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VEGF Axis by miR-125b during Cholestatic Liver Injury in Mice
The American Journal of Pathology, Vol. 184, No. 3, March 2014
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GASTROINTESTINAL, HEPATOBILIARY, AND PANCREATIC PATHOLOGY
Regulation of the Histamine/VEGF Axis by miR-125b during Cholestatic Liver Injury in Mice Fanyin Meng,*yz Paolo Onori,x Laura Hargrove,y Yuyan Han,z Lindsey Kennedy,y Allyson Graf,y Kyle Hodges,y Yoshiyuki Ueno,{k Taylor Francis,y Eugenio Gaudio,x and Heather L. Francis*yz From the Department of Research,* Central Texas Veteran’s Health Care System, the Scott and White Digestive Disease Research Center,y Scott and White Healthcare, and the Department of Internal Medicine,z Texas A&M Health Science Center, Temple, Texas; the Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences,x Sapienza, University of Rome, Rome, Italy; CREST,{ Japan Science and Technology Agency, Yamagata, Japan; and the Department of Gastroenterology,k Yamagata University Faculty of Medicine, Yamagata, Japan Accepted for publication November 15, 2013. Address correspondence to Heather L. Francis, Ph.D., Department of Internal Medicine, Texas A&M Health Science Center College of Medicine, Central Texas Veteran’s Health Care System, Digestive Disease Research Center, Scott and White Healthcare, 1901 S Veterans Memorial Dr, Bldg 205, Office 1R52, Temple, TX 76504. E-mail: [email protected].
Histamine is formed by the conversion of L-histidine into histamine by histidine decarboxylase (HDC). We have previously shown that inhibition of HDC blocks cholangiocyte proliferation and silencing of HDC decreases vascular endothelial growth factor (VEGF) expression. We hypothesized that increased HDC expression during cholestatic liver injury is mediated by the down-regulation of the specific miRNA miR125b, a post-transcriptional regulator. Mice were subjected to sham surgery or bile duct ligation (BDL), which induces large cholangiocyte proliferation, and subsequently treated with either saline or a-methylDL-histidine (an HDC inhibitor) for 7 days. Liver blocks, serum, and large cholangiocytes were obtained, and intrahepatic bile duct mass, cholangiocyte proliferation (proliferating cellular nuclear antigen expression), and expression of both HDC and VEGF were measured. miRNA profiling was performed in isolated cholangiocytes. In vitro, miR-125b was overexpressed (or inhibited) or HDC was silenced before measuring HDC and VEGF-A/C expression and cholangiocyte proliferation. After BDL plus a-methyl-DLhistidine, expression of intrahepatic bile duct mass, proliferating cellular nuclear antigen, VEGF-A/C, and HDC and levels of histamine all decreased compared with those of BDL alone. miR-125b was significantly down-regulated after BDL. In vitro, overexpression of miR-125b and knockdown of HDC both decreased HDC and VEGF expression and cholangiocyte proliferation. Manipulation of miR-125beregulated HDC/ VEGF expression may, thus, be a therapeutic approach for the treatment of aberrant cholangiocyte growth in biliary disorders. (Am J Pathol 2014, 184: 662e673; http://dx.doi.org/10.1016/j.ajpath.2013.11.008)
Primary biliary cirrhosis and primary sclerosing cholangitis are liver diseases targeting cholangiocytes.1 A hallmark feature of cholangiopathies is dysregulation of the balance between biliary proliferation and loss.1 In animal models such as bile duct ligation (BDL),2,3 cholangiocytes proliferate in response to cholestasis, increasing intrahepatic bile duct mass.3,4 After BDL for 1 week, only large cholangiocytes proliferate, leading to enhanced intrahepatic bile duct mass, and increased secretion of several growth factors, such as vascular endothelial growth factor (VEGF) and histamine.3,5 Histamine is formed by the conversion of histidine, by the enzyme, L-histidine decarboxylase (HDC).3,6 After conversion, histamine is readily used or stored in cells, such as mast cells.7,8 Patients with cholangiopathies have increased serum levels of histamine, suggesting that histamine plays a critical role in liver pathological conditions.9 Histamine acts through Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2013.11.008
four G-proteinecoupled receptors (H1 to H4) that induce both stimulatory (H1 and H2 histamine receptors) and inhibitory (H3 and H4 histamine receptors) effects on cellular function.10,11 We have shown that the H3 histamine receptor inhibits BDL-induced large cholangiocyte proliferation by down-regulating cAMP signaling.3 Stimulation with an H1 Portions of this work were supported by a Veteran’s Affairs Career Development Award and a Scott and White Research Advancement Award (H.L.F.); the Scott and White Health Care Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology (G.A.); University of Rome Sapienza and a Minestro dell’Instruzione dell’universita e della Ricerca grant 2009X84L84 and Futuro in Ricera grant RBAP10Z7FS (E.G.); the Ministry of Health, Labor, and Welfare of Japan grant-in-aid (Y.U.); and Scott and White Health Care internal support money and a Veteran’s Affairs Merit Award (F.M.). F.M. and P.O. contributed equally to this work. Disclosures: None declared.
miR-125b Regulation of Biliary Growth histamine receptor agonist increases small cholangiocyte proliferation.12 We have also shown that inhibition of HDC decreases cholangiocarcinoma growth in vitro and in vivo.10 Cholangiocytes express VEGF-A and VEGF-C and secrete VEGF through an autocrine mechanism, thus providing the support needed to the proliferating biliary system after BDL.5,13 VEGF-A induces many cellular effects, including increasing cellular migration, mitogenesis, and angiogenesis and promoting cellular proliferation while inhibiting apoptosis.13,14 VEGF-C is an important mediator of cell growth and survival, as well as being a regulator of angiogenesis.15,16 HDC regulates both VEGF-A and VEGFC expression.10,15 In HDC/ mice, angiogenesis is defective and histamine derived from nonmast cells regulates the angiogenesis of the inflammatory tissue.16 miRNAs are post-transcriptional regulators that alter cellular translation. After 70% partial hepatectomy, biliary miR-181b expression is significantly up-regulated compared with shameoperated on animals.17 Also, miR-125b is
significantly down-regulated in hepatocellular cancer18 and cholangiocarcinoma.19 Another study found that miR-125b inhibits hepatocellular cell migration and invasion,20 suggesting that this miRNA is involved in angiogenesis. On the basis of this background, we proposed to demonstrate that miR-125b regulates large biliary hyperplasia by modulating the HDC/VEGF axis.
