Mechanisms of cholangiocyte responses to injury

Abstract

Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis.

1. Introduction

The intrahepatic and extrahepatic biliary epithelium is lined with bile duct epithelial cells, i.e., cholangiocytes [1]. Cholangiocytes are active secretory cells that modify the composition of canalicular bile by secreting water, bicarbonate, and chloride ions [2, 3]. Although cholangiocytes are normally quiescent, these cells can also respond to liver or biliary damage and show various specific reactions in response to experimental triggers including partial hepatectomy, bile duct ligation (BDL), and bile acid feeding [4, 5]. Various genetically modified mouse models, such as MDR2^−/− and Tgfbr2^−/− mice, are also used for experimental cholestatic liver injury [6]. Cholangiocytes are the main target of cholangiopathies, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Characteristic responses of cholangiocytes during cholestatic liver injury include enhanced cholangiocyte proliferation, related changes in biliary apoptosis and liver fibrosis, and upregulation of the cytokine expression such as interleukin (IL)-6 [7–9]. In addition, recent studies have shown that cholangiocyte senescence is a characteristic of PSC and that it is associated with other cholangiopathies [10–12]. Cholangiocytes are functionally heterogeneous cells and respond differently to specific pathological triggers. The gastrointestinal peptide hormone, secretin (SCT), binds to secretin receptor (SR), and the SCT/SR axis is critically involved in functions of cholangiocytes and pathophysiology of cholestatic liver injury [13]. Although various extracellular messengers and their receptors have been reported to regulate cholangiocyte proliferation, detailed mechanisms of regulation of cholangiocyte responses are not fully understood [14, 15]. This review summarizes current understandings of mechanisms of cholangiocyte responses and their regulations with particular emphasis on the SCT/SR axis that is only expressed in cholangiocytes in the liver [16, 17].

2. Cholangiocyte heterogeneity

Intrahepatic bile ducts are heterogeneous in external diameter (5–200 μm) and individual cholangiocytes are also heterogeneous in diameter [17]. In rats, bile ducts with a diameter greater than 15 μm are termed large bile ducts and cholangiocytes lining those ducts are termed large cholangiocytes. Small bile ducts have a diameter less than 15 μm and consist of small cholangiocytes [18]. Small and large cholangiocytes have differences not only in diameter but also in protein expression and functions [17, 19]. Large but not small cholangiocytes express SR, cystic fibrosis transmembrane conductance regulator (CFTR), Cl⁻/HCO₃⁻ anion exchanger 2 (AE2), and somatostatin receptor (SSTR₂) [16, 20–22]. Large but not small cholangiocytes perform ductular secretion induced by SCT [16]. In addition, previous studies have shown that small and large cholangiocytes respond differently to various experimental biliary damage.

BDL is a surgical obstruction of the common bile duct and is widely used in rodents as an animal model of cholestasis and cholestatic liver injury in humans [23]. During BDL, cholangiocytes extensively proliferate and biliary hyperplasia is observed in rodent livers [24–26]. However, only large cholangiocytes proliferate during BDL. After one week of BDL, large bile duct mass increases but small bile duct mass remains pre-BDL levels [20]. Carbon tetrachloride (CCl₄) administration by oral gavage is also commonly used to induce chronic liver damage, fibrosis, and hepatocellular carcinoma in rodents [27]. During CCl₄ administration, large but not small cholangiocytes are damaged by enhanced apoptosis, and small cholangiocytes de novo proliferate to compensate for the functional loss of large cholangiocytes [28, 29]. Interestingly, these studies have also shown that small cholangiocytes, which do not express endogenous SR at normal conditions, de novo express SR when large cholangiocytes are damaged. Later studies also demonstrated that γ-aminobutyric acid damages large cholangiocytes but induces differentiation of small into large cholangiocytes in a Ca²⁺-dependent pathway [30, 31]. These studies suggest that small cholangiocytes have characteristics of progenitor cells and differentiate into large cholangiocytes when large bile ducts are damaged and need to be repaired.

