following order of potency: chenodeoxycholic acid > taurolithocholic acid > lithocholic acid > deoxycholic acid.⁴⁵

Since TGR5 is present in primary cilia and the apical membrane, the loss of primary cilia leads to dysregulation of bile acid signaling, contributing to cholangiopathies.¹⁹ TGR5 activation has opposing effects depending on whether it is linked to Gs or Gi proteins and the presence of primary cilia in cholangiocytes. When linked to Gαs proteins in the apical domain of nonciliated cholangiocytes, TGR5 activation increases intracellular cAMP levels via adenylate cyclase activation, which subsequently increases cell proliferation. In contrast, in ciliated cholangiocytes, ciliary TGR5 is coupled to Gαi proteins, resulting in decreased cAMP levels through adenylate cyclase inhibition, which reduces cell proliferation.^19,46,47 Individuals with primary sclerosing cholangitis exhibit reduced levels of TGR5 protein and mRNA in their livers and bile ducts. TGR5 activation increases cholangiocyte growth in cholangiocarcinoma and polycystic liver disease cells. The activation of TGR5 stimulates cell proliferation and affects cells’ sensitivity to apoptosis, which is essential for the progression of cholangiocarcinoma and polycystic liver disease. TGR5 signaling facilitates oncogenic processes in cholangiocarcinoma by increasing pro-proliferative pathways like ERK and AKT, while simultaneously affecting pro-apoptotic mechanisms. Furthermore, targeting TGR5 is a potential therapeutic approach to inhibit the proliferation of malignant cholangiocytes and mitigate the advancement of liver cysts in polycystic liver disease.^{18,19,48–50}

Receptor Tyrosine Kinase Signaling

Primary cilia play a crucial role in various tyrosine kinase receptors signaling pathways, particularly in the platelet-derived growth factor (PDGF) signaling pathway, which includes two receptors, PDGFRα and PDGFRβ, in conjunction with four ligands. Ligand binding to these receptors induces dimerization and autophosphorylation of tyrosine residues, activating downstream signaling molecules such as ERK1/2, MEK1/2, and AKT, which ultimately facilitate cell cycle entry. PDGFRα is localized to cilia, and the proper functioning of primary cilia is essential for mediating activity induced by PDGF ligands.^51,52 The lack of functional primary cilia greatly affects the PDGF signaling pathway. PDGFRα is frequently observed in cilia, where it regulates cell growth via receptor dimerization and autophosphorylation of tyrosine residues. This process activates downstream signaling molecules such as ERK1/2, MEK1/2, and AKT, which are important in cellular proliferation and differentiation. The absence of primarycilia in PLD disrupts PDGFR signaling, resulting in uncontrolled cell proliferation.^51,52

The TGF-β signaling pathway is essential for the regulation of cellular responses in both cholangiocarcinoma and polycystic liver disease. TGF-β receptors (TGFβ-RI and TGFβ-RII) are situated at the base of primary cilia,⁵³ where they regulate signaling effectors, including ERK1/2 and SMAD3. Following TGF-β activation, these effectors enhance the development of a procystic, fibronectin-enriched extracellular matrix, thereby supporting cyst growth in polycystic liver disease.⁵⁴ TGF-β functions as a tumor suppressor in the early stages of disease and as a tumor promoter in later stages.

Dysfunctional signaling of epidermal growth factor receptor (EGFR) results from defective cilia in both cholangiocarcinoma and polycystic liver disease. EGFR is typically subject to regulation and is degraded postactivation to prevent uncontrolled growth. In cholangiocytes lacking functional primary cilia, EGFR activation persists, resulting in continuous growth signaling that facilitates cyst cell growth and aids in tumor development in cholangiocarcinoma.⁴⁶ Histone deacetylase 6 inhibitors, including tubastatin-A and ACY-1215, can restore EGFR localization to primary cilia and improve its degradation. This method may alleviate the detrimental impacts of receptor dysregulation in both cholangiocarcinoma and polycystic liver disease.^46,51

Dynamic Interplay between Primary Cilia and Biliary Hyperplasia

Ciliary dysfunction dysregulates pathways essential for controlled growth and tissue repair. These changes drive excessive biliary proliferation and fibrosis, impairing liver function. Several liver disorders (►Fig. 4) are associated with biliary hyperplasia and involve ciliary signaling alterations.

