Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes in rats

Authors

  • Anastasia Renzi,

    1. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    2. Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, University “Sapienza,” Rome, Italy
    Search for more papers by this author
  • Sharon DeMorrow,

    1. Scott & White Digestive Disease Research Center, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    2. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Paolo Onori,

    1. Department of Biotechnological and Applied Clinical Sciences, State University of L'Aquila, L'Aquila, Italy
    Search for more papers by this author
  • Guido Carpino,

    1. Department of Health Sciences, University of Rome “Foro Italico,” Rome, Italy
    Search for more papers by this author
  • Romina Mancinelli,

    1. Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, University “Sapienza,” Rome, Italy
    Search for more papers by this author
  • Fanyin Meng,

    1. Scott & White Digestive Disease Research Center, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    2. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    3. Division of Research and Education, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Julie Venter,

    1. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Mellanie White,

    1. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Antonio Franchitto,

    1. Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, University “Sapienza,” Rome, Italy
    2. Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy
    Search for more papers by this author
  • Heather Francis,

    1. Scott & White Digestive Disease Research Center, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    2. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    3. Division of Research and Education, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Yuyan Han,

    1. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Yoshiyuki Ueno,

    1. Department of Gastroenterology, Yamagata University Faculty of Medicine, Yamagata, Japan
    Search for more papers by this author
  • Giuseppina Dusio,

    1. Division of Research and Education, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Kendal J. Jensen,

    1. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • John J. Greene Jr.,

    1. Pathology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
  • Shannon Glaser,

    1. Division of Research, Central Texas Veterans Health Care System, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    2. Scott & White Digestive Disease Research Center, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    3. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    Search for more papers by this author
    • Drs. Alpini, Glaser and Gaudio share the last authorship.

  • Eugenio Gaudio,

    1. Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences, University “Sapienza,” Rome, Italy
    Search for more papers by this author
    • Drs. Alpini, Glaser and Gaudio share the last authorship.

  • Gianfranco Alpini

    Corresponding author
    1. Division of Research, Central Texas Veterans Health Care System, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    2. Scott & White Digestive Disease Research Center, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    3. Department of Medicine, Division of Gastroenterology, Scott & White Healthcare and Texas A&M Health Science Center, College of Medicine, Temple, TX
    • Ph.D., Scott & White Digestive Diseases Research Center, Central Texas Veterans Health Care System, Texas A & M Health Science Center College of Medicine, Olin E. Teague Medical Center, 1901 South 1st Street, Building 205, 1R60, Temple, TX 76504
    Search for more papers by this author
    • Drs. Alpini, Glaser and Gaudio share the last authorship.

    • fax: 743-0378 or 743-0555


  • Potential conflict of interest: Nothing to report.

Abstract

Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3′,5′-monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl/HCOmath image AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic bile-duct–ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down-regulation of cAMP-dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N-acetyltransferase (AANAT; the rate-limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl/HCOmath image AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl/HCOmath image AE2 and ablated secretin-stimulated biliary secretion in these cells. Conclusion: Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013)

Cholangiocytes modify canalicular bile before it reaches the duodenum through a series of secretory/absorptive events regulated by gastrointestinal hormones, including secretin.1, 2 Secretin stimulates bile secretion by interaction with secretin receptor (SR; expressed only by large cholangiocytes in the liver).3 Binding of secretin to its receptor induces an increase in cyclic adenosine 3′,5′-monophosphate (cAMP) levels,1, 4 activation of protein kinase A (PKA), which results in the efflux of Cl through the cystic fibrosis transmembrane conductance regulator (CFTR),4 and subsequent activation of the chloride bicarbonate anion exchanger 2 (Cl/HCOmath image AE2)5 stimulating bicarbonate secretion.2

Cholangiocytes are the target cells in human cholangiopathies6 and animal models of cholestasis, such as bile duct ligation (BDL), a maneuver that induces proliferation of large, but not small, cholangiocytes.2 Subsequent to BDL, biliary hyperplasia is coupled with enhanced functional expression of SR, CFTR, and Cl/HCOmath image AE2 and increased secretory responses to secretin.2, 3, 7 In the BDL model, small cholangiocytes proliferate de novo to compensate for the functional damage of large cholangiocytes (e.g., after CCl4 administration).8 The balance between biliary proliferation and damage is regulated by several autocrine factors, including vascular endothelial growth factor A/C (VEGF-A/C) and serotonin.9, 10