Although small and large cholangiocytes respond to BDL and CCl₄ differently, both cells respond similarly to bile acids. Small and large cholangiocytes isolated from rats show increased proliferation and expression of Na⁺-dependent apical bile acid transporter against taurocholate and taurolithocholate in vitro [32, 33]. Taurocholate feeding also attenuates large cholangiocyte damage caused by CCl₄ by inducing proliferation and inhibiting apoptosis in rats [34]. Both small and large cholangiocytes also respond similarly to α-naphthylisothiocyanate (ANIT). ANIT feeding damages bile ducts and increases apoptosis as well as proliferation in both small and large cholangiocytes in rats [35]. Histamine is a mediator for the local immune system inducing inflammation. Studies have shown that histamine induces cell proliferation in both small and large cholangiocytes [36, 37]. These findings suggest that while small and large cholangiocytes have different functions and mechanisms for proliferation and injury response, these two subsets of cholangiocytes share the same response (or similar response) to specific triggering agents.

3. Mechanisms of cholangiocyte proliferation and functions

Adenosine 3′,5′-cyclic monophosphate (cAMP) is a key messenger in cholangiocytes for their proliferation and function. The neuropeptide hormone SCT binds to SR that is expressed only in the basolateral membrane of cholangiocytes, and this SCT binding and SR activation elevate intracellular cAMP levels leading to enhanced cell proliferation, exocytosis and ductular secretion in cholangiocytes [38, 39]. Elevated intracellular cAMP levels increase bicarbonate secretion from cholangiocytes in bile through CFTR-dependent ATP release [40]. Triggering agents that induce cAMP outputs, such as follicle stimulating hormone or forskolin, increase intracellular cAMP levels in cholangiocytes leading to proliferation via cAMP-dependent PKA/MEK/ERK1/2/Elk-1 signaling [41, 42]. As mentioned above, large but not small cholangiocytes express CFTR and SR, and therefore only large cholangiocytes perform CFTR- or SR-dependent proliferation and bicarbonate secretion in the liver. Another study has also shown that large but not small cholangiocytes are involved in SCT-induced ductular secretion [16].

In small cholangiocytes, cAMP levels are involved in proliferation, but Ca²⁺ signaling is also important. During histamine-induced small cholangiocyte proliferation, inositol 1,4,5-trisphosphate (IP₃) levels are increased [37]. IP₃ binds to IP₃ receptor (IP₃R), which is a calcium channel located on the endoplasmic reticulum (ER), a calcium storage organelle in the cell. Activated IP₃R by IP₃ binding releases Ca²⁺ from the ER into the cytosol. This Ca²⁺ release in the cytosol activates calcineurin (CN) and calmodulin (CaM). CN phosphorylates nuclear factor of activated T-cells (NFAT) proteins, and CaM leads to activation of CaM-dependent kinase (CaMK) [43, 44]. Studies have shown that small cholangiocytes proliferate through this IP₃/Ca²⁺/CaMK signaling [36, 37]. Another study has shown that activation and nuclear translocation of NFAT2 (also known as NFATc1) are increased in proliferating small cholangiocytes [45]. This Ca²⁺/CN/NFATc1 pathway is also associated with cell proliferation in hepatocellular carcinoma [46]. In large cholangiocytes, however, Ca²⁺ signaling may not be critical for cell proliferation during cholestatic liver injury. A study has shown that expression levels of IP₃R are decreased in large cholangiocytes during BDL [47]. Although cAMP pathways may be more important for large cholangiocyte proliferation than IP₃/Ca²⁺ pathways, Ca²⁺ signaling may be more important than cAMP pathways for ductular secretion in large cholangiocytes. Large but not small cholangiocytes perform ductular secretion induced by triggers [16], and a previous study has demonstrated that trigger-induced Cl⁻ secretion is Ca²⁺-dependent [48]. This study has also shown that the impact of Ca²⁺-induced ductular secretion is significantly greater than cAMP-induced secretion mediated by Ca²⁺-activated K⁺ channel in cholangiocytes. These findings suggest that cAMP as well as Ca²⁺ signaling pathways are involved in cholangiocyte proliferation and secretion depending on its heterogeneity. Figure 1 summarizes the mechanisms of cell proliferation in small and large cholangiocytes.

Figure 1. Mechanisms of cholangiocyte proliferation.