Fig. 4.

Primary cilia and liver diseases. Bile duct abnormalities linked to hepatic biliary hyperplasia are depicted. i) The absence of primary cilia in cholangiocarcinoma results in the unregulated proliferation of bile duct cells and the development of tumoral growth. ii) Malfunctioning cilia in polycystic liver disease leads to bile duct hyperplasia and the development of cysts. iii) Primary biliary cholangitis is characterized primarily by inflammation, oxidative stress, and autophagy in the smaller bile ducts. Cholangiocyte hyperplasia is a secondary, less prominent response to ongoing bile duct injury, but no links to primary cilia defects have been described to date. iv) The clinical presentation of primary sclerosing cholangitis includes the restriction of bile ducts, infiltration of immune cells, and cholangitis; potential links to primary cilia involvement are emerging. v) Biliary atresia is a rare, life-threatening liver disease in infants where the bile ducts become inflamed, blocked, or absent, preventing bile flow from the liver to the intestines and leading to liver damage. (Created with BioRender).

Primary Cilia in Polycystic Liver Disease

Polycystic liver disease is a genetic condition characterized by bile duct deformation and cyst formation, resulting from cholangiocyte hyperproliferation and excessive secretion. It may arise alone or in association with polycystic kidney disease and acquired in a dominant or recessivefashion. The most prevalent symptoms and sequelae of polycystic liver disease include hypertension, back pain, bloating and stomach discomfort, dyspnea, gastroesophageal reflux, bleeding, infection, and cyst rupture. A spiration, sclerotherapy, fenestration, and/or segmental liver resection have limited therapeutic effects, and the only curative therapy is liver transplantation.^55,56

Autosomal dominant polycystic liver disease is a rare genetic disorder characterized by the development of numerous cysts in the liver without significant involvement of other organs. ADPLD is inherited in a dominant manner, but it is not clear how heterozygous mutations are involved. Single allele mutations have no effect on cholangiocyte function and polycystic liver disease only emerges once heterozygosity is lost. Second-hit events, such as loss of heterozygosity, have been identified in ADPKD patients with PKD1 and PKD2 germline mutations.⁵⁷ Protein kinase C substrate 80K-H (PRKCSH), SEC63, and LDL receptor-related protein 5 (LRP5) genes also are linked to the etiology of ADPLD. PKD1 mutations are notably more prevalent than PKD2 mutations and are linked to more severe disorders. PRKCSH, SEC63, or LRP5 mutations are present in 20% of ADPLD patients, and PRKCSH is detected in roughly 15% of all ADPLD patients.⁵⁷

Hepatic cystogenesis is believed to be linked to sensory dysfunction of the cilia, which arises from structural and functional abnormalities associated with aberrant development of components related to polycystic liver disease and cilia. Cystic cholangiocytes with shortened, excessively long, or entirely absent cilia have been observed in animal models of polycystic liver disease and in patients with ADPKD.^58,59 Anomalies in the primary cilium are associated with partially formed centrosomes, aberrant centrosome organization, and multipolar spindles.⁵⁹ Cholangiocytes with hyper-expanded centrosomes make up a significant proportion of the cells lining hepatic cysts. This connection between centrosome amplification and renal cystogenesis highlights the role of centrosome malfunction in hepatic cystogenesis.⁵⁹ Dysregulation of the calcium channel TRPV4⁶⁰ or the absence of proteins related to polycystic liver disease such as fibrocystin, polycystin-1, polycystin-2, and TGR5 cause functional abnormalities in cholangiocyte cilia and increased proliferation and fluid secretion, which aid in cyst progression.⁵⁹ Polycystin-1 is a key regulator of cyst growth that links cyst formation directly to primary cilia function, and its degree of expression seems to be involved in determining the severity of ADPKD,⁶¹ ARPKD, and ADPLD phenotypes.^62–64

Biliary Atresia

Biliary atresia is a rare, life-threatening disease where the bile ducts become inflamed and blocked, causing bile accumulation and liver damage. It often results in liver cirrhosis and requires transplantation if not promptly treated.