Melatonin is an indole formed enzymatically from L-tryptophan by the enzymes, serotonin N-acetyltransferase (AANAT) and N-acetylserotonin O-methyltransferase (ASMT),11 and is produced by the pineal gland as well as the small intestine and liver.12, 13 Melatonin ameliorates liver fibrosis and systemic oxidative stress (OS) in cholestatic rats.14, 15 Melatonin inhibits biliary hyperplasia and secretin-stimulated choleresis in BDL rats by interaction with melatonin type 1 (MT1) receptor by decreased PKA phosphorylation.16 No information exists regarding the role of melatonin in the autocrine regulation of biliary growth. We proposed to evaluate the (1) expression of AANAT by cholangiocytes and (2) effects of in vivo and in vitro modulation of biliary AANAT and melatonin secretion on the proliferative and secretory responses of cholangiocytes by autocrine signaling.

Abbreviations

AANAT, serotonin N-acetyltransferase or arylalkylamine N-acetyltransferase; ALP, alkaline phosphatase; ASMT, N-acetylserotonin O-methyltransferase; BDL, bile duct ligation; BSA, bovine serum albumin; BW, body weight; cAMP, cyclic adenosine 3′,5′-monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; CK-19, cytokeratin-19; Cl/HCOmath image AE2, chloride bicarbonate anion exchanger 2; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBDM, intrahepatic bile duct mass; H&E, hematoxylin and eosin; IHC, immunohistochemistry; MCL, mouse cholangiocyte line; MT1, melatonin type 1; mRNA, messenger RNA; NCBI, National Center for Biotechnology Information; OS, oxidative stress; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; PKA, protein kinase A; SEM, standard error of the mean; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamate pyruvate transaminases; SR, secretin receptor; TBIL, total bilirubin; VEGF-A/C, vascular endothelial growth factor A/C.

Materials and Methods

Materials.

All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. Antibodies used are detailed in the Supporting Materials. The RNeasy Mini Kit for RNA purification was purchased from Qiagen (Valencia, CA). Radioimmunoassay kits for measurement of cAMP levels were purchased from GE Healthcare (Arlington Heights, IL).

Animal Models.

Male Fischer 344 rats (150-175 g; from Charles River Laboratories, Wilmington, MA) were housed at 22°C with 12-hour light/dark cycles and had free access to chow and drinking water. In addition to healthy (sham) rats, we used animals that, immediately after BDL, had free access to drinking water (vehicle) or melatonin (20 mg/L in drinking water)16 for 1 week. This dose corresponds to a melatonin intake of approximately 2 mg/g body weight (BW)/day/rat.16 This model of melatonin administration to rats has been previously validated and results in increased melatonin serum levels.16 Animal experiments were performed in accordance with a protocol approved by the Scott & White and Texas A&M Health Science Center Institutional Animal Care and Use Committee (Temple, TX).

In separate experiments, healthy or BDL (immediately after surgery)2 rats (n = 9 per group) were treated with Vivo-Morpholino sequences of AANAT (5′-GTTCCCCAGCTTTGGAAGTGGTCCC, to reduce hepatic expression of AANAT) or mismatched Morpholino (5′-GTTCCCGACCTTTGCAACTCGTCCC) (Gene Tools LCC, Philomath, OR) for 1 week by an implanted portal vein catheter (Supporting Materials). Serum, liver tissue, cholangiocytes, pineal gland, kidney, spleen, small intestine, stomach, and heart were collected. Because we aimed to selectively knock down AANAT expression in the liver, we used a lower dose (1.0 mg/kg BW/day) of Vivo-Morpholino than that previously described (3.0 mg/kg/day).17 This approach minimizes the amount of Vivo-Morpholino that circulates outside of the liver after slow infusion into the portal vein.

Freshly Isolated and Immortalized Cholangiocytes.

Pure small and large cholangiocytes were isolated by immunoaffinity separation.4 In vitro studies were performed in immortalized large cholangiocytes (mouse cholangiocyte line [MCL]; from large bile ducts)18 that are functionally similar to freshly isolated large cholangiocytes.7, 19 MCLs were cultured as previously described.7

Measurement of AANAT Expression and Melatonin Levels.