Materials and Methods All reagents were obtained from Sigma-Aldrich, Co (St. Louis, MO), unless otherwise indicated. All primers and transfection plasmids were obtained from SA Biosciences (Frederick, MD), unless stated otherwise. Antibodies for immunohistochemistry (IHC) and Western blot analysis were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), unless indicated otherwise. To determine histamine levels, we used an enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI).
Figure 1 Evaluation of HDC expression. A: HDC expression in bile ducts compared with normal mice and after treatment with the HDC inhibitor, a-methylDHis. Original magnification, 20. Inset: Increased resolution of image. B: HDC gene expression in large cholangiocytes compared with BDL mice and after amethyl-DHis treatment. Data are the means SEM of eight experiments from cumulative preparations of large cholangiocytes from at least eight animals from each treatment group. *P < 0.05 versus normal; yP < 0.05 versus BDL. C: After BDL, histamine secretion in serum compared with normal and after treatment with the HDC inhibitor compared with both normal and BDL. D: In isolated large cholangiocyte supernatant, histamine secretion after BDL compared with normal, and after BDL plus a-methyl-DHis treatment compared with normal and BDL. Data are the means SEM of 12 experiments from cholangiocyte supernatants collected from cumulative preparations of large cholangiocytes from at least six animals from each treatment group. *P < 0.05 versus normal; y P < 0.05 versus BDL.
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Animal Models and Isolated Cholangiocytes Mice were subjected to sham surgery (normal) or BDL and treated, immediately after surgery, by daily i.p. injections with vehicle (0.9% NaCl) or a-methyl-DL-histidine (amethyl-DHis; 150 mg/body weight; M8628; SigmaAldrich)10,21; after 3 and 7 days, liver blocks (paraffin and frozen, 4 to 5 mm thick), serum, organs, and isolated large cholangiocytes were collected. Large cholangiocytes were provided by Dr. Gianfranco Alpini (Scott and White, Central Texas Veterans Health Care System and Texas A&M Health Science Center, Temple).4,22
LMCC supernatants were measured by commercially available enzyme immunolink assays (Cayman Chemical).10 Intrahepatic bile duct mass and cholangiocyte proliferation were determined in all animal groups by semiquantitative IHC staining for cytokeratin-19 and proliferating cellular nuclear antigen (PCNA), respectively.11,25 In isolated large cholangiocytes from all animal groups and LMCC, protein lysates were collected and Western blot analysis was performed for PCNA to determine cholangiocyte proliferation. Western by scanning video densitometry using the ImageQuant TL Software version 2003.02.
In Vitro Cell Lines In vitro studies were performed in SV-40etransformed large mouse cholangiocyte lines (LMCC)23 and human intrahepatic epithelial cells (HiBECs). HiBECs were purchased from Sciencell Research Laboratories (Carlsbad, CA).10 Cells were cultured according to previous studies.10,12,23
Evaluation of HDC Expression and Histamine Secretion To determine the expression of HDC in the liver, we performed IHC in liver sections (4 to 5 mm thick) and real-time and standard RT-PCR in isolated large cholangiocytes, hepatocytes, and LMCC. IHC was performed in paraffinembedded liver sections. The antibody for HDC (sc-34458; Santa Cruz Biotechnology) was used at 1:50. Negative controls were performed in parallel using nonimmune serum as the primary antibody. Light microscopy (Leica Microsystems, Wetzlar, Germany) was performed to obtain and quantify images. Positive cells were counted in six nonoverlapping fields (20 magnification) for each slide, and the data expressed as percentage of positive cells. Light microscopy and IHC observation were performed in a blinded manner by two independent morphologists. Real-time PCR was performed in total RNA extracted from isolated large cholangiocytes from all animal groups to determine gene expression of HDC. For all PCRs, mouse primers (SA Biosciences) were used and a DDCT analysis was performed. Total cellular RNA (1 mg) was used for PCRs, and cytokeratin-19 (a cholangiocyte-specific marker)14 or glyceraldehyde-3-phosphate dehydrogenase14 was used as the control. Standard RT-PCR was performed in LMCC to determine mRNA levels of HDC, as described.24 Western blot analysis for HDC expression was performed on protein from whole cell lysates from purified large cholangiocytes, hepatocytes, and LMCC. Cells were trypsinized and lysed for immunoblots and SDS-PAGE, as described.10 Western blot analysis was performed with 10 to 20 mg of protein, and band intensity was determined ImageQuant TL Software version 2003.02 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Histamine levels in serum, isolated large cholangiocyte supernatants (after incubation for 6 hours at 37 C), and
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Figure 2 Evaluation of intrahepatic bile duct mass after a-methyl-DHis treatment. Ductal mass after BDL compared with normal shown by IHC (A). Quantification of IHC (B). Original magnifications: 20; 40 (inset). *P < 0.05 versus normal; yP < 0.05 versus BDL.
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Determination of VEGF Expression VEGF cholangiocyte expression was evaluated by IHC in paraffin-embedded liver sections (4 mm thick), and realtime and standard RT-PCR in purified large cholangiocytes and LMCC, respectively. Sections were incubated overnight at 4 C with the following antibodies from Santa Cruz Biotechnology: VEGF-A (sc-152; 1:1000) and VEGF-C (sc-9047; 1:200). The immunoreactivity of VEGF-A/C in cholangiocytes was automatically quantified by ImageScope (Aperio Scanscope CS System, Vista, CA). Bile ducts were randomly selected by the operator, and an image analysis algorithm that automatically analyzes the selected areas was used to quantify the number of positive pixels in cholangiocytes. The algorithm was applied to the entire section of at least three different sections from each liver fragment. The percentage of positive pixels and the average intensity of positivity were obtained from the software, and quantification of IHC was obtained by multiplying the percentage of positive pixels by the average intensity of positivity. Immunofluorescence and Western blot analysis were performed in LMCC to detect VEGF-A/C protein levels, as
described.11,15 Cells were seeded on coverslips in a six-well plate (500,000 cells per well) and allowed to adhere overnight. Immunofluorescence was performed, as described, using a specific antibody for VEGF-A and VEGF-C (diluted 1:50 in 1% bovine serum albumin/PBS with Tween or nonimmune serum for negative controls).10,12 Images were obtained using an inverted Olympus IX-71 confocal microscope (Olympus, Tokyo, Japan). Quantification of immunofluorescent staining was performed manually and expressed as the percentage of VEGF-A/Cepositive cholangiocytes per area (six areas were counted by a blinded operator). Primary antibodies for Western blot analysis were diluted at 1:50.