Small and large cholangiocytes have different mechanisms for proliferation. Proliferation for small cholangiocytes, which are smaller in size and have a larger nucleus to cytosol ratio compared to large cholangiocytes, is dependent on intracellular Ca²⁺ release induced by IP₃ while large cholangiocyte proliferation is cAMP-dependent. Binding of messengers such as histamine for small cholangiocytes and SCT for large cholangiocytes to their corresponding receptors triggers these pathways leading to cholangiocyte proliferation. When large cholangiocytes are damaged by triggers such as γ–aminobutyric acid or CCl₄, small cholangiocytes begin to express SR and differentiate into large cholangiocytes in order to maintain bile duct homeostasis and function. Signaling mechanisms on how damaged large cholangiocytes initiate differentiation in small cholangiocytes are still unknown.

4. Pathways associated with cholangiocyte responses and their regulations

Cholangiocytes are responsive cells and various messengers and regulators for cholangiocyte proliferation have been identified to date [15]. Recent studies have shown that some messengers and their receptors are critically involved in cholangiopathies and cholangiocyte responses to injury. This section summarizes specific pathways that are associated with cholangiocyte responses in cholestatic liver injury.

4.1. The secretin/secretin receptor axis

As described above, large cholangiocytes express various membrane proteins and channels, which small cholangiocytes do not express endogenously, such as SR, CFTR, SSTR₂, and AE2 [19]. The signaling pathway of SCT and its receptor SR is one of the most studied messengers/receptors in cholestatic liver injury. It has been suggested that the SCT/SR axis is associated with cholestatic liver injury because rats show elevated expression of SR after BDL [49]. SCT administration into rats increases expression of SR and CFTR as well as intracellular cAMP levels and cholangiocyte proliferation in vivo [50]. SCT is expressed by cholangiocytes and S cells which are located primarily in the mucosa of the duodenum [51]. During BDL, both large cholangiocytes and S cells show elevated SCT expression [52]. SCT^−/− mice show attenuated bile duct hyperplasia after BDL compared to wild-type mice suggesting that SCT is a key messenger in regulating large cholangiocyte proliferation in an autocrine/paracrine manner [52]. SR^−/− mice also show reduced intracellular cAMP levels, ERK1/2 phosphorylation and cholangiocyte proliferation during BDL [53]. In human PSC, bile duct hyperplasia as well as liver fibrosis are often observed as common characteristics [54]. A recent study has shown that PSC patients show higher expression levels of SCT and SR in the liver compared to healthy individuals [55]. Mdr2^−/− mice are widely used as a mouse model of human PSC [56, 57]. This study has also demonstrated that administration of SR antagonist into BDL or Mdr2^−/− mice attenuated both bile duct hyperplasia and liver fibrosis compared to respective controls [55]. These findings suggest that the SCT/SR axis is an important signaling pathway for cholangiocyte responses in cholangiopathies. In addition, we have demonstrated that SR^−/− mice show less liver steatosis with high fat diet compared to wild-type mice suggesting that the SCT/SR axis may play a key role not only in cholangiopathies but also in non-alcoholic steatohepatitis (NASH) [58]. Further studies will reveal functional roles of cholangiocytes in lipid metabolisms and liver steatosis during the development of non-alcoholic fatty liver disease (NAFLD) and NASH. Figure 2 summarizes functions of the SCT/SR axis in liver diseases.

Figure 2. The function of the SCT/SR axis in liver diseases.

SCT binds to SR that is expressed only in large cholangiocytes in the liver, and SCT binding and SR activation lead to elevated exocytosis and ductular secretion. Recent studies have shown that human patients with cholangiopathies such as PSC show elevated expression of SCT and SR indicating an association between the SCT/SR axis and cholestatic liver injury. Studies using SCT^−/− and SR^−/− mice have demonstrated that bile duct hyperplasia and liver fibrosis are attenuated in those knockout mice during BDL suggesting the functional contribution of the SCT/SR axis to the pathogenesis of cholangiopathies. A recent study has also demonstrated that the SCT/SR axis may be responsible for liver steatosis leading to NAFLD and NASH.