We evaluated the (1) expression of AANAT in liver sections (4 μm thick) by immunohistochemistry (IHC)20 and RNA (1 μg) and protein (10 μg) (by real-time polymerase chain reaction [PCR] and immunoblottings, respectively) from total liver, pooled, small, and/or large cholangiocytes (Supporting Materials)16, 21 and (2) effectiveness of AANAT Vivo-Morpholino in altering AANAT protein expression in liver sections by IHC16 in total liver, cholangiocytes, pineal gland, and small intestine by immunoblottings16 and melatonin levels by enzyme-linked immunosorbent assay (ELISA) kits in cholangiocytes from the selected groups of animals. IHC observations were taken in a coded fashion by a BX-51 light microscope (Olympus, Tokyo, Japan) with a Videocam (Spot Insight; Diagnostic Instruments, Inc., Sterling Heights, MI) and were analyzed with an image analysis system (IAS 2000; Delta Sistemi, Rome, Italy). Negative controls were included. A previously described method was used to quantify, in liver sections, the percent of bile ducts positive for AANAT.18 When 0%-5% of bile ducts were positive, we assigned a negative score; a plus/minus score was assigned when 6%-10% of ducts were positive; and a plus score was assigned when 11%-30% of bile ducts were positive.18 Melatonin levels in serum and medium of primary of cultures (after 6 hours of incubation at 37°C)22 of cholangiocytes were determined by ELISA kits (Genway, San Diego, CA). We evaluated protein expression of cytokeratin-19 (CK-19) by immunoblottings16 in cholangiocytes from healthy rats and BDL rats treated with vehicle or melatonin.

Evaluation of Histomorphology, Biliary Proliferation, Apoptosis, and Serum Chemistry.

Connective tissue was quantified by Sirius red staining by analyzing liver sections with an image analysis system (IAS 2000; Delta Sistemi), and morphological changes in spleen, kidney, heart, stomach, and small intestine by hematoxylin and eosin (H&E) staining was measured. Biliary proliferation was determined by measurement of the percentage of proliferating cell nuclear antigen (PCNA)-positive cholangiocytes, with intrahepatic bile duct mass (IBDM) by IHC for CK-19.20 Biliary apoptosis was evaluated by a semiquantitative terminal deoxynucleotidyl transferase dUTP nick-end labeling kit (Chemicon International, Inc., Temecula, CA).20 Levels of serum glutamate pyruvate transaminases (SGPTs), serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), and total bilirubin (TBIL) were measured by a Dimension RxL Max Integrated Chemistry system (Dade Behring Inc., Deerfield IL) by the Chemistry Department at the Scott & White Digestive Diseases Research Center.

Effect of AANAT Knockdown on Expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2.

We evaluated, by real-time PCR and/or immunoblottings, expression of PCNA, CK-19, SR, CFTR, and Cl/HCOmath image AE2 in liver tissue and/or cholangiocytes from healthy and BDL rats treated with mismatch or AANAT Vivo-Morpholino. A delta delta of the threshold cycle analysis was obtained using normal total liver or healthy cholangiocytes, respectively, as control samples. Primers for rat PCNA, SR, CFTR, Cl/HCOmath image AE2, and CK-19 (SABiosciences) were designed according to the following National Center for Biotechnology Information (NCBI) GenBank accession numbers: NM_022381 (PCNA); NM_031115 (SR); NM_017048 (Cl/HCOmath image AE2); XM_001059206 (CFTR); and NM_199498 (CK-19). Messenger RNA (mRNA) data are expressed as ratio to CK-19 mRNA levels.

In Vitro Effect of Melatonin on Proliferation and Protein Expression of SR, CFTR, and Cl/HCOmath image AE2 of Large Cholangiocytes.

After trypsinization, MCLs were treated at 37°C for 24, 48, or 72 hours with 0.2% bovine serum albumin (BSA) or melatonin (10−11 M)16 before evaluating cell proliferation by PCNA immunoblottings or MTS assays16 and protein expression of SR, CFTR, Cl/HCOmath image AE2 by fluorescence-activated cell sorting (FACS) analysis.16

Overexpression of AANAT in MCL and Measurement of Biliary Proliferative and Secretory Activities.