Evaluation of miR-125b To determine whether HDC is a target of the predicted miR125b, we extracted the mRNA sequence of HDC and scanned potential binding sites for miRNAs using both Targetscan (Whitehead Institute for Biomedical Research, Cambridge, MA) and PicTar 4-way software (Center for Comparative Functional Genomics, New York, New York) and performed miRNA profiling by microarray analysis.
Evaluation of PCNA expression after a-methyl-DHis treatment. A: The number of PCNA-positive cholangiocytes after BDL and after a-methyl-DHis treatment shown by IHC. Original magnifications: 20; 40 (inset). Quantification of IHC (B). PCNA protein expression after BDL and after treatment with amethyl-DHis compared with BDL mice (C). Data are the means SEM of at least four experiments from cumulative preparations of large cholangiocytes from at least eight animals from each treatment group. *P < 0.05 versus normal; yP < 0.05 versus BDL.
Figure 3
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Meng et al After identification of the target miR-125b, we measured miR-125b expression in isolated cholangiocytes from normal and BDL mice by microarray analysis performed by the Center for Targeted Therapy, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center (Houston) and verified by real-time PCR analysis, as previously described, using a specific TaqMan miRNA assay (Life Technologies, Carlsbad, CA).
Luciferase Reporter Assay An intact putative miR-125b recognition sequence from the 30 euntranslated region (UTR) of HDC/VEGF (pMIRe HDCewte30 -UTR or pGL3eVEGFe30 -UTR) or with random mutations (pMIReHDCemute30 -UTR) was cloned downstream of the firefly luciferase reporter gene. For the miR-125beHDC target relationship, luciferase assays were performed at 72 hours after transfection with control and miR-125b precursor or inhibitor (50 nmol/L) in HiBEC cells using the Dual Luciferase Reporter Assay system (Promega, Madison, WI). To determine whether miR-125b directly targets VEGF, HiBEC cells were plated (2 106 cells per well) in 6-well plates. The pGL3eVEGFe30 -UTR firefly luciferase expression construct (1 mg) and a Renilla luciferase expression construct, pRL-TK (1 mg; Promega), were cotransfected with the miR-125b or control precursor or
inhibitor. Luciferase assays were performed 48 hours after transfection using the dual Luciferase Reporter Assay System (Promega).
Genetic Modification of HDC and miR-125b in LMCC To demonstrate that the effects of HDC and miR-125b are specific to cholangiocyte proliferation, we performed in vitro independent experiments to genetically knock down HDC or overexpress miR-125b in LMCC. By using an HDC shRNA plasmid, we generated a stably transfected HDC LMCC knockdown cell line (LMCC-HDC), along with a negative (LMCC-neg) cell line, as described.10 Overexpression of miR125b (preemiR-125b) was performed in standard media containing 10% fetal bovine serum and using preemiR-125b (50 nmol/L) from Ambion Inc. (Austin, TX), along with appropriate controls (pre-miRNA), and miR-125b expression was measured by TaqMan real-time PCR assay (Applied Biosystem Inc., Grand Island, NY). In LMCC-HDC or miR-125b overexpressed cells (and respective controls), HDC, VEGF-A, and VEGF-C gene and protein levels were determined. In LMCC-HDC and respective control cells, we also evaluated the expression of the mRNA for H1 to H4 histamine receptors by real-time PCR. Histamine secretion was measured in conditioned media from genetically modified cells using EIA, and cholangiocyte
Figure 4 In vivo expression of VEGF-A and VEGF-C in liver sections. VEGF-A and VEGF-C expression after BDL compared with normal, and after treatment with the HDC inhibitor, a-methyl-DHis, compared with BDL. Original magnifications: 20; 40 (inset). Data are the means SEM from at least four animals per group. *P < 0.05 versus normal; yP < 0.05 versus BDL.
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Figure 5
In vivo evaluation of miR-125b expression and verification by PCR. miR-125b levels after BDL compared with normal by microarray and confirmed by real-time PCR (A) and in isolated large cholangiocytes with total RNA extracted from control and BDL mice (B). Data are the means SEM of six experiments. *P < 0.05 versus normal. C: Targeting of HDC 30 -UTR by miR-125b. The sequences of the mutated target site of HDC with mutations to disrupt base pairing between the specific binding sites and miR-125b are displayed. D: Luciferase reporter constructs containing the miR-125b recognition sequence from the 30 -UTR of HDC inserted downstream of the luciferase gene were generated in HiBEC cholangiocytes. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample transfected with control and miR-125b precursor (50 nmol/L) for 72 hours. Data are the means SEM of six experiments. *P < 0.05 versus control.
proliferation was determined by MTT assays12 and Western blot analyses12 for PCNA. Apical and basolateral membranes were collected from LMCCs, according to our previous studies.26,27 Specifically, cells were placed in transwell chambers and brought to confluency, and transepithelial resistance was measured before experimental analysis of histamine levels by EIA.
otherwise. Differences between groups were analyzed by the Student’s unpaired t-test when two groups were analyzed, and by analysis of variance when more than two groups were analyzed, followed by an appropriate post hoc test. The null hypothesis was rejected at P < 0.05.
Statistical Analysis
HDC Expression and Histamine Release in Vivo
Data are expressed as means SEM from at least three separate experiments performed in triplicate, unless noted
Prolonged administration (3 and 7 days) of a-methyl-DHis to normal mice induced no changes in intrahepatic bile duct
Results
Figure 6
Standard RT-PCR evaluation of HDC, VEGF-A, and VEGF-C expression in LMCCs. LMCCs express HDC, VEGF-A, and VEGF-C. Total RNA from liver was used as a positive control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 140 bp; HDC, 162 bp; VEGF-A, 62 bp; VEGF-C, 118 bp.