In a Pkd1l1-deficient mouse model displaying a biliary atresia phenotype, ciliary polycystin 1 like 1 (Pkd111) deficiency correlates with reduced ciliary presence in cholangiocytes and increased cell proliferation.⁶⁵ The liver-specific deletion of Pkd1l1 leads to bile duct dysmorphogenesis and ciliopathy. Cilia also have a role in bile duct development and flow, and changes in ciliary function may result in biliary atresia. Biliary organoids have shed new light on the relationship between cilia and bile duct formation, in addition to the function of cilia in bile duct integrity.⁶⁶ Cholangiocyte cilia have significant abnormalities in specimens from patients with biliary atresia, indicating that these structural defects may contribute to both syndromic and nonsyndromic types of the disease. Specific developmental defects, including laterality abnormalities such as situs in versus, are seen in 10 to 20% of biliary atresia cases. Patients with situs in versus have shorter and more sparse cilia, suggesting a potential shared pathogenic mechanism that may impact disease progression.⁶⁷

Subsequent studies show associations between primary cilia and the development of renal cysts in biliary atresia.⁶⁷ These findings demonstrate that ciliary abnormalities are not only isolated occurrences but may have wider consequences for related diseases, such as renal problems.

Whole exome sequencing has revealed rare mutations in ciliary genes in over 30% of patients, suggesting that intrinsic ciliary defects play a crucial role in the genesis of nonsyndromic biliary atresia.⁶⁸ This genetic predisposition was corroborated by a genome-wide association study that identified significant single nucleotide polymorphisms in genes such as actin filament-associated protein 1 and tumor suppressor candidate 3 that are implicated in ciliogenesis.⁶⁸ A polygenic risk linked to uncommon polymorphisms in the ciliogenesis and planar cell polarity effectors genes could be linked to developmental defects in biliary atresia, linking genetic predispositions to specific liver disorders.⁶⁹ These results collectively illustrate the multidimensional character of biliary atresia, emphasizing the critical roles of cilia and genetic factors in its development and progression.

Cholangiocarcinoma

Primary cilia act as a critical hub for cell cycle regulation, with their basal body, composed of centrioles, essential for spindle pole formation during mitosis. Their disassembly serves as a prerequisite for cell cycle re-entry, highlighting their significance in cell cycle dynamics. Primary cilia are thought to function as tumor suppressor organelles, as they harbor signaling pathways associated with cancer, and the loss of primary cilia is observed in many types of tumors, including cholangiocarcinoma. Primary cilia expression decreases during hyperplasia, while the absence of primary cilia typically elevates proliferation rates. Cells that are quiescent often harbor primary cilia, whereas the dismantling of these structures marks a cell’s re-entry into the cell cycle.

Notably, cholangiocarcinoma exhibits reduced ciliary expression in vivo and in vitro.⁷⁰ Removing primary cilia in normal cholangiocytes (by knocking out IFT88, IFT20, or KIF3A, or using drugs such as chloral hydrate) enhances malignancy, characterized by increased cell proliferation and anchorage-independent growth and invasion.⁷⁰ It also leads to the activation of the Hedgehog and MAPK pathways, which are typically suppressed by cilia and are implicated in promoting malignant cholangiocarcinoma phenotypes. In vivo studies demonstrate that deciliating CK19+ cells through KIF3A knockout in mice treated with thioacetamide results in cancer-like growth patterns, particularly biliary cell proliferation, and cysticliverlesions.⁷¹ In this model, the loss of primary cilia in vivo led to enhanced ERK signaling. Inhibition of ERK signaling subsequently reduced the expansion of primary cilia-deficient biliary progenitor cells in vitro. The mechanisms underlying the loss of cilia in cholangiocytes during cholangiocarcinoma include the overexpression of HDAC6 and silent information regulator sirtuin 1 (SIRT1).^70,72