MCLs were transfected using an AANAT complementary DNA clone vector from OriGene Technologies, Inc. (Rockville, MD), that confers resistance to geneticin for the selection of stable transfected cells. Transfected cells were selected by the addition of geneticin into the media, and the selection process was allowed to continue for 4-7 days.23 Surviving cells (MCL-AANAT) were assessed for relative expression of AANAT, compared to control transfected cells (MCL-puro), by real-time PCR.21 In the selected clone with the greatest degree of overexpression, we measured protein expression of AANAT (by FACS)21, 24 and melatonin secretion (after 6-hour incubation) by ELISA kits, compared to MCL-puro. In the two cell lines, we measured basal proliferative activity by (1) immunoblottings for PCNA (after 48-hour incubation)21 and MTS assays (after 24-72 hours of incubation),7 (2) determination of cell number by a hemocytometer chamber and the Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA)25 (after incubation for 24-72 hours), and (3) mRNA and protein expression for SR, CFTR, and Cl/HCOmath image AE2 were evaluated by real-time PCR and FACs analysis, respectively.21, 24 Effects of secretin (10−7 M for 5 minutes) on cAMP levels18, 26 and Cl efflux, a functional index of CFTR activity,4 were also evaluated. Primers for mouse SR, CFTR, Cl/HCOmath image AE2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SABiosciences) were designed according to the following NCBI GenBank accession numbers: NM_001012322 (SR); NM_021050 (CFTR); NM_009207 (Cl/HCOmath image AE2); NM_009591 (AANAT); and NM_008084 (GAPDH). mRNA data are expressed as ratio to GAPDH mRNA levels.

Statistical Analysis.

All data are expressed as mean ± standard error of the mean (SEM). Differences between groups were analyzed by Student unpaired t test when two groups were analyzed. Analysis of variance was utilized when more than two groups were analyzed, which was followed by an appropriate post-hoc test.

Results

Expression of AANAT.

By IHC in liver sections, AANAT was expressed by small (red arrows) and large (yellow arrows) bile ducts from healthy and BDL rats (Figs. 1A and 2A). AANAT expression increased in bile ducts from BDL, compared to healthy rats, and in BDL rats treated with melatonin, compared to BDL rats (Fig. 1A). Healthy hepatocytes were negative for AANAT, whereas scattered hepatocytes from BDL rats showed weak positivity for AANAT (Figs. 1A and 2A). By real-time PCR, AANAT was expressed by total liver as well as pooled, small, and large cholangiocytes from healthy and BDL rats (Fig. 1B). By both real-time PCR and/or immunoblottings, AANAT expression increased in total liver and pooled (which included small and large cholangiocytes) biliary epithelial cells from BDL, compared to healthy rats and from BDL rats treated with melatonin, compared to BDL rats (Fig. 1 B,C). CK-19 expression increased in cholangiocytes from BDL, compared to healthy rats and decreased in BDL rats treated with melatonin, compared to BDL rats (Fig. 1D).

Figure 1.

(A) By IHC, AANAT was expressed by both small (red arrows) and large (yellow arrows) bile ducts from healthy and BDL rats. AANAT expression increased in bile ducts from BDL, compared to healthy, rats and in BDL rats treated with melatonin, compared to BDL rats. Values were obtained from IHC evaluation of 10 randomly selected fields of three slides obtained from 3 rats for each group. *P < 0.05 versus the corresponding value of healthy rats; #P < 0.05 versus the value of BDL rats. Original magnification, ×40. (B) By real-time PCR, AANAT was expressed by total liver and pooled, small, and large cholangiocytes from healthy and BDL rats. (B and C) By real-time PCR and/or immunoblottings, AANAT expression increased in total liver and pooled cholangiocytes from BDL rats, compared to healthy rats, and from BDL rats treated with melatonin, compared to BDL rats. (B) Data are mean ± SEM of six real-time reactions performed in cumulative preparations (resulting from the low cell yield from 1 rat) of cholangiocytes obtained from 6 rats. (C) Data are mean ± SEM of six immunoblottings performed in cumulative preparations of cholangiocytes obtained from 6 rats. *P < 0.05 versus the values of healthy rats; #P < 0.05 versus the value of BDL rats. (D) CK-19 expression increased in cholangiocytes from BDL, compared to healthy rats, and decreased in BDL rats treated with melatonin, compared to BDL rats. Data are mean ± SEM of six immunoblottings performed in cumulative preparations of cholangiocytes obtained from 6 rats. *P < 0.05 versus the corresponding value of healthy rats; #P < 0.05 versus the value of BDL rats.

Figure 2.