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Meng et al mass, liver weight, body weight, and liver/body weight ratio (data not shown). Therefore, we performed all studies in normal and BDL mice treated for 7 days with vehicle or a-methyl-DHis. HDC was expressed mainly in large cholangiocytes (Figure 1A) and increased after BDL. Realtime PCR (Figure 1B) for HDC expression demonstrates that HDC gene expression is increased in large cholangiocytes after BDL and reduced in BDL þ a-methylDHisetreated mice. By immunoblotting, a slight expression of HDC was found in mouse hepatocytes, whereas a strong expression was found in isolated mouse cholangiocytes, thus strengthening the autocrine role of HDC in biliary proliferation (Supplemental Figure S1). Histamine levels increase in serum and cholangiocyte supernatant obtained from BDL mice when compared with normal mice, whereas treatment with a-methyl-DHis significantly decreased histamine release in serum and cholangiocyte supernatant (Figure 1, C and D).
Evaluation of Intrahepatic Bile Duct Mass and Cholangiocyte Proliferation After BDL, intrahepatic bile duct mass increases compared with normal mice; treatment with a-methyl-DHis significantly decreases intrahepatic bile duct mass when compared with BDL alone (Figure 2). The number of PCNA-positive cholangiocytes increased after BDL and was significantly reduced when animals were treated with a-methyl-DHis (Figure 3A). There was a significant decrease in PCNA protein expression in large cholangiocytes from a-methylDHisetreated BDL mice compared with BDL mice (Figure 3B). We evaluated lobular damage, necrosis, and inflammation in the liver and other major organs (heart, lung, intestine, spleen, and stomach) in all groups and found
that a-methyl-DHis has no pathological effects in our animal model, and no significant morphological changes were noted (data not shown). These data demonstrate that HDC is an important regulator of cholangiocyte proliferation.
VEGF Expression in Vivo The expression of VEGF-A and VEGF-C significantly increased in cholangiocytes from mouse liver after BDL when compared with normal (Figure 4). Furthermore, after HDC inhibition, cholangiocyte VEGF-A/C expression decreased, suggesting that HDC regulates the expression of VEGF during biliary proliferation.
In Vivo Evaluation of miR-125b and Its Target Proteins in BDL Mice The potential miRNAs predicted to target HDC were selected from the list of miRNAs in which expression levels were reduced in BDL mouse liver (<0.05-fold, P < 0.01). miR-125b is significantly decreased in BDL liver versus normal mice (Figure 5A) (ArrayExpress database; http:// www.ebi.ac.uk/arrayexpress, accession number E-MTAB2267). Real-time PCR analysis revealed that, in cholangiocytes isolated from BDL and normal mouse liver, miR-125b gene expression was significantly downregulated after BDL compared with normal (Figure 5B). HDC is a predicted target of miR-125b, as shown by bioinformatic analysis (Figure 5C). To verify that HDC is a direct target of miR-125b, we used luciferase reporter constructs containing the miR-125b recognition sequence from the 30 -UTR of HDC inserted downstream of the luciferase gene (Figure 5C). Transfection with precursors or inhibitors of miR-125b significantly modulated reporter activity of
Figure 7
Effects of overexpression of miR-125b in LMCCs. A: miR-125b expression compared with control pre-miRNA (top panel). HDC expression after overexpression of miR-125b (bottom panel). B: By realtime PCR, VEGF-A, VEGF-C, and H1 to H4 histamine receptor gene expression levels were measured after miR-125b overexpression compared with control premiRNA. Data are the means SEM of six experiments. *P < 0.05 versus control transfected cells.
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miR-125b Regulation of Biliary Growth HDC in human cholangiocytes (Figure 5D). Studies were repeated with random mutations in the shared recognition sequence (Figure 5D), which resulted in abolition/ enhancement of the HDC reporter activation by miR-125b overexpression or inhibition (Figure 5D). To determine that miR-125b also targets VEGF, we performed a comparison of bp between mature human miR-125b, human VEGF-A, mouse mmu-miR-125b, and mouse VEGF-A, and found a sequence conservation between the species (Supplemental Figure S2). Furthermore, by using luciferase assays, we found that both overexpression (miR-125b precursor) and inhibition (antiemiR-125b) of miR-125b induced a significant change of VEGF-A luciferase activity (Supplemental Figure S2), which demonstrated that VEGF-A is also a direct target of miR-125b in biliary epithelial cells.
(Figure 5B), we overexpressed miR-125b in LMCCs. Overexpression of miR-125b decreased HDC, H1 to H4 histamine receptors, and VEGF-A/C gene expression when compared with pre-miRNA control-transfected LMCCs (Figure 7). HDC and VEGF-A/C protein expression and histamine release were significantly reduced after miR-125b overexpression compared with miRNA control precursor (Figure 8, AeC). Cholangiocyte proliferation was reduced after miR-125b overexpression (Figure 8, D and E) compared with control precursor-transfected cells. To further confirm that miR-125b is an important regulator of HDC (and downstream targets), we silenced miR-125b (Supplemental Figure S3) and performed real-time PCR for HDC. We found that, after knockdown of miR-125b, there is an increase in HDC expression compared with control-transfected cells (Supplemental Figure S3). These data suggest that miR-125b is an important target and regulator of the HDC/VEGF axis.
In Vitro Evaluation of miR-125b Effects on HDC and VEGF Expression and Cholangiocyte Proliferation
Validation of HDC Effects in Vitro
The LMCCs23 express HDC and VEGF-A/C (Figure 6). Because miR-125b expression is greatly reduced after BDL
To validate that the effects of miR-125b can be mimicked by silencing HDC, we generated a stably transfected HDC
Figure 8
Effects of miR-125b in vitro. Effects of miR125b overexpression in LMCCs on HDC, VEGF-A, and VEGF-C protein expression, histamine secretion, and cholangiocyte proliferation. HDC (A) and histamine secretion in conditioned medium from miR-125b overexpressed cells compared with control pre-miRNA transfected cells (B) C: Protein expression of VEGF-A/C after miR-125b overexpression compared with control pre-miRNA transfected cells. D and E: PCNA protein expression (D) and MTT assay (E) after miR-125b overexpression compared with control pre-miRNAe transfected cells. Data are the means SEM of 12 experiments for secretion, the means SEM of 8 experiments for Western blot analysis, and the means SEM of 16 experiments for MTT assay (24-hour time point). *P < 0.05 versus control pre-miRNAetransfected cholangiocytes.