The upregulation of HDAC6 and SIRT1 in normal cholangiocytes triggers deciliation and increases proliferation. Furthermore, the dysregulation of microRNAs (miRNAs) in cholangiocarcinoma contributes to HDAC6 overexpression, resulting in ciliary disassembly.⁷³ Conversely, inhibiting deacetylases like SIRT1^72,74 and HDAC6,^70,75 which destabilize the microtubules forming the primary cilia, promote ciliogenesis in tumor cells and reduce malignancy.^70,72–76 SIRT1 activity in cholangiocarcinoma is regulated by the availability of NAD +. Consequently, inhibiting nicotinamide phosphoribosyl transferase, an enzyme responsible for NAD+ production, also increases both the length and prevalence of cilia in cholangiocarcinoma cells.^76–78 Autophagy appears to play a crucial role in ciliary disassembly.^76,77,79 Overexpression of HDAC6 enhances autophagic flux, while its inhibition reduces it. Inhibiting both autophagy and HDAC6 leads to the restoration of cilia and a reduction in cell proliferation. HDAC6 and ciliary proteins interact with LC3, and in this context, NBR1 is an autophagy cargo receptor involved in directing ciliary components to the autophagy machinery.⁷⁹ Additionally, mice that overexpress the autophagy transcription factor TFEB have a cholangiocarcinoma-like phenotype and fewer cilia, further linking autophagy, cilia regulation, and cholangiocarcinoma.^79–81

Another study, although not focused on cholangiocarcinoma, highlights the importance of HDAC in cilia. In glioblastoma, suppressing HDAC6 significantly increased the percentage of ciliated cells that were positive for ADP-ribosylation factor-like protein 13B, thereby enhancing anti-proliferative and differentiation effects. However, the anti-proliferative effect of HDAC6 inhibition was mediated by primary cilia, as the therapeutic effectiveness of HDAC6 inhibitors decreased when ciliary production was genetically altered. This suggests that effective ciliary signaling is crucial for the efficacy of HDAC6-targeted cancer therapies.⁸²

In cholangiocarcinoma, LKB1 dysfunction is associated with tumor progression and poor prognosis, as mutations in the LKB1 gene impair cilia formation, thereby disrupting the signaling pathways essential for proper cholangiocyte function. This disruption activates carcinogenic pathways, such as mTOR and PI3K/AKT, promoting increased cell proliferation, invasion, and resistance to apoptosis.^41,42 The lack of functioning cilia resulting from LKB1 loss affects the cells’ sensitivity to growth stimuli and environmental cues, leading to tumor progression.

The chemosensory functions of primary cilia in cholangiocytes act as a significant tumor suppressor mechanism. Upon activation by extracellular nucleotides, these cilia inhibit the migration and invasion of cholangiocytes via a signaling cascade that involves the P2Y11 ciliary receptor and LKB1. The stimulation of deciliated cholangiocytes results in higher migration and invasion, suggesting that cilia are required for preserving the anti-tumorigenic characteristics of these cells. This underscores the crucial role of cilia and LKB1 function in regulating cellular activity and its potential implications for developing treatment strategies for cholangiocarcinoma.¹⁴

Primary Sclerosing Cholangitis

Primary sclerosing cholangitis is a chronic disease characterized by progressive inflammation and fibrosis of the bile ducts, thought to have both autoimmune and genetic components. It primarily affects young males with inflammatory bowel disease.⁸³ Its autoimmune aspect is suggested by its strong association with inflammatory bowel disease, particularly ulcerative colitis, and the presence of immune-mediated features such as aberrant T-cell responses. However, classical autoantibodies like ANCA are not considered definitive markers of disease pathogenesis.⁸⁴

Primary sclerosing cholangitis may involve autoimmune-mediated processes since patients often exhibit antibodies against neutrophil cytoplasmic and nuclear antigens with a perinuclear staining pattern.^85,86 While the exact etiology of the disease remains unclear, studies have linked it to the interleukin-2 pathway, particularly genes such as CD28 and the HLA-DRB1 and HLADQB1 haplotypes. Characteristic symptoms include fever, upper abdominal discomfort, fatigue, pruritus, and jaundice, which are triggered by inflammatory and cholestatic mechanisms that promote fibrosis and cirrhosis.⁸⁶ Primary sclerosing cholangitis is a risk factor for cholangiocarcinoma.⁸⁵ Cholangiograms are the diagnostic gold standard for primary sclerosing cholangitis, since biochemical tests may not reliably reflect disease progression.⁸⁵ While liver transplantation is the only treatment option, it is associated with high rates of acute cellular rejection, recurrence, and progression of inflammatory bowel disease.^85,86