(A) AANAT was expressed by both small (red arrows) and large (yellow arrows) bile ducts from healthy and BDL rats. (A-C) AANAT protein expression decreased in bile ducts (in liver sections), total liver samples, and cholangiocytes from both healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. (A) Original magnification, ×40. Values were obtained from IHC evaluation of 10 randomly selected fields of three slides from 3 rats for each group. (B) Data are mean ± SEM of six immunoblottings performed in three different total liver samples obtained from 6 individual rats. (C) Data are mean ± SEM of six immunoblottings performed in cumulative preparations of cholangiocytes obtained from 6 rats. *P < 0.05 versus the corresponding value of rats treated with mismatch Morpholino. #P < 0.05 versus the corresponding value of healthy rats. (D and E) CK-19 protein expression increased in total liver and cholangiocytes from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. (D) Data are mean ± SEM of six immunoblottings performed in three different total liver samples obtained from 6 individual rats. (E) Data are mean ± SEM of six immunoblottings performed in cumulative preparations of cholangiocytes obtained from 6 rats. *P < 0.05 versus the corresponding value of rats treated with mismatch Morpholino. #P < 0.05 versus the corresponding value of healthy rats.

AANAT protein expression decreased in bile ducts (in liver sections), total liver samples, and cholangiocytes from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls (Fig. 2A-C). AANAT protein expression increased in pineal gland and small intestine from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls (not shown). CK-19 protein expression increased in total liver and cholangiocytes from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls (Fig. 2 D,E).

Melatonin Levels in Serum and Cholangiocyte Supernatant.

Melatonin levels were higher in supernatant of cholangiocytes from BDL, compared to healthy, rats and increased in cholangiocyte samples from BDL rats treated with melatonin (Supporting Table 1). Consistent with previous studies,16 melatonin serum levels were higher in BDL, compared to healthy, rats (Table 1). Melatonin serum levels increased in healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to rats treated with mismatch Morpholino (Table 1). Although AANAT biliary expression decreased in rats treated with AANAT Vivo-Morpholino (Fig. 2 A-C), the increase in melatonin serum levels observed in these rats was likely a result of enhanced expression of AANAT (and subsequent increased melatonin secretion) in the pineal gland and small intestine, which also express AANAT.13, 27 Melatonin levels decreased in supernatant of cholangiocytes from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls (Table 1).

Table 1. Evaluation of Melatonin Levels, Percentage of PCNA-Positive or Apoptotic Cholangiocytes, and Intrahepatic Bile Duct Mass and Serum Chemistry
ParametersNormal Rats + Mismatch MorpholinoNormal Rats + AANAT Vivo-MorpholinoBDL Rats + Mismatch MorpholinoBDL Rats + AANAT Vivo-Morpholino
  • Data are mean ± SEM. IBDM represents area occupied by CK-19-positive bile duct/total area × 100.

  • *

    P < 0.05 versus the corresponding value of normal rats treated with mismatch Morpholino.

  • P < 0.05 versus the corresponding value of normal rats treated with mismatch Morpholino AANAT Vivo-Morpholino.

  • P < 0.05 versus all other groups.

  • §

    P < 0.05 versus the corresponding value of BDL rats treated with mismatch Morpholino.

Serum melatonin levels (pg/mL)70.9 ± 1.1 (n = 5)77.6 ± 2.9* (n = 5)97.5 ± 2.8 (n = 5)174.1 ± 11.7 (n = 5)
Cholangiocyte melatonin levels (pg/ml)43.9 ± 4 (n = 4)26.9 ± 4.01* (n = 4)61.5 ± 3.5 (n = 4)39.5 ± 1.44 (n = 4)
% PCNA-positive cholangiocytes6.80 ± 2.1310.24 ± 1.17*58.07 ± 7.2568.10 ± 4.94
IBDM (%)0.22 ± 0.10.41 ± 0.08*2.32 ± 0.414.75 ± 1.60
% apoptotic cholangiocytesNot detectedNot detected4.12 ± 2.30Not detected
SGPT (Units/L)62.6 ± 7.7 (n = 9)70 ± 1.25 (n = 9)194.4 ± 17 (n = 9)121.2 ± 19§ (n = 9)
SGOT (Units/L)129 ± 7 (n = 9)148 ± 5.1 (n = 9)614.5 ± 68.3 (n = 9)312 ± 52§ (n = 9)
ALP (Units/L)203 ± 6.6 (n = 9)202.8 ± 8 (n = 9)395 ± 9.8 (n = 9)343.2 ± 18.7§ (n = 9)
TBIL (mg/L)<0.1 (n = 9)<0.1 (n = 9)8.7 ± 0.5 (n = 9)7.5 ± 0.6§ (n = 9)

Evaluation of Histomorphology, Biliary Proliferation, Apoptosis, and Serum Chemistry.