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Meng et al knockdown LMCC cell line (LMCC-HDC) lacking HDC and both shRNA control-transfected LMCC (LMCC-neg) and nontransfected LMCC (LMCC basal) cells as controls and measured the following: (i) HDC and VEGF-A/C expression, (ii) histamine secretion, and (iii) cholangiocyte proliferation. Loss of HDC (Figure 9, A and B) induced a decrease in these components. VEGF-A/C gene and protein expression was significantly decreased in LMCC-HDC (cells deficient in HDC expression) compared with controltransfected LMCC (LMCC-neg) (Figure 10) and nontransfected LMCC (data not shown) cells. LMCC secretes histamine at both the apical and basolateral membrane (Figure 9C), and histamine release (Figure 9D) and cholangiocyte proliferation (Figure 9, E and F) were diminished after knockdown of HDC when compared with controls. To determine whether histamine rescues the effects of genetic manipulation of HDC inhibition and further demonstrate an autocrine regulation by cholangiocytes, histamine was added back into the LMCC-HDC cells, secretion was measured by EIA, and the expression of H1 to H4 histamine receptors was
evaluated by real-time PCR. Addition of histamine to LMCC-HDC increased histamine secretion (Figure 9D). Finally, we found that after knockdown of HDC (LMCCHDC), the expression of all histamine receptors (H1 to H4) decreased compared with LMCC-neg (Figure 11), and when histamine was added to LMCC-HDC, H1 to H4 gene expression was significantly increased (Figure 11). These data demonstrate a role for HDC in regulating cholangiocyte proliferation and VEGF expression, potentially via one of the four histamine receptors.
Discussion In the present study, we demonstrated the autocrine role of HDC on the modulation of cholangiocyte hyperplasia during extrahepatic bile duct obstruction, and that these effects are specific to cholangiocytes and regulated by miR-125b. We demonstrated that HDC expression is primarily expressed by cholangiocytes and increases after BDL that is coupled with a significant reduction in miR-125b expression. Traditionally,
Figure 9
Overall effects of HDC knockdown on biliary HDC expression, histamine secretion, and proliferation. By real-time PCR (A) and Western blot analysis (B), HDC gene and protein expression after stable knockdown transfection of HDC shRNA (LMCC-HDC) compared with control-transfected, LMCC-neg. Data are the means SEM of three experiments for real-time PCR and eight experiments for Western blot analysis. Histamine at the apical and basolateral membrane of LMCC (nontransfected, basal treatment) (C); histamine release in LMCC lacking HDC (LMCC-HDC); effects of addition of histamine (10 mmol/L) in LMCC-HDC histamine secretion (gray bar) compared with LMCC-neg and LMCC-HDC (D). By MTT assay (E) and PCNA protein expression (F), cholangiocyte proliferation after loss of HDC (LMCC-HDC) compared with cells with normal amounts of HDC (LMCC-neg). Data are the means SEM of 12 experiments. *P < 0.05 versus LMCC-neg, control-transfected cholangiocytes; yP < 0.05 versus LMCC-HDC.
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Figure 11
Evaluation of loss of histamine receptors in LMCCs. After stable transfection and knockdown of HDC, histamine receptor (H1 to H4) expression compared with LMCC-neg cells. Effects of addition of histamine on expression of H1 to H4 compared with LMCC-HDCetransfected cells. Data are the means SEM of six experiments for real-time PCR. *P < 0.05 versus LMCC-neg (control)etransfected cholangiocytes; yP < 0.05 versus LMCC-HDCetransfected cholangiocytes.
this concept, demonstrating that histamine is a trophic, growth-promoting factor.11 Our study shows that cholangiocytes contain the machinery to produce histamine and secrete increased levels of histamine after BDL, likely to sustain the increased ductal mass. Also, preliminary data from BDL rats show that histamine levels are significantly increased in bile [0.756 0.1307 (normal) versus 47.85 1.116 (BDL) nmol/L], which supports our autocrine hypothesis for histamine regulation of cholangiocyte proliferation. In genetically modified, HDC knockout mice, wound-healing time is slower when compared with mice
Figure 10
Evaluation of loss of HDC in LMCCs. After stable transfection and knockdown of HDC, VEGF-A, and VEGF-C gene (left panel) and protein (right panels) expression compared with LMCC-neg cells. Original magnification, 20. Inset: Increased resolution of image (arrows). Data are the means SEM of three experiments for real-time PCR. *P < 0.05 versus LMCC-neg (control)etransfected cholangiocytes.
HDC is expressed primarily by mast cells.28,29 However, in support of our findings, previous studies have shown that HDC expression and histamine synthesis are also found in gastric enterochromaffin-like cells.30 Histamine synthesis plays a role in neuroregulation, and HDC expression regulates day-night rhythms by the changes in the fluctuation of HDC expression in the hypothalamus.31 Numerous factors regulate cholangiocyte proliferation via an autocrine loop, including histamine, melatonin, and VEGF.5,32e34 We have shown that HDC is critical in regulating cholangiocyte hyperplasia, as shown by the loss of intrahepatic bile duct mass and cholangiocyte proliferation after inhibition of HDC. Previous work also supports
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Figure 12
Proposed overall working model of the effects of miR125beregulated HDC expression in BDL mice treated with the HDC inhibitor, a-methyl-DL-histidine.