Although primary sclerosing cholangitis is not officially classified as a ciliopathy, some researchers suggest it could be, as cholangiocytes in this condition have larger primary cilia compared with healthy ones⁸⁷; however, experimental confirmation is required. Furthermore, elegant studies in human tissues have shown that the percentage of primary cilia expression is significantly reduced in dysplastic peribiliary glands in PSC-affected ducts, while primary cilia almost completely disappear in neoplastic glands in PSCCCA samples versus normal and PSC-affected ducts.⁸⁸ Ciliary dysfunction is associated with impaired signaling and abnormal cellular responses, leading to cholangiocyte hyper-proliferation, a key characteristic of primary sclerosing cholangitis.⁸⁹ Changes in ciliary structure and function contribute to inflammation and fibrosis in the bile ducts in patients with primary sclerosing cholangitis.^89,90 Reduced ciliary function increases the expression of pro-inflammatory cytokines and growth factors, worsening cholestasis and liver injury. Given the critical role of cilia in cholangiocyte activity, restoring normal ciliary function or targeting ciliary-related pathways could offer promising therapeutic approaches for primary sclerosing cholangitis.⁹⁰ Another immune-related condition is primary biliary cholangitis, in which the immune system attacks the bile ducts; however, no significant link to primary cilia has been established.

Dysregulated signaling pathways appear to interact with ciliary function to exacerbate the progression of primary sclerosing cholangitis. For instance, reduced expression of TGR5, a bile acid receptor, impairs cholangiocyte cilia-mediated regulation of intracellular cAMP levels, disrupting bile acid signaling and promoting inflammation in patients with primary sclerosing cholangitis.^17,47 This dysregulation enhances the release of pro-inflammatory cytokines such as IL-6 and TNF-α, which drive fibrotic responses.⁹¹ Additionally, Hedgehog signaling, typically modulated by primary cilia, becomes hyperactivated in primary sclerosing cholangitis, contributing to epithelial-to-mesenchymal transition in cholangiocytes.²³ These findings underscore the importance of cilia-mediated signaling in primary sclerosing cholangitis and highlight potential therapeutic targets for intervention.

Clinical Implications and Therapeutic Prospects

Cilia regulate cell proliferation, but cancer cells often inhibit ciliogenesis, promoting growth. Thus, restoring ciliary function in tumors is a promising strategy. In cholangiocarcinoma, HDAC6 inhibition partially restores cilia and reverses malignancy. HDAC6 is overexpressed in CCA, and its inhibition leads to ciliary restoration, reduced cell proliferation, decreased anchorage-independent growth, and the downregulation of oncogenic pathways, including Hedgehog and MAPK. These effects depend on the presence of cilia, as shown in experiments where the knockdown of the ciliary protein IFT88 via shRNA inhibited ciliogenesis, rendering the malignant phenotype resistant to reversal by the HDAC6 inhibitor tubastatin-A.^70,92 Tubastatin-A reduced proliferation, restored cilia, and decreased tumor growth in a rat model of cholangiocarcinoma.⁷⁰ Additionally, sirtinol, a SIRT1 inhibitor, reduced tumor size; decreased expression of tumorigenic proteins such as GLI1, phosphorylated ERK, and IL-6; and increased expression of the tumor suppressor p53.⁷² The disassemblyof primarycilia, driven by HDAC6 and SIRT1, is linked to degradative pathways, including ubiquitination and autophagy.⁷⁹

Autophagy maintains the structural integrity of primary cilia through the degradation and recycling of proteins required for ciliogenesis. In polycystic liver disease, increased autophagy contributes to the ubiquitination and degradation of essential ciliogenic proteins, including ARL3 and ARL13B.⁹³ This process hinders ciliogenesis and disrupts the localization of important receptors such as TGR5, PDGFR, and TGF-β receptors.⁹³ Pharmacologic inhibition of autophagy with agents such as mefloquine and verteporfin restored ciliogenesis, reestablished receptor localization, and reduced cyst formation.⁹³