In liver sections from healthy and BDL rats treated with AANAT Vivo-Morpholino, there was increased percentage of PCNA-positive cholangiocytes and IBDM, compared to controls (Fig. 3A,B; Table 1). No changes in biliary apoptosis (Table 1) were observed between healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to healthy rats treated with mismatch Morpholino. No difference in lobular damage or necrosis was observed for healthy versus BDL rats treated with AANAT Vivo-Morpholino, compared to controls (not shown). A similar degree of portal inflammation was observed between healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls (not shown). Serum levels of transaminases, ALP, and TBIL decreased in BDL rats treated with Vivo-Morpholino, compared to rats treated with mismatch-Morpholino (Table 1). In BDL Mismatch-treated rats, we found that connective tissue represents approximately 1.5% of the liver, whereas in BDL rats treated with AANAT Vivo-Morpholino, collagen tissues represented approximately 3% of liver mass (not shown). None of the organs analyzed by H&E staining showed structural damage, necrosis, or inflammation (not shown).

Figure 3.

Evaluation of the percentage of (A) PCNA-positive cholangiocytes and (B) IBDM in liver sections from the selected groups of animals. In rats treated with AANAT Vivo-Morpholino, there was an enhanced percentage of PCNA-positive cholangiocytes and IBDM, compared to controls (for semiquantitative analysis, see Table 1). (A) Percentage of PCNA-positive cholangiocytes was assessed in 10 randomly selected fields of three slides obtained from 3 different animals for each group. Positive cells were counted in six nonoverlapping fields for each slide. Original magnification, ×40. (B) Percentage surface occupied by CK-19-positive cholangiocytes (IBDM) was assessed in 10 randomly selected fields of three slides. *P < 0.05 versus the corresponding value of healthy rats treated with mismatch Morpholino. Original magnification, ×20.

Effect of AANAT Knockdown on Expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2.

There was increased expression of mRNA (Fig. 4A) and protein (Fig. 4B) of PCNA, SR, CFTR, and Cl/HCOmath image AE2 in cholangiocytes from rats treated with AANAT Vivo-Morpholino, compared to controls (Fig. 4B).

Figure 4.

(A) Effect of AANAT knockdown on mRNA expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2 in lysate from total liver samples and isolated cholangiocytes. Increased mRNA expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2 was observed in total liver and cholangiocytes from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. Data are mean ± SEM of six real-time reactions performed in cumulative preparations (resulting from the low cell yield from 1 rat) of cholangiocytes obtained from 6 rats. *P < 0.05 versus the corresponding value of healthy and BDL rats treated with mismatch Morpholino. #P < 0.05 versus the corresponding value of healthy rats treated with mismatch Morpholino. (B) Effect of AANAT knockdown on protein expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2 in lysate from isolated cholangiocytes. Increased expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2 was observed in cholangiocytes from normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. Data are mean ± SEM of six immunoblottings performed in cumulative preparations of cholangiocytes obtained from 6 rats. *P < 0.05 versus the corresponding value of healthy and BDL rats treated with mismatch Morpholino. #P < 0.05 versus the corresponding value of healthy rats treated with mismatch Morpholino.

In Vitro Effect of Melatonin on the Proliferation and Protein Expression of SR, CFTR, and Cl/HCOmath image AE2 of Large Cholangiocytes.

In vitro, melatonin inhibited biliary proliferation (by MTS assays and PCNA immunoblottings; Supporting Fig. 1) and protein expression (by FACS analysis) of SR, CFTR, and Cl/HCOmath image AE2, compared to large cholangiocytes treated with 0.2% BSA (Supporting Fig. 1).

Effect of Overexpression of AANAT in MCL on Expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2.

Enhanced mRNA and protein expression for AANAT and increased melatonin secretion were observed in AANAT-transfected cholangiocytes, compared to controls (Supporting Fig. 2A-C). In cholangiocytes overexpressing AANAT, there was (1) decreased biliary proliferation shown by PCNA immunoblottings and MTS assays (Fig. 5A,B) and a reduced number of cholangiocytes (Supporting Table 2) and (2) reduced mRNA (Fig. 5C-E) and protein (by FACS analysis; Fig. 5F) expression for SR, CFTR, and Cl/HCOmath image AE2, compared to control cholangiocytes. Secretin did not increase cAMP levels (a functional index of SR expression)4, 28 and Cl efflux (a functional parameter of CFTR activity)4 at 360 seconds after treatment with secretin in stably AANAT-overexpressing cholangiocytes (Supporting Fig. 3A,B). Secretin stimulated cAMP and Cl efflux in large cholangiocytes transfected with the control vector (Supporting Fig. 3A,B).

Figure 5.