4.2 The substance P/neurokinin-1 receptor axis

Substance P (SP) is a neuropeptide that binds to neurokinin-1 receptor (NK-1R). It is known that SP induces inflammatory responses by elevating cytokine expression [59]. In cholestatic liver injury, elevated serum levels of SP are observed in patients with chronic liver disease as well as in cholestatic rat models compared to respective controls suggesting an association between SP production and cholestatic liver damage [60]. Elevated SP secretion is also identified in cholangiocarcinoma (CCA) cell lines and administration of NK-1R antagonist L-733, 060 inhibits proliferation of CCA cells [61]. A study has demonstrated that BDL mice show elevated NK-1R expression in large cholangiocytes, and NK-1R^−/− mice show attenuated large cholangiocyte proliferation and bile duct hyperplasia [62]. This study has also demonstrated that SP administration elevates intracellular cAMP levels followed by cell proliferation in large cholangiocytes suggesting that the SP/NK-1R axis is associated with large cholangiocyte proliferation. A recent study has shown that serum levels of SP and NK-1R expression in the liver are elevated in human PSC patients compared to healthy individuals [63]. In addition, SP administration induces bile duct hyperplasia and liver fibrosis in wild-type mice, and administration of NK-1R antagonist attenuates liver fibrosis in Mdr2^−/− mice and BDL mice [63]. These findings suggest that the SP/NK-1R axis plays a key role in bile duct proliferation and fibrosis in cholangiopathies.

4.3 The gonadotropin-releasing hormone and its receptor

Gonadotropin-releasing hormone (GnRH) is a tropic peptide hormone that modulates cell proliferation. GnRH has stimulatory and inhibitory effects on cell proliferation depending on the cell type [64]. A recent study has shown that cholangiocytes express GnRH receptor 1 (GnRHR₁) and its expression levels are elevated during BDL in rats [65]. This study has also demonstrated that administration of GnRH enhances bile duct mass as well as intracellular cAMP levels, expression of SR, CFTR and AE2 in cholangiocytes in vivo. GnRH Vivo-Morpholino attenuates bile duct hyperplasia and fibrosis induced by BDL suggesting that GnRH is a key messenger for cholangiocyte responses in cholestatic liver injury [65]. A recent study has shown that serum levels of GnRH and GnRHR₁ expression in the liver are elevated in human PSC patients compared to healthy individuals [66]. This study has also demonstrated that GnRH and GnRHR₁ expression is enhanced in cholangiocytes isolated from Mdr2^−/− mice, and GnRH Vivo-Morpholino attenuates bile duct hyperplasia and fibrosis in these mice suggesting that the GnRH/GnRHR₁ axis is associated with cholangiocyte proliferation and liver fibrosis in cholestatic liver injury.

4.4 Mast cell-derived histamine and histamine receptors

As described above, histamine induces small and large cholangiocyte proliferation [36, 37]. Histamine interacts with G protein-coupled histamine receptors: H1 to H4 [67]. Interestingly, agonists for H1 histamine receptor induce small cholangiocyte proliferation, and agonists for H2 induce large cholangiocyte proliferation [36, 37]. It is known that mast cells secrete histamine and the number of mast cells in the liver is increased during cholestatic liver injury such as PBC [68]. A recent study has shown that inhibition of mast cell-derived histamine secretion attenuates bile duct hyperplasia during BDL in vivo [69]. Another study has shown that serum levels of histamine are increased in Mdr2^−/− mice, and inhibition of mast cell-derived histamine secretion decreases serum histamine levels as well as bile duct hyperplasia and fibrosis in these mice [70]. Mast cell-deficient mice show less liver damage, bile duct mass, and fibrosis during BDL compared to wild-type [71]. These studies suggest that histamine secreted from mast cells infiltrated during cholestatic liver injury interacts with histamine receptors and regulates cholangiocyte responses leading to bile duct hyperplasia and fibrosis.