Similarly, SIRT1 promotes the degradation of ciliary proteins, including α-tubulin, ARL13B, and KIF3A, through a proteasome-mediated pathway. In an orthotopic rat model of cholangiocarcinoma, treatment with ACY-1215, chloroquine (which inhibits autophagy), or both reduced tumor growth.⁷⁹ In in vitro models of cholangiocarcinoma and polycystic liver disease, HDAC6 and EGFR inhibitors demonstrated synergistic anti-proliferative effects, highlighting the potential of cilia-targeted therapies to modulate key oncogenic pathways and improve treatment outcomes.⁴⁶

In a mouse model of liver cold storage, damage to primary cilia led to biliary injury and reduced regenerative capacity in the context of ischemia-reperfusion injury during liver transplantation. Tubastatin-A, known for its role in stabilizing primary cilia, increased levels of acetylated α-tubulinin biliary endothelial cells, indicating enhanced ciliary stability.⁹⁴ Ex vivo experiments using bile duct organoids from livers treated with tubastatin-A during cold storage showed significantly enhanced growth rates compared with control groups. Subsequent studies in wild-type mice before ischemia-reperfusion induced hepatic injury demonstrated that tubastatin-A reduced apoptosis and decreased biliary damage in biliary epithelial cells. These findings suggest that stabilizing primary cilia may protect against bile duct damage and apoptosis of biliary epithelial cells. These data suggest that cilia-targeted strategies with HDAC6 inhibitors like tubastatin-A could be therapeutic options for preserving bile duct function during organ storage and transplantation.⁹⁴

In glioblastoma, inhibition of HDAC6 with ACY-1215 or ACY-738 disrupted the G2/M transition, increasing cell death and promoting cell cycle exit and differentiation of tumor cells in a cilia-dependent manner.⁸² Additionally, butyrate enhances primary cilia expression, and it decreases proliferation in cholangiocarcinoma cells. Butyrate also significantly potentiated the effects of HDAC6 inhibitors on cholangiocarcinoma.⁷⁴ In an orthotopic rat model of cholangiocarcinoma, hesperidin methyl chalcone, an activator of cytoplasmic LKB1, reduced tumor growth and increased apoptosis, compensating for the absence of primary cilia.¹⁴

Conclusion

Despite advances in understanding cholangiopathies, current treatments remain limited. The ability to study and model key aspects of cholangiocyte signaling pathways, along with evidence that several druggable pathways are disrupted, has sparked a growing interest in cholangiopathies, which are often considered rare and less frequently characterized diseases. Primary cilia are essential organelles for maintaining normal biliary health. In cholangiopathies, ciliary defects trigger a ductular response that leads to biliary dysfunction. Research into the pathophysiology of cholangiocarcinoma and polycystic liver disease has revealed a close connection between fundamental processes and specific ciliary dysfunctions, which in turn impact key morphogenetic pathways, including β-catenin, Hedgehog, Notch, EGFR/IGF, and TGR5.

Additionally, cholangiocyte ciliotherapy appears to play a critical role in hepatic cystogenesis and the control of cholangiocarcinoma and polycystic liver disease. Restoring primary cilia in cholangiocytes has emerged as a promising therapeutic strategy, with recent advances in cholangiopathy research highlighting the potential of ciliotherapies. Drugs that restore ciliary function—such as ACY1215 and SIRT1 inhibitors in cholangiocarcinoma and polycystic liver disease—have suggested additional signaling pathways as promising pharmacologic targets. However, the practical application of ciliotherapies still faces significant hurdles. One major challenge is the risk of side effects, such as off-target actions or toxicity, which can limit their use in patients. Additionally, delivering these therapies specifically to cholangiocytes without affecting other cells remains a technical challenge. Developing targeted delivery systems, such as nanoparticle-based carriers or cell-specific drug formulations, could help address these issues, but requires further research and innovation. Finally, more extensive clinical trials are needed to ensure that ciliotherapies are both effective and safe. Balancing the therapeutic benefits with potential risks will be crucial as these therapies move closer to clinical application.