In cholangiocytes overexpressing AANAT, (A and B) decreased biliary proliferation was observed, as shown by PCNA immunoblottings and MTS assays as well as reduced (C-E) mRNA and (F) protein expression (by FACS analysis) for SR, CFTR, and Cl/HCOmath image AE2, compared to control cholangiocytes. (A and B) Data are mean ± SEM of six immunoblottings and MTS reactions performed in six different samples obtained from cholangiocyte lines. (C-E) Data are mean ± SEM of six real-time PCR reactions performed in six different samples obtained from cholangiocyte lines. (F) Data are mean ± SEM of four different FACS analyses performed in four different samples obtained from cholangiocyte lines. *P < 0.05 versus the corresponding value of cholangiocytes transfected with control vector.

Discussion

The data demonstrate that (1) AANAT is expressed by both small and large cholangiocytes and (2) local modulation of AANAT expression alters large cholangiocyte growth and secretin-stimulated ductal secretion. We demonstrated that (1) AANAT is expressed by bile ducts, and AANAT expression is up-regulated after BDL and by the administration of melatonin to BDL rats; weak immunoreactivity is present in BDL hepatocytes and (2) AANAT expression is decreased in liver samples and cholangiocytes from both healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. Concomitant with reduced AANAT biliary expression, there was increased proliferation and IBDM in liver sections and enhanced expression of PCNA, SR, CFTR, and Cl/HCOmath image AE2 in cholangiocytes from healthy and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. Serum levels of transaminases, ALP, and total bilirubin decreased in AANAT Vivo-Morpholino–treated BDL rats, confirming the improvement of cholestasis after modulation of AANAT, likely the result of increased melatonin serum levels. In support of our findings, we have previously shown that serum levels of transaminases and bilirubin increased in BDL, compared to healthy rats and decreased after administration of melatonin.16 In vitro overexpression of AANAT in large cholangiocytes decreased (1) biliary proliferation, mitosis, and expression of SR, CFTR, and Cl/HCOmath image AE2 and (2) secretin-stimulated cAMP levels and Cl efflux, a functional index of CFTR activity.4, 29

Growing information is evident regarding autocrine regulation of cholangiocyte growth and damage by autocrine factors.9, 10 Serotonin regulates hyper- and neoplastic biliary growth, both in vivo and in vitro.30, 31 Blocking VEGF secretion decreases cholangiocyte proliferation, revealing an autocrine loop wherein cholangiocytes secrete VEGF interacting with VEGF receptors 2 and 3 to increase biliary proliferation.10 In cholangiocytes from polycystic liver-disease samples, VEGF expression is up-regulated and VEGF supports cholangiocyte proliferation by autocrine mechanisms.32 Although melatonin synthesis is dys-regulated in cholangiocarcinoma,33 no data exist regarding the autocrine role of melatonin (secreted by cholangiocytes) in the regulation of biliary hyperplasia.

Morpholinos are free of off-target effects because they are not degraded in biological systems and do not generate degradation products toxic to cells.34 Phosphorodiamidate Morpholino oligomers have been used to evaluate the role of β-catenin in cell proliferation and apoptosis and early biliary lineage commitment of bipotential stem cells in the developing liver.35 In zebrafish, Morpholino antisense oligonucleotide-mediated knockdown of planar-cell polarity genes led to developmental biliary abnormalities as well as localization defects of the liver.36

