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Cholangiocytes are ephitilial cells which line the biliary tree and that in normal condition are mitotically quiescient. Under experimental conditions, such as bile duct ligation (BDL) or in some human cholangiopathies (eg, primary biliary cirrhosis-PBC) cholangiocytes undergo a marked proliferation. A number of factors including Vascular endothelial growth factor (VEGF) and Follicle-stimulating hormone (FSH) regulate biliary tree growth. Melatonin is secreted by the pineal gland and other tissues where it acts as a local regulator of growth, as in the gastrointestinal tract. Melatonin exerts its effects through two membrane receptors (MT1 and MT2) and is synthesized from tryptophan by four enzymes: tryptophan-hydroxylase (TPH), aromatic-amino acid decarboxylase (AADC), serotonin N-acetyltransferase (AANAT) and acetilserotonin- O-methyltransferase (ASMT). Melatonin acts by modulating the expression of Clock genes in the pineal gland. In the liver melatonin reduces oxidative damage. Little information exists about the effects of melatonin on biliary epithelium, therefore the aim of this study was to evaluate the role of melatonin in the regulation of proliferation of biliary epithelium. To this end, we used normal and BDL rats treated or not with: i) melatonin, ii) Arilalkilamina-N-acetyltransferase (AANAT) mistatch and iii) AANAT Vivo Morpholino for 7days and iiii) nude mice injected with the cholangiocarcinoma cell line (Mz-Cha-1) treated or not with melatonin. On liver samples of the experimental models we evaluated: i) the expression of melatonin, the melatonin receptors (MT1 and MT2) and Clock genes, cholangiocytes proliferation and apoptosis, ii) basal and secretin Cyclic adenosine monophosphate (cAMP) levels and Protein Chinase A (PKA) phosphorylation. In addition, we evaluated the expression of melatonin, its receptors and AANAT also in fragments of normal human liver, cholangiocarcioma and PBC. In vitro, large mouse cholangiocytes (LMC), normal human cholangiocytes (H69) and cholangiocarcinoma cell line (Mz-Cha-1) were treated with melatonin 10-11 M for 48h, plus or minus Luzindole (MT1 antagonist) or cis-4-Phenyl-2-propionamidotetralin(4P-PDOT) (MT1 and MT2 antagonist); Finally, we evaluated the effects of AANAT overexpression in LMC and in Mz-Cha-1 on proliferation, apoptosis and cAMP pathway. The results obtained showed that: cholangiocytes expess AANAT, melatonin and its receptors MT1 and MT2 and Clock genes. Chronic administration of melatonin in vivo decreased cholangiocytes proliferation after BDL, and the down-regulation of AANAT in Normal and BDL AANAT Morpholino treated rats increased proliferation. In vitro, melatonin decreased LMC proliferation, decreased which is blocked by Luzindole but not by (4P-PDOT). Overexpression of AANAT in LMC decreased cholangiocytes proliferation, and the intracellular cAMP levels. The expression of AANAT and melatonin decreased in cholangiocarcinoma, instead increased is the expression of MT1 and MT2. In nude mice injected with CCA cell lines the tumor decreased after treatment with melatonin. Overexpression of AANAT in Mz-Cha-1 decreased proliferation and increased apoptosis. In PBC the expression of AANAT, melatonin and its receptors increased if compared to normal. Based on the results obtained, we can say that melatonin plays an important role in the regulation of cholangiocytes proliferation during cholestatic liver diseases and cholangiocarcinoma.

Domenico Alvaro, Adelaide Continenza, Giuseppe Ricciardi