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SummaryCholangiocytes, the cells lining bile ducts, are a heterogenous, highly dynamic population of epithelial cells. While these cells comprise a small fraction of the total cellular component of the liver, they perform the essential role of bile modification and transport of biliary and blood constituents. From a pathophysiological standpoint, cholangiocytes are the target of a diverse group of biliary disorders, collectively referred to as the cholangiopathies. To date, the cause of most cholangiopathies remains obscure. It is known, however, that cholangiocytes exist in an environment rich in potential mediators of cellular injury, express receptors that recognize potential injurious insults, and participate in portal tract repair processes following hepatic injury. As such, cholangiocytes may not be only a passive target, but are likely directly and actively involved in the pathogenesis of cholangiopathies. Here, we briefly summarize the characteristics of the reactive cholangiocyte and cholangiocyte responses to potentially injurious endogenous and exogenous molecules, and in addition, present emerging concepts in our understanding of the etiopathogenesis of several cholangiopathies.

Cholangiocytes, which line the biliary epithelium, are hormone-responsive and responsible for the modification of canalicular bile before reaching the duodenum. Cholangiocytes are the target cells in cholangiopathies such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) that are characterized by cholangiocyte proliferation/loss [1]. The biliary epithelium is morphologically and functionally heterogeneous and is lined by small ( 15 lm in diameter) cholangiocytes lining small and large bile ducts, respectively [2]. Cholangiocyte responses are regulated by both Ca2+- and cAMP-dependent signaling [2–5]. Histamine is a biogenic amine that is mostly recognized for its role in allergy-related diseases. After histamine is formed from the decarboxylation of the amino acid histidine, by histidine decarboxylase (HDC), it is rapidly stored or degraded. Histamine exerts its effects through four G-protein coupled receptors (GPCRs), H1–H4 histamine receptors (HRs) [6]. Activation/inhibition of HRs triggers downstream signaling pathways to elicit cellular responses such as proliferation, apoptosis, and differentiation. It has been shown that histamine plays a critical role in liver repair and regeneration. The H1 histamine receptor affects gamma-glutamyltransferase (c-GT) levels during liver regeneration after partial hepatectomy in rats [7]. Blocking H1HR increased serum c-GT activity, whereas inhibition of H2HR did not have a significant effect in this model [7]. Histamine, via the H4HR, protects against ischemia/reperfusion liver injury by decreasing cytokine release [8]. These studies demonstrate the varying role of histamine in liver injury and the potential benefits of histamine during the progression of liver damage. Our group has demonstrated that histamine, via the four histamine receptors, H1–H4HRs, induces a differential response in cholangiocyte proliferation via diverse signaling pathways as shown in Fig. 1 [4,5,9,10]. We have found that all HRs are present on cholangiocytes and that these receptors are both inhibitory (H3–H4HRs) and stimulatory (H1–H2HRs) during cholangiocyte proliferation [4,5,9,10]. Fig. 1 Top panel: In normal and during cholestatic injury, histamine receptors are found in small and large cholangiocyte populations. Expression levels of HRs have not been found to have a significant impact on cholangiocyte response, however, activation of ... In a rodent model of cholangiocyte proliferation induced by bile duct ligation (BDL), we have demonstrated that stimulation of the inhibitory H3HR by an H3HR agonist decreases BDL-induced cholangiocyte hyperplasia [4]. Activation of H3HR (that couples to Gαi) inhibits cAMP signaling with a subsequent decrease in the phosphorylation of ERK1/2 and the transcription factor, Elk1 [4]. Inhibition of cholangiocyte proliferation may be a useful therapy for treatment of cholangiopathies characterized by increased biliary hyperplasia (Fig. 1, top panel). We also found that histamine, via H1HR (coupled to Gαq) regulates small cholangiocyte proliferation without inducing any effects in large cholangiocytes [5]. Using cultured mouse cholangiocytes, we found that stimulation of H1HR increases the proliferation of small (but not large) cholangiocytes via activation of IP3/Ca2+/PKC/CaMKI/CREB-dependent pathway as shown in Fig. 1, top panel [5]. The H3HR signals either via Gαi (inhibition of cAMP) or Gαo (activation of Ca2+). Using a specific agonist for the H3HR, we evaluated the effects of this agonist on cholangiocarcinoma growth [9]. We demonstrated that activation of H3HR decreases cholangiocarcinoma growth both in vitro and in vivo [9]. Surprisingly, the effects by RAMH were mediated not by inhibition of cAMP signaling, but by activation of the IP3/Ca2+/PKCα [9]. We demonstrated that activation of PKCα led to decrease ERK1/2 phosphorylation and subsequent decreased biliary proliferation as shown in Fig. 1, bottom panel. In a separate study, we have evaluated the specific role of the H4HR agonist, clobenpropit, on cholangiocarcinoma growth and metastatic potential [10]. We demonstrated that by activation of H4HR cholangiocarcinoma growth decreases (both in vitro and in a xenograft tumor model) and epithelial–mesenchymal transition is also inhibited. Our findings suggest that the activation of H4HR decreases the expression of extracellular matrix degrading enzymes, matrix metalloproteinases preserving the extracellular matrix and making tumor spreading more difficult [10]. Further, we found that H4HR allows cholangiocarcinoma cells to maintain more of an epithelial cell-type phenotype by preserving the expression of numerous hepatobiliary markers including the cytokeratins, CK-7, -8, and -19. The actions of H4HR were mediated by activation of a Ca2+/PKC-dependent pathway similar to what we found using an agonist for H3HR as depicted in Fig. 1, bottom panel [10]. Studies have shown that cholangiocytes (that are normally) dormant, proliferate after BDL, a model that mimics human cholestatic liver disease [3,4]. While these studies above document the role of histamine and HRs during diseased states, preliminary data show that normal cholangiocytes can be activated to proliferate via histamine and two stimulatory histamine receptors, H1HR and H2HR. We demonstrated that histamine increases normal cholangiocyte proliferation via activation of both Ca2+- and cAMP-dependent pathways. When elucidating the specific histamine receptors, we discovered that H1HR increases normal cholangiocyte growth via a Ca2+/PKCα-dependent pathway, whereas H2HR activates cAMP synthesis to increase normal cholangiocyte proliferation. We have data demonstrating that H1HR-induced cholangiocyte proliferation occurs in small cholangiocytes (regulated by Ca2+-dependent signaling) whereas H2HR-induced cholangiocyte proliferation exists in large cholangiocytes that signal via cAMP activation. Overall, these findings are important in understanding the differential response induced by histamine that may regulate cholangiocyte proliferation during the progression of cholangiopathies or liver regeneration. Modulation of H1–H4HRs may be key for the management of the balance between biliary growth/loss in cholangiopathies.

Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A–dependent activation of the extracellular signal–regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)–mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2–/– knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum–resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2–/– mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2. Conclusion: In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases. (Hepatology 2015)

The scientific framework concerning estrogen effects on different tissues has expanded enormously during the last decades, when estrogen receptor (ER) subtypes were identified. Estrogens are not only essential for the female reproductive system, but they also control fundamental functions in other tissues including the cardiovascular system, bone, brain and liver. Recently, estrogens have been shown to target the biliary tree, where they modulate the proliferative and secretory activities of cholangiocytes, the epithelial cells lining bile ducts. By acting on both estrogen receptors (ER-alpha) and (ER-beta) subtypes, and by activating either genomic or non-genomic pathways, estrogens play a key role in the complex loop of growth factors and cytokines, which modulates the proliferative response of cholangiocytes to damage. Specifically, estrogens activate intracellular signalling cascades [ERK(1/2) (extracellular regulated kinases (1/2), PI3- kinase/AKT (phosphatidylinositol-3' kinase/AKT)] typical of growth factors such as insulin like growth factor (IGF1), nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), thus potentiating their action. In addition, estrogens stimulate the secretion of different growth factors in proliferating cholangiocytes. This review specifically deals with the recent advances related to the role and mechanisms by which estrogens modulate cholangiocyte functions in normal and pathological conditions.