Matthew Quinn

Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat

Cholestatic patients often present with clinical features suggestive of adrenal insufficiency. In the bile duct-ligated (BDL) model of cholestasis, the hypothalamic-pituitary-adrenal (HPA) axis is suppressed. The consequences of this suppression on cholangiocyte proliferation are unknown. We evaluated 1) HPA axis activity in various rat models of cholestasis and 2) effects of HPA axis modulation on cholangiocyte proliferation. Expression of regulatory molecules of the HPA axis was determined after BDL, partial BDL, and α-naphthylisothiocyanate (ANIT) intoxication. The HPA axis was suppressed by inhibition of hypothalamic corticotropin-releasing hormone (CRH) expression by central administration of CRH-specific Vivo-morpholinos or by adrenalectomy. After BDL, the HPA axis was reactivated by 1) central administration of CRH, 2) systemic ACTH treatment, or 3) treatment with cortisol or corticosterone for 7 days postsurgery. There was decreased expression of 1) hypothalamic CRH, 2) pituitary ACTH, and 3) key glucocorticoid synthesis enzymes in the adrenal glands. Serum corticosterone and cortisol remained low after BDL (but not partial BDL) compared with sham surgery and after 2 wk of ANIT feeding. Experimental suppression of the HPA axis increased cholangiocyte proliferation, shown by increased cytokeratin-19- and proliferating cell nuclear antigen-positive cholangiocytes. Conversely, restoration of HPA axis activity inhibited BDL-induced cholangiocyte proliferation. Suppression of the HPA axis is an early event following BDL and induces cholangiocyte proliferation. Knowledge of the role of the HPA axis during cholestasis may lead to development of innovative treatment paradigms for chronic liver disease.

Monoamine oxidase A expression is suppressed in human cholangiocarcinoma via coordinated epigenetic and IL-6-driven events

The secretion of dopamine and serotonin is increased in cholangiocarcinoma, which has growth-promoting effects. Monoamine oxidase A (MAOA), the degradation enzyme of serotonin and dopamine, is suppressed in cholangiocarcinoma via an unknown mechanism. The aims of this study were to (i) correlate MAOA immunoreactivity with pathophysiological parameters of cholangiocarcinoma, (ii) determine the mechanism by which MAOA expression is suppressed and (iii) evaluate the consequences of restored MAOA expression in cholangiocarcinoma. MAOA expression was assessed in cholangiocarcinoma and nonmalignant controls. The control of MAOA expression by promoter hypermethylation was evaluated and the contribution of interleukin-6 (IL-6) signaling to the suppression of MAOA expression was determined. The effects of MAOA overexpression on cholangiocarcinoma growth and invasion were also assessed. MAOA expression is correlated with differentiation, invasion and survival in cholangiocarcinoma. The MAOA promoter was hypermethylated immediately upstream of the start codon in cholangiocarcinoma samples and cell lines but not in nonmalignant counterparts. IL-6 signaling also decreased MAOA expression via a mechanism independent of hypermethylation, involving the regulation of the balance between SP-1 transcriptional activity and its inhibitor, R1 repressor. Inhibition of both IL-6 signaling and DNA methylation restored MAOA levels to those observed in cholangiocytes. Forced MAOA overexpression inhibited cholangiocarcinoma growth and invasion. MAOA expression is suppressed by the coordinated control of promoter hypermethylation and IL-6 signaling. MAOA may be a useful prognostic marker in the management of cholangiocarcinoma, and therapies designed to increase MAOA expression might prove beneficial in the treatment of cholangiocarcinoma.

