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Polycystic liver disease phenotypes arise from two distinct inherited diseases, autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PCLD). ADPKD, caused by mutations in PKD1 or PKD2 genes, is characterized by polycystic kidneys (1). In many patients with ADPKD, there is the development of a polycystic liver manifestation. On the other hand, PCLD is caused by mutations in PRKCSH or SEC63 genes and is characterized by the presence of an isolated polycystic liver without the kidney phenotype (2, 3). The diagnosis of polycystic liver is usually made during the third or fourth decade of life with hepatic capacity preserved in the great majority of patients (4, 5). This disease is usually asymptomatic, but the progressive growth of the liver cysts may cause dyspnoea, gastrooesophageal reflux, nausea and mechanical low back pain arise because of the mass effect of the polycystic liver (6). Severe ADPKD primarily affects women and is characterized by the massive cystic liver disease. The number and size of hepatic cysts correlate with the occurrence of pregnancy, female gender, increased age and severity of the renal lesion (7). Treatment is initiated only in those with the symptoms and all interventional procedures are aimed to reduce liver volume (5). In the last few years, the number of studies to discover viable medical options has increased with indications that somatostatin analogues or mTOR inhibitors may slow cyst growth (8–10). Many experimental and clinical studies have demonstrated that cholangiocytes respond to hormones, growth factors, neuropeptides and cytokines increasing their proliferative capacity (11–13). In particular, oestrogens play a key role in sustaining cholangiocyte growth, cyst formation and progression in ADPKD patients. Oestrogens act not only directly, but also by promoting the synthesis and release of other growth factors from the cystic epithelium (14). Additional sex hormones such as prolactin (15), progesterone (16) and follicle-stimulating hormone (FSH) (17) regulate biliary function. Many events in the adult ovary are controlled by two hormones, FSH and luteinizing hormone (LH) secreted from the anterior pituitary gland under the control of gonadotropin-releasing hormone (GnRH) from the hypothalamus. FSH is required for granulosa cell differentiation and facilitates the follicular growth (18). In the classical cascade, occupancy of FSH receptor (FSHR) causes activation of the heterotrimeric GS protein, which stimulates the effector adenylyl cyclase with the consequent increase in the synthesis of the second messenger cAMP (19, 20). One of the most characterized components of the MAPK family is the extracellular-regulated kinase (ERK). The ERK pathway regulates cell proliferation, differentiation and cell survival (21). C-myc represents a key downstream target of this mechanism (22). Others have demonstrated that c-myc participates in the progression of the G1-cell cycle phase by enhancing cyclin expression (23) and CDK/cyclin complex activities (24). Lastly, both c-myc and ERK, as a consequence of their marked capacity to promote proliferation, play a pivotal role in the control of the differentiation programme in several cell types (25–27). We have previously shown that the cAMP/ERK-dependent signalling mechanism is activated in proliferating cholangiocytes (13, 28). In particular, in the hyperplastic BDL model, cholangiocyte proliferation is closely associated with increased cAMP levels (29–32). It has been demonstrated that FSH plays an important role in stimulating rat cholangiocyte proliferation through an autocrine mechanism that is associated with increased cAMP-dependent phosphorylation of ERK1/2 and Elk-1 both in vivo and in vitro (17). However, no information exists regarding the role of FSH and its receptors in the regulation of epithelial cell growth in the hepatic cysts. The aim of this study was to evaluate the hypothesis that FSH regulates hepatic cysts growth during the course of ADPKD.

Small cholangiocytes proliferate via activation of calcium (Ca2+)-dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate–dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca2+-dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an α1-adrenergic agonist stimulating intracellular D-myo-inositol-1,4,5-triphosphate (IP3)/Ca2+ levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP3/Ca2+-dependent signaling. Phenylephrine stimulated the production of intracellular IP3. The Ca2+-dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca2+-dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca2+-dependent activation of NFAT2 and Sp1. Conclusion: Selective stimulation of Ca2+-dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins. (HEPATOLOGY 2010;53:628-639)