4.5 Melatonin and melatonin 1A receptor

Melatonin is a hormone produced by the pineal gland, small intestine and liver [72]. Melatonin regulates sleep as a part of the circadian rhythm as well as cell proliferation [73]. A previous study has demonstrated that melatonin administration in rats reduces liver fibrosis and serum cytokine levels for IL-1β and IL-6 during BDL [74]. Other studies have also shown that melatonin administration protects BDL- or ANIT-induced liver damage in vivo suggesting that melatonin has therapeutic effects on cholestatic liver injury [75, 76]. A recent study has demonstrated that melatonin administration inhibits GnRH secretion and reduces bile duct hyperplasia and liver fibrosis in BDL rats (McMillin et al. in press). Melatonin interacts with G protein-coupled melatonin 1A (MT1), MT2, and MT3 receptors [77]. A study has demonstrated that melatonin administration reduces intracellular cAMP levels and large cholangiocyte proliferation in BDL rats by activation of MT1 but not MT2 melatonin receptor [78]. Melatonin is an indole produced by arylalkylamine N-acetyltransferase (AANAT) [79]. A recent study has shown that melatonin administration increases AANAT expression in cholangiocytes [80]. This study has also demonstrated that AANAT Vivo-Morpholino exacerbates BDL-induced bile duct hyperplasia, and overexpression of AANAT in cholangiocytes reduces cell proliferation in vitro suggesting an association between AANAT followed by melatonin synthesis and cholangiocyte proliferation. Melatonin secretion is induced when exposed to darkness. BDL rats housed in complete darkness for one week show elevated AANAT expression and melatonin production compared to BDL rats with 12:12-hour light-dark cycles (Wu et al. in press #1). This study has demonstrated that complete darkness reduces bile duct mass and liver fibrosis during BDL by reducing intracellular cAMP levels. These findings suggest that the AANAT/melatonin/MT1 receptor axis could be a potential target for novel treatments of cholestatic liver injury.

4.6 Vascular endothelial growth factors and their receptors

Vascular endothelial growth factor (VEGF) is a protein family of growth factors including VEGF-A, -B, -C, -D, and -E [81, 82]. Three VEGF receptors (VEGFR-1 to -3) have been identified to date, and it is known that VEGF-A interacts with VEGFR-2 and VEGF-C interacts with VEGFR-3 [83, 84]. A previous study has shown that cholangiocytes express VEGF-A and -C as well as VEGFR-2 and -3, and these VEGF-A/VEGFR-2 and VEGF-C/VEGFR-3 axes are upregulated during BDL in rats [85]. This study has also demonstrated that administration of VEGF-A/C induces cholangiocyte proliferation by elevating intracellular IP₃ levels and ERK1/2 phosphorylation. AANAT expression is correlated with VEGF-A/C expression and AANAT overexpression reduces VEGF-A/C expression in vitro, and AANAT Vivo-Morpholino increases VEGF-A/C expression in vivo [86]. Pancreatic duodenal homeobox-1 (PDX-1) is a transcription factor and expression levels of PDX-1 are upregulated in cholangiocytes isolated from BDL rats [87]. Expression levels of PDX-1 are associated with VEGF expression in cholangiocytes and siRNA transfection for PDX-1 inhibits VEGF expression [87]. Another study has demonstrated that PDX-1^+/− mice show attenuated bile duct mass and liver fibrosis during BDL compared to wild-type [88]. These studies suggest that the PDX-1/VEGF-A/C/VEGFR-2/3 axis is associated with cholangiocyte proliferation and liver fibrosis in cholestatic liver diseases.

4.7 Galanin and galanin receptor 1

Galanin is a neuropeptide that interacts with three G protein-coupled receptors: galanin receptor 1 (GalR1), GalR2 and GalR3 [89]. Serum levels of galanin and GalR1 expression in cholangiocytes are elevated in BDL rats [90]. Administration of galanin induces bile duct hyperplasia in normal rats and galanin Vivo-Morpholino attenuates BDL-induced bile duct hyperplasia and fibrosis [90]. Although there are limited numbers of studies, the galanin/GalR1 axis may be associated with cholestatic liver injury.

4.8 Receptors activated by bile acids

As mentioned above, bile acids induce cholangiocyte proliferation. During cholestasis, accumulated bile acids stimulate cholangiocytes leading to bile duct hyperplasia. TGR5 (also known as G protein-coupled bile acid receptor 1, GPBAR1) is a specific receptor for bile acids. It is known that activation of TGR5 is associated with intracellular cAMP elevation in various cells [91]. Several mutations of the TGR5 gene have been identified from human PCS patients suggesting an association between TGR5 and cholangiopathies [92]. In cholangiocytes, the majority of expressed TGR5 is localized on the primary cilium [93]. A recent study has shown that taurolithocholic acid and other TGR5 agonists induce cholangiocyte proliferation via ERK1/2 phosphorylation [94]. This study has also demonstrated that TGR5^−/− mice show reduced bile duct hyperplasia during BDL as well as cholic acid feeding compared to wild-type or control feeding suggesting that TGR5 is required for cholestasis- or bile acid-induced cholangiocyte proliferation. Another study, however, has demonstrated that TGR5 agonists inhibit cholangiocyte proliferation [95]. Further studies are required to elucidate the mechanisms of regulation of cholangiocyte proliferation by bile acids and TGR5 activation.