The novel growth factor, progranulin, stimulates mouse cholangiocyte proliferation via sirtuin-1-mediated inactivation of FOXO1

Progranulin (PGRN), a secreted growth factor, regulates the proliferation of various epithelial cells. Its mechanism of action is largely unknown. Sirtuin 1 (Sirt1) is a protein deacetylase that is known to regulate the transcriptional activity of the forkhead receptor FOXO1, thereby modulating the balance between proapoptotic and cell cycle-arresting genes. We have shown that PGRN is overexpressed in cholangiocarcinoma and stimulates proliferation. However, its effects on hyperplastic cholangiocyte proliferation are unknown. In the present study, the expression of PGRN and its downstream targets was determined after bile duct ligation (BDL) in mice and in a mouse cholangiocyte cell line after stimulation with PGRN. The effects of PGRN on cholangiocyte proliferation were assessed in sham-operated (sham) and BDL mice treated with PGRN or by specifically knocking down endogenous PGRN expression using Vivo-Morpholinos or short hairpin RNA. PGRN expression and secretion were upregulated in proliferating cholangiocytes isolated after BDL. Treatment of mice with PGRN increased biliary mass and cholangiocyte proliferation in vivo and in vitro and enhanced cholangiocyte proliferation observed after BDL. PGRN treatment decreased Sirt1 expression and increased the acetylation of FOXO1, resulting in the cytoplasmic accumulation of FOXO1 in cholangiocytes. Overexpression of Sirt1 in vitro prevented the proliferative effects of PGRN. Conversely, knocking down PGRN expression in vitro or in vivo inhibited cholangiocyte proliferation. In conclusion, these data suggest that the upregulation of PGRN may be a key feature stimulating cholangiocyte proliferation. Modulating PGRN levels may be a viable technique for regulating the balance between ductal proliferation and ductopenia observed in a variety of cholangiopathies.

Neuropeptide Y inhibits biliary hyperplasia of cholestatic rats by paracrine and autocrine mechanisms

Neuropeptide Y (NPY) exerts its functions through six subtypes of receptors (Y₁-Y₆). Biliary homeostasis is regulated by several factors through autocrine/paracrine signaling. NPY inhibits cholangiocarcinoma growth; however, no information exists regarding the autocrine/paracrine role of NPY on biliary hyperplasia during cholestasis. The aims of this study were to determine: 1) the expression of NPY and Y₁-Y₅ in cholangiocytes and 2) the paracrine/autocrine effects of NPY on cholangiocyte proliferation. Normal or bile duct ligation (BDL) rats were treated with NPY, neutralizing anti-NPY antibody, or vehicle for 7 days. NPY and NPY receptor (NPYR) expression was assessed in liver sections and isolated cholangiocytes. NPY secretion was assessed in serum and bile from normal and BDL rats, as well as supernatants from normal and BDL cholangiocytes and normal rat cholangiocyte cell line [intrahepatic normal cholangiocyte culture (NRICC)]. We evaluated intrahepatic bile ductal mass (IBDM) in liver sections and proliferation in cholangiocytes. With the use of NRICC, the effects of NPY or anti-NPY antibody on cholangiocyte proliferation were determined. The expression of NPY and all NPYR were increased after BDL. NPY levels were lower in serum and cholangiocyte supernatant from BDL compared with normal rats. NPY secretion from NRICC was detected at both the basolateral and apical domains. Chronic NPY treatment decreased proliferating cellular nuclear antigen (PCNA) expression and IBDM in BDL rats. Administration of anti-NPY antibody to BDL rats increased cholangiocyte proliferation and IBDM. NPY treatment of NRICC decreased PCNA expression and increased the cell cycle arrest, whereas treatment with anti-NPY antibody increased proliferation. Therapies targeting NPY-mediated signaling may prove beneficial for the treatment of cholangiopathies.

The Role of the Tumor Microenvironment in the Pathogenesis of Cholangiocarcinoma

Cholangiocarcinoma is a type of liver cancer arising from the neoplastic transformation of the epithelial cells that line the intraand extrahepatic bile ducts. Symptoms are usually evident only after blockage of the bile duct by the tumor. This is an extremely aggressive tumor, which has very poor prognosis and limited treatment options. Cholangiocarcinoma is relatively resistant to chemotherapy and radiation therapy leaving conventional treatment like surgery as the only option. Therefore, further understanding into the factors that are involved in tumor initiation, promotion and progression is required for designing alternate therapies to combat this devastating disease.