We first measured the expression of AANAT in liver sections, total liver and cholangiocytes, and melatonin serum levels in our models. The reason why we measured AANAT expression in healthy and BDL rats, and BDL rats treated with melatonin, was to demonstrate a link between AANAT expression and cholangiocyte proliferation in well-established models of biliary hyperplasia (BDL)2, 16 and reduced biliary hyperplasia (BDL + melatonin).16 The increase of AANAT biliary expression and melatonin secretion after BDL are likely the result of a compensatory mechanism and correlates with increased melatonin serum levels observed in cholestatic rats,16 an increase that may result from enhanced secretion of melatonin not only from cholangiocytes, but also from the small intestine and pineal gland.12, 13 The increase in AANAT expression by the pineal gland may result from a compensatory mechanism to ameliorate cholestatic-induced OS.37 The enhanced biliary expression of AANAT in melatonin-treated BDL rats is supported by studies in rats and humans.16, 38 The reduction of biliary AANAT expression and melatonin secretion in cholangiocytes (after AANAT Vivo-Morpholino administration) supports the validity of the model and the hypothesis that the AANAT expression⇒melatonin secretion axis may be an important autocrine loop regulating locally biliary proliferation. The increase in melatonin serum levels observed in rats treated with AANAT Vivo-Morpholino was likely a result of the higher expression of AANAT (and likely melatonin secretion) by other sites, such as pineal glands and intestine, to compensate for the loss of biliary AANAT expression. The current findings do not exclude that other paracrine pathways (e.g., melatonin released from the pineal gland and/or hyperplastic hepatocytes) are important for the regulation of biliary function. In the healthy state, cholangiocytes represent approximately 2%-4% of the total liver cell population; however, after BDL cholangiocytes proliferate, and 1 week after BDL, biliary mass represents approximately 25%-30% of the total liver mass.2 Hepatocytes may have a role (scattered) in the modulation of biliary proliferation, but in light of our findings (the reduced AANAT biliary expression by Morpholino enhances IBDM in vivo, and overexpression of AANAT in vitro decreases biliary proliferation), we propose that AANAT has a role in the modulation of biliary proliferation, which probably is not the main, or the only one, acting factor on cholangiocytes. Studies aimed to evaluate the effects of changes in the central synthesis of melatonin (e.g., after pinealectomy and exposure to dark to stimulate melatonin release from the pineal gland) in the regulation of biliary functions are ongoing.

Having demonstrated that AANAT is expressed by cholangiocytes and that AANAT expression is up-regulated by BDL and melatonin administration, we proposed to demonstrate that reduction of biliary AANAT expression (by Vivo-Morpholino) increases cholangiocyte proliferation and IBDM as well as the expression of SR, CFTR, and Cl/HCOmath image AE2 in cholangiocytes. After BDL, the increase of biliary proliferation and IBDM is followed by the extension of the peribiliary plexus and the increase of surrounding connective tissue, which is organized around bile ducts and vessels.10 In our model, we only observed a slight difference in collagen tissue content in BDL rats treated with mismatch Morpholino versus BDL rats treated with AANAT Vivo-Morpholino. This low increase of connective tissue cannot determine a clear hepatic fibrosis in our model of short time of BDL. Further studies are needed to evaluate the long-term effects of BDL on the modulation of melatonin synthesis on liver fibrosis. Also, the novel concept that AANAT regulates SR⇒CFTR⇒Cl/HCOmath image AE2 expression is supported by our previous study16 showing that in vivo administration of melatonin to BDL rats decreases secretin-induced choleresis.

To determine that the effects of down-regulation of AANAT on biliary growth depend on direct effects on bile ducts, cholangiocytes were treated in vitro with melatonin that decreased the biliary proliferation and expression of SR, CFTR, and Cl/HCOmath image AE2. We overexpressed AANAT in cholangiocytes and demonstrated a decrease in biliary proliferation and secretin-stimulated cAMP levels and Cl efflux. In vitro, overexpression of AANAT in cholangiocytes leading to decreased biliary proliferation and secretin receptor-dependent ductal secretion (in the absence of intestinal secretin supply) was likely the result of the fact that cholangiocytes express SR and express the message for secretin and secrete secretin,7, 39, 40 which (similar to what is observed in vivo) is an important autocrine factor sustaining biliary proliferation. We propose that the modulation of biliary melatonin secretion (by chronic administration of melatonin or changes in AANAT expression; Fig. 6) may be a valuable therapeutic approach for regulating the balance between biliary growth/apoptosis. In support of this view, we have shown that in the first stage of primary biliary cirrhosis, there is an increase of cholangiocyte proliferation that resulted in a positive balance between growth and apoptosis.41 By contrast, the end stage is characterized by the collapse of the proliferative capacity of cholangiocytes, resulting in the reduction (high apoptosis rate) of the number of bile ducts (vanishing bile duct syndrome).41 Because, in our model, we have shown that the modulation of melatonin synthesis is involved in the balance between biliary growth and apoptosis, modulation of melatonin synthesis can be proposed as a possible strategy for the management of cholangiopathologies.

Figure 6.

Working model of changes induced by local modulation of AANAT in large biliary proliferation. Top: exogenous melatonin, by interaction with MT1 receptor, inhibits large cholangiocyte proliferation (hypoplasia) by increasing biliary AANAT expression and then melatonin secretion. Bottom: down-regulation of biliary AANAT levels by Vivo-Morpholino stimulates the proliferation of large cholangiocytes by reduction of biliary AANAT expression and melatonin secretion, which induces subsequent activation of biliary proliferation (hyperplasia).

Ancillary