Sphingosine 1-phosphate receptor 2 (S1PR2) is activated by bile acids as well as TGR5, and accumulating evidence suggests that S1PR2 signaling is associated with biliary diseases [96]. A recent study has shown that S1PR2 expression is elevated in cholangiocytes during BDL, and taurocholate induces cholangiocyte proliferation by ERK1/2 activation and enhanced S1PR2 expression [97]. This study has also demonstrated that S1PR2^−/− mice show reduced bile acid secretion, bile duct mass, and liver fibrosis during BDL. These studies suggest that bile acids followed by activation of receptors may be an important signaling pathway in pathogenesis of cholestatic liver diseases.

4.9 Yes-associated protein

Yes-associated protein (YAP) is a transcriptional regulator for development, repair and proliferation of liver cells [98]. A recent study has demonstrated that patients with biliary atresia (BA) show bile duct hyperplasia and enhanced YAP expression in cholangiocytes compared to non-BA patients, and hence YAP expression levels in bile ducts can be a useful biomarker for diagnosis of BA [99]. Another study has shown that YAP expression in bile ducts is upregulated in human PSC and PBC patients, and YAP^−/− mice show attenuated cholangiocyte proliferation and bile duct hyperplasia during BDL [100]. YAP is a major mediator of the Hippo signaling pathway, and a recent study has shown that the loss of Hippo-YAP signaling enhances the expression of Notch signaling pathway receptor Notch2 (Wu, et al. in press #2). This study has also demonstrated that the loss of both Hippo and Notch signaling results in excessive bile duct development suggesting that Hippo-YAP signaling is associated with Notch signaling and bile duct development. These studies suggest that YAP signaling is associated with cholangiocyte proliferation and bile duct development although further studies are required to understand the detailed mechanisms and functional roles of YAP signaling in cholangiopathies. Figure 3 illustrates regulations of cholangiocyte responses by messengers and their receptors.

Figure 3. Working model of cholangiocyte responses during cholangiopathies.

Cholangiocytes are quiescent in healthy conditions (left). During cholestatic liver injury, expression levels of messengers such as SCT and SP as well as corresponding receptors such as SR and NK-1R are elevated in cholangiocytes (right). Binding of messengers to receptors in an autocrine/paracrine manner triggers signaling pathways for proliferation and fibrogenesis leading to bile duct hyperplasia and liver fibrosis. Although specific or primary locations are not identified for all proteins, some proteins such as TGR5 are located on primary cilia or apical membrane, and some proteins such as SR and CFTR are located on the basolateral membrane.

5 Regulation of cholangiocyte responses by extracellular vesicles

Extracellular vesicles (EVs) are membrane-bound vesicles secreted by various cell types. Recent studies suggest that EVs may play an important role in liver diseases [101, 102]. EVs contain various functionally active mediators including proteins, mRNAs and microRNAs (miRNAs). Secreted EVs can be transferred from donor cells to recipient cells and regulate pathophysiological events by delivering cargo mediators. Physiology and functions of cholangiocytes are regulated by other cholangiocytes or other liver cells during cholestatic liver injury. For example, Kupffer cells secrete inflammatory cytokines such as IL-6 during biliary damage and elevated IL-6 stimulates cholangiocytes to proliferate [103, 104]. Cholangiocyte proliferation and response could be regulated by EVs secreted from other cholangiocytes or liver cells. Cholangiocytes have primary cilia, which are chemosensory organelles, to detect and maintain bile homeostasis [105]. As described above, bile acids induce cholangiocyte proliferation and bile acid receptor TGR5 is expressed primarily in cilia in cholangiocytes. A previous study has shown that bile contain EVs and those biliary EVs interact with cholangiocyte cilia to downregulate cholangiocyte proliferation [106]. Another study has demonstrated that this downregulation of cholangiocyte proliferation caused by biliary EVs is dependent on TGR5 located on primary cilia [95]. These findings suggest that cholangiocyte response and proliferation may be regulated by EVs and their cargo mediators. EV transfer and pathophysiological regulation may be performed between cholangiocytes or cholangiocytes and other liver cells in an autocrine or paracrine manner. Further studies are required to elucidate mediators and detailed mechanisms of cholangiocyte regulation by EVs during cholestatic liver diseases.