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Meng et al with normal histamine levels.35,36 In our current and previously published studies, we have demonstrated that the HDC/histamine axis is a trophic pathway increasing cholangiocyte proliferation. In data presented herein and in preliminary data using HDC/ mice (data not shown), we found that inhibition of HDC (by pharmacological treatment or genetic manipulation) decreases cholangiocyte proliferation via decreased VEGF expression. In contrast, a recent study has shown that, in HDC/ mice, there is an increased rate of colon cancer and skin carcinogenesis.37 This study focused on the paracrine contribution of immature myeloid cell production of HDC and proposes that the balance of histamine synthesis is dysregulated in the tumor microenvironment of skin and colon cancer. Our study focuses on the autocrine release of HDC from the biliary epithelium, which is an unusual tissue. For example, gastrin (which has trophic effects in gastric, colonic, and pancreatic cancer cell lines) inhibits both biliary hyperplasia in BDL rats and cholangiocarcinoma growth of several human cell lines.38,39 We speculate that targeting HDC locally (by nanoparticles containing HDC inhibitors) may be an effective therapeutic approach for modulating HDC directly in the liver. After BDL, the intrahepatic biliary epithelium experiences rapid cholangiocyte proliferation (that occurs before the growth of the peribiliary vascular plexus) that is characterized by increased proliferative activities, including the increased release of histamine and VEGF.5,40 In our study, systemic treatment of a-methyl-DHis decreased cholangiocyte VEGF-A/C expression after biliary injury, presumably to compensate for the decrease in biliary demand. To support our findings, VEGF and angiogenesis are decreased in HDC/ mice granulation tissue when compared with normal controls.16 When HDC/ mice are treated with an H2 histamine receptor agonist, wound healing is recovered and angiogenesis is restored.16 Furthermore, our data demonstrate that, after loss of HDC (or in miR-125beoverexpressed cells), H1 to H4 histamine receptor expression decreases in a similar manner. Addition of histamine to cells with diminished HDC expression resulted in increased histamine secretion and increased receptor expression. We have shown, in our previous work, that there is a differential cholangiocyte response that results from specific receptor stimulation or inhibition, whereas in this study, it appears that knockdown of HDC affects all receptors, leading to a decrease in cholangiocyte proliferation. The exact role of the individual receptors will need to be further investigated. Consistent with our previous findings in human cholangiocytes,10 our present study demonstrates that HDC regulates cholangiocyte VEGF-A/C expression; to expand this, we evaluated the effects of the specific miRNA miR-125b, on HDC. miR-125b expression is decreased in hepatocellular carcinoma, as well as cholangiocarcinoma tissues and cells, whereas overexpression of miR-125b decreases tumor growth.19 After BDL, miR-125b was significantly decreased, which may lead to up-regulation of HDC. After
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overexpression of miR-125b, the HDC/VEGF axis and cholangiocyte proliferation are all down-regulated. Further studies will focus on evaluating other targets (in addition to HDC) for miR-125b that may affect liver functions. In summary, we have provided evidence that HDC plays a critical role in cholangiocyte proliferation. The HDC/ VEGF axis that mediates cholangiocyte proliferation loses function when histamine is no longer being synthesized after the loss of HDC. Furthermore, we found that miR125b regulates the HDC/VEGF axis and may be a potential target for future studies. The current study, coupled with our previous work regarding the role of histamine and VEGF on cholangiocyte proliferation, provides substantial evidence that histamine and HDC (regulated by miR-125b) are key mediators of cholangiocyte response during cholestatic liver injury (Figure 12 depicts an overall working model).
Acknowledgment We thank Dr. Gianfranco Alpini for supplying freshly isolated large mouse cholangiocytes and generously contributing his time in the editing of this work.
Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2013.11.008.
References 1. Alpini G, Prall RT, LaRusso NF: The pathobiology of biliary epithelia. Edited by Arias IM, Boyer JL, Chisari FV, Fausto N, Jakoby W, Schachter D, Shafritz DAs. The Liver: Biology & Pathobiology. ed 4. Philadelphia, Lippincott Williams & Wilkins, 2001, pp 421e435 2. Mancinelli R, Onori P, Gaudio E, Franchitto A, Carpino G, Ueno Y, Alvaro D, Annarale LP, DeMorrow S, Francis H: Taurocholate feeding to bile duct ligated rats prevents caffeic acid-induced bile duct damage by changes in cholangiocyte VEGF expression. Exp Biol Med 2009, 234:462e474 3. Francis H, Franchitto A, Ueno Y, Glaser S, DeMorrow S, Venter J, Gaudio E, Alvaro D, Fava G, Marzioni M, Vaculin B, Alpini G: H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway. Lab Invest 2007, 87:473e487 4. Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV, Colegio O, LeSage GD, Miller LJ, LaRusso NF: Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 1997, 272:G289eG297 5. Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Ueno Y, Meininger CJ, Franchitto A, Onori P, Marzioni M, Taffetani S, Fava G, Stoica G, Venter J, Reichenbach R, De Morrow S, Summers R, Alpini G: Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism. Gastroenterology 2006, 130:1270e1282 6. Jutel M, Akdis M, Akdis CA: Histamine, histamine receptors and their role in immune pathology. Clin Exp Allergy 2009, 39:1786e1800 7. Beaven MA: Our perception of the mast cell from Paul Ehrlich to now. Eur J Immunol 2009, 39:11e25 8. Francis H, Meininger CJ: A review of mast cells and liver disease: what have we learned? Dig Liver Dis 2010, 42:529e536
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miR-125b Regulation of Biliary Growth 9. Gittlen SD, Schulman ES, Maddrey WC: Raised histamine concentrations in chronic cholestatic liver disease. Gut 1990, 31:96e99 10. Francis H, DeMorrow S, Venter J, Onori P, White M, Gaudio E, Francis T, Greene JF Jr., Tran S, Meininger CJ, Alpini G: Inhibition of histidine decarboxylase ablates the autocrine tumorigenic effects of histamine in human cholangiocarcinoma. Gut 2012, 61:753e764 11. Francis HL, DeMorrow S, Franchitto A, Venter JK, Mancinelli RA, White MA, Meng F, Ueno Y, Carpino G, Renzi A, Baker KK, Shine HE, Francis TC, Gaudio E, Alpini GD, Onori P: Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2þ and cAMP-dependent signaling mechanisms. Lab Invest 2012, 92:282e294 12. Francis H, Glaser S, DeMorrow S, Gaudio E, Ueno Y, Venter J, Dostal D, Onori P, Franchitto A, Marzioni M, Vaculin S, Vaculin B, Katki K, Stutes M, Savage J, Alpini G: Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway. Am J Physiol Cell Physiol 2008, 295:C499eC513 13. Gaudio E, Franchitto A, Pannarale L, Carpino G, Alpini G, Francis H, Glaser S, Alvaro D, Onori P: Cholangiocytes and blood supply. World J Gastroenterol 2006, 12:3546e3552 14. Gaudio E, Barbaro B, Alvaro D, Glaser S, Francis H, Franchitto A, Onori P, Ueno Y, Marzioni M, Fava G, Venter J, Reichenbach R, Summers R, Alpini G: Administration of r-VEGF-A prevents hepatic artery ligation-induced bile duct damage in bile duct ligated rats. Am J Physiol Gastrointest Liver Physiol 2006, 291:G307eG317 15. Francis H, Onori P, Gaudio E, Franchitto A, DeMorrow S, Venter J, Kopriva S, Carpino G, Mancinelli R, White M, Meng F, Vetuschi A, Sferra R, Alpini G: H3 histamine receptor-mediated activation of protein kinase Calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo. Mol Cancer Res 2009, 7:1704e1713 16. Ghosh AK, Hirasawa N, Ohtsu H, Watanabe T, Ohuchi K: Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice. J Exp Med 2002, 195:973e982 17. Meng F, Francis H, Glaser S, Han Y, DeMorrow S, Stokes A, Staloch D, Venter J, White M, Ueno Y, Reid LM, Alpini G: Role of stem cell factor and granulocyte colony-stimulating factor in remodeling during liver regeneration. Hepatology 2012, 55:209e221 18. Alpini G, Glaser S, Zhang JP, Francis H, Han Y, Gong J, Stokes A, Francis T, Hughart N, Hubble L, Zhuang SM, Meng F: Regulation of placenta growth factor by microRNA-125b in hepatocellular cancer. J Hepatol 2011, 55:1339e1345 19. Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT, Jiang J, Schmittgen TD, Patel T: Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology 2006, 130:2113e2129 20. Liang L, Wong CM, Ying Q, Fan DN, Huang S, Ding J, Yao J, Yan M, Li J, Yao M, Ng IO, He X: MicroRNA-125b suppressesed human liver cancer cell proliferation and metastasis by directly targeting oncogene LIN28B2. Hepatology 2010, 52:1731e1740 21. Malmberg-Aiello P, Lamberti C, Ghelardini C, Giotti A, Bartolini A: Role of histamine in rodent antinociception. Br J Pharmacol 1994, 111: 1269e1279 22. LeSage GD, Glaser SS, Marucci L, Benedetti A, Phinizy JL, Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM, Holcomb LA, Alpini G: Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver. Am J Physiol Gastrointest Liver Physiol 1999, 276:G1289eG1301 23. Ueno Y, Alpini G, Yahagi K, Kanno N, Moritoki Y, Fukushima K, Glaser S, LeSage G, Shimosegawa T: Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice. Liver Int 2003, 23:449e459 24. Patsenker E, Popov Y, Stickel F, Jonczyk A, Goodman SL, Schuppan D: Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression. Gastroenterology 2008, 135:660e670
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25. Marzioni M, Glaser S, Francis H, Marucci L, Benedetti A, Alvaro D, Taffetani S, Ueno Y, Roskams T, Phinizy JL, Venter J, Fava G, LeSage G, Alpini G: Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin. Gastroenterology 2005, 128:121e137 26. Han Y, DeMorrow S, Invernizzi P, Jing Q, Glaser S, Renzi A, Meng F, Venter J, Bernuzzi F, White M, Francis H, Lleo A, Marzioni M, Onori P, Alvaro D, Torzilli G, Gaudio E, Alpini G: Melatonin exerts by an autocrine loop antiproliferative effects in cholangiocarcinoma: its synthesis is reduced favoring cholangiocarcinoma growth. Am J Physiol Gastrointest Liver Physiol 2011, 301:G623eG633 27. Vroman B, LaRusso NF: Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells. Lab Invest 1996, 74:303e313 28. Boyce JA: Mast cells: beyond IgE. J Allergy Clin Immunol 2003, 111: 24e32. quiz 33 29. Boyce JA: The biology of the mast cell. Allergy Asthma Proc 2004, 25:27e30 30. Lambrecht NW, Yakubov I, Sachs G: Fasting-induced changes in ECL cell gene expression. Physiol Genomics 2007, 31:183e192 31. Shan L, Hofman MA, van Wamelen DJ, Van Someren EJ, Bao AM, Swaab Dick F: Diurnal fluctuation in histidine decarboxylase expression, the rate limiting enzyme for histamine production, and its disorder in neurodegenerative diseases. Sleep 2012, 35:713e715 32. Francis H, LeSage G, DeMorrow S, Alvaro D, Ueno Y, Venter J, Glaser S, Mancino MG, Marucci L, Benedetti A, Alpini G: The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretinstimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats. Am J Physiol Cell Physiol 2007, 293: C1252eC1262 33. Glaser SS, Onori P, Wise C, Yang F, Marzioni M, Alvaro D, Franchitto A, Mancinelli R, Alpini G, Munshi MK, Gaudio E: Recent advances in the regulation of cholangiocyte proliferation and function during extrahepatic cholestasis. Dig Liver Dis 2010, 42:245e252 34. Renzi A, Glaser S, DeMorrow S, Mancinelli R, Meng F, Franchitto A, Venter J, White M, Francis H, Han Y, Alvaro D, Gaudio E, Carpino G, Ueno Y, Onori P, Alpini G: Melatonin inhibits cholangiocyte hyperplasia in cholestatic rats by interaction with MT1 but not MT2 melatonin receptors. Am J Physiol Gastrointest Liver Physiol 2011, 301: G634eG643 35. Numata Y, Terui T, Okuyama R, Hirasawa N, Sugiura Y, Miyoshi I, Watanabe T, Kuramasu A, Tagami H, Ohtsu H: The accelerating effect of histamine on the cutaneous wound-healing process through the action of basic fibroblast growth factor. J Invest Dermatol 2006, 126: 1403e1409 36. Ohtsu H: Pathophysiologic role of histamine: evidence clarified by histidine decarboxylase gene knockout mice. Int Arch Allergy Immunol 2012, 158(Suppl 1):2e6 37. Yang XD, Ai W, Asfaha S, Bhagat G, Friedman RA, Jin G, Park H, Shykind B, Diacovo TG, Falus A, Wang TC: Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11bþLy6Gþ immature myeloid cells. Nat Med 2011, 17:87e95 38. Kanno N, Glaser S, Chowdhury U, Phinizy JL, Baiocchi L, Francis H, LeSage G, Alpini G: Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2þ-dependent protein kinase C-alpha. J Hepatol 2001, 34:284e291 39. Glaser S, Benedetti A, Marucci L, Alvaro D, Baiocchi L, Kanno N, Caligiuri A, Phinizy JL, Chowdury U, Papa E, LeSage G, Alpini G: Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myoinositol 1,4,5-triphosphate-, Ca(2þ)-, and protein kinase C alphadependent mechanisms. Hepatology 2000, 32:17e25 40. Onori P, Gaudio E, Franchitto A, Alpini G, Francis H: Histamine regulation of hyperplastic and neoplastic cell growth in cholangiocytes. World J Gastroenterol 2010, 1:38e49
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GASTROINTESTINAL, HEPATOBILIARY, AND PANCREATIC PATHOLOGY
Regulation of the Histamine/VEGF Axis by miR-125b during Cholestatic Liver Injury in Mice Fanyin Meng,*yz Paolo Onori,x Laura Hargrove,y Yuyan Han,z Lindsey Kennedy,y Allyson Graf,y Kyle Hodges,y Yoshiyuki Ueno,{k Taylor Francis,y Eugenio Gaudio,x and Heather L. Francis*yz From the Department of Research,* Central Texas Veteran’s Health Care System, the Scott and White Digestive Disease Research Center,y Scott and White Healthcare, and the Department of Internal Medicine,z Texas A&M Health Science Center, Temple, Texas; the Department of Anatomical, Histological, Forensic Medicine and Orthopedic Sciences,x Sapienza, University of Rome, Rome, Italy; CREST,{ Japan Science and Technology Agency, Yamagata, Japan; and the Department of Gastroenterology,k Yamagata University Faculty of Medicine, Yamagata, Japan Accepted for publication November 15, 2013. Address correspondence to Heather L. Francis, Ph.D., Department of Internal Medicine, Texas A&M Health Science Center College of Medicine, Central Texas Veteran’s Health Care System, Digestive Disease Research Center, Scott and White Healthcare, 1901 S Veterans Memorial Dr, Bldg 205, Office 1R52, Temple, TX 76504. E-mail: [email protected].
Histamine is formed by the conversion of L-histidine into histamine by histidine decarboxylase (HDC). We have previously shown that inhibition of HDC blocks cholangiocyte proliferation and silencing of HDC decreases vascular endothelial growth factor (VEGF) expression. We hypothesized that increased HDC expression during cholestatic liver injury is mediated by the down-regulation of the specific miRNA miR125b, a post-transcriptional regulator. Mice were subjected to sham surgery or bile duct ligation (BDL), which induces large cholangiocyte proliferation, and subsequently treated with either saline or a-methylDL-histidine (an HDC inhibitor) for 7 days. Liver blocks, serum, and large cholangiocytes were obtained, and intrahepatic bile duct mass, cholangiocyte proliferation (proliferating cellular nuclear antigen expression), and expression of both HDC and VEGF were measured. miRNA profiling was performed in isolated cholangiocytes. In vitro, miR-125b was overexpressed (or inhibited) or HDC was silenced before measuring HDC and VEGF-A/C expression and cholangiocyte proliferation. After BDL plus a-methyl-DLhistidine, expression of intrahepatic bile duct mass, proliferating cellular nuclear antigen, VEGF-A/C, and HDC and levels of histamine all decreased compared with those of BDL alone. miR-125b was significantly down-regulated after BDL. In vitro, overexpression of miR-125b and knockdown of HDC both decreased HDC and VEGF expression and cholangiocyte proliferation. Manipulation of miR-125beregulated HDC/ VEGF expression may, thus, be a therapeutic approach for the treatment of aberrant cholangiocyte growth in biliary disorders. (Am J Pathol 2014, 184: 662e673; http://dx.doi.org/10.1016/j.ajpath.2013.11.008)
Primary biliary cirrhosis and primary sclerosing cholangitis are liver diseases targeting cholangiocytes.1 A hallmark feature of cholangiopathies is dysregulation of the balance between biliary proliferation and loss.1 In animal models such as bile duct ligation (BDL),2,3 cholangiocytes proliferate in response to cholestasis, increasing intrahepatic bile duct mass.3,4 After BDL for 1 week, only large cholangiocytes proliferate, leading to enhanced intrahepatic bile duct mass, and increased secretion of several growth factors, such as vascular endothelial growth factor (VEGF) and histamine.3,5 Histamine is formed by the conversion of histidine, by the enzyme, L-histidine decarboxylase (HDC).3,6 After conversion, histamine is readily used or stored in cells, such as mast cells.7,8 Patients with cholangiopathies have increased serum levels of histamine, suggesting that histamine plays a critical role in liver pathological conditions.9 Histamine acts through Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2013.11.008
four G-proteinecoupled receptors (H1 to H4) that induce both stimulatory (H1 and H2 histamine receptors) and inhibitory (H3 and H4 histamine receptors) effects on cellular function.10,11 We have shown that the H3 histamine receptor inhibits BDL-induced large cholangiocyte proliferation by down-regulating cAMP signaling.3 Stimulation with an H1 Portions of this work were supported by a Veteran’s Affairs Career Development Award and a Scott and White Research Advancement Award (H.L.F.); the Scott and White Health Care Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology (G.A.); University of Rome Sapienza and a Minestro dell’Instruzione dell’universita e della Ricerca grant 2009X84L84 and Futuro in Ricera grant RBAP10Z7FS (E.G.); the Ministry of Health, Labor, and Welfare of Japan grant-in-aid (Y.U.); and Scott and White Health Care internal support money and a Veteran’s Affairs Merit Award (F.M.). F.M. and P.O. contributed equally to this work. Disclosures: None declared.
miR-125b Regulation of Biliary Growth histamine receptor agonist increases small cholangiocyte proliferation.12 We have also shown that inhibition of HDC decreases cholangiocarcinoma growth in vitro and in vivo.10 Cholangiocytes express VEGF-A and VEGF-C and secrete VEGF through an autocrine mechanism, thus providing the support needed to the proliferating biliary system after BDL.5,13 VEGF-A induces many cellular effects, including increasing cellular migration, mitogenesis, and angiogenesis and promoting cellular proliferation while inhibiting apoptosis.13,14 VEGF-C is an important mediator of cell growth and survival, as well as being a regulator of angiogenesis.15,16 HDC regulates both VEGF-A and VEGFC expression.10,15 In HDC/ mice, angiogenesis is defective and histamine derived from nonmast cells regulates the angiogenesis of the inflammatory tissue.16 miRNAs are post-transcriptional regulators that