6 Cholangiocyte regulation by microRNAs

Accumulating evidence suggests that miRNAs could be the key mediators for regulation of cholangiocyte response. During SCT-induced proliferation, miRNAs 125b and let7a, which regulate VEGF expression, are downregulated and Vivo-Morpholino for these miRNAs exacerbate bile duct hyperplasia in BDL mice by elevating VEGF expression and cholangiocyte proliferation [52]. Another study has demonstrated that miR-124 is downregulated during BDL and this leads to cholangiocyte proliferation in an IL-6-dependent mechanism [107]. This study has also found that miR-200 families are upregulated in BDL mice. A recent study has demonstrated that human PSC patients show elevated miR-200b expression in the liver compared to healthy individuals and miR-200b Vivo-Morpholino attenuates bile duct hyperplasia and liver fibrosis in Mdr2−/− mice (Wu et al. in press #1). Another study has also demonstrated that miR-200b expression is upregulated during GnRH-induced cell proliferation suggesting an association between miR-200b and cholangiocyte responses in cholangiopathies [66]. As described above, the PDX-1/VEGF-A/C axis is associated with cholangiocyte proliferation and liver fibrosis. A recent study has demonstrated that upregulation of miR-7a is associated with VEGF-A/C expression and bile duct hyperplasia during biliary damage [108]. Several miRNAs have been identified as candidate miRNAs that are associated with cholestatic liver diseases [109]. Expression of these miRNAs can be regulated endogenously or by EVs in an autocrine or paracrine manner. Further studies are required to elucidate detailed mechanisms of miRNA-mediated cholangiocyte responses.

7 Conclusion and future perspectives

Cholangiocytes are the main target cells in cholestatic liver diseases and current studies have shown that various pathways and axes are associated with cholangiocyte proliferation and response to biliary damage. These pathways and axes could be potential therapeutic targets for novel treatments. It is largely unclear, however, how these axes are regulated and what mediators are involved. Although EVs and miRNAs may be involved in regulation of those axes, further studies are required.

Highlights.

• Cholangiocytes are heterogeneous showing different responses to injury

• Typical cholangiocyte responses are proliferation and fibrogenesis

• The secretin/secretin receptor axis is important for cholangiocyte responses

• There are several other pathways associated with cholangiocyte proliferation

Abbreviation

AANAT

arylalkylamine N-acetyltransferase

AE2

Cl⁻/HCO₃⁻ anion exchanger 2

ANIT

α-naphthylisothiocyanate

BA

biliary atresia

BDL

bile duct ligation

cAMP

adenosine 3′,5′-cyclic monophosphate

CCA

cholangiocarcinoma

CCl₄

carbon tetrachloride

CFTR

cystic fibrosis transmembrane conductance regulator

CaM

calmodulin

CaMK

CaM-dependent kinase

CN

calcineurin

ER

endoplasmic reticulum

EVs

extracellular vesicles

GalR1

galanin receptor 1

GnRH

gonadotropin-releasing hormone

GnRHR₁

GnRH receptor 1

GPBAR1

G protein-coupled bile acid receptor 1

IL

interleukin

IP₃

inositol 1,4,5-trisphosphate

IP₃R

IP₃ receptor

miRNAs

microRNAs

MT1

melatonin 1A

NAFLD

non-alcoholic fatty liver diseases

NASH

non-alcoholic steatohepatitis

NFAT

nuclear factor of activated T-cells

NK-1R

neurokinin-1 receptor

PDX-1

pancreatic duodenal homeobox-1

PBC

primary biliary cholangitis

PSC

primary sclerosing cholangitis

SCT

secretin

SP

substance P

S1PR2

sphingosine 1-phosphate receptor 2

SR

secretin receptor

SSTR₂

somatostatin receptor

VEGF

vascular endothelial growth factor

VEGFR

VEGF receptor

YAP

Yes-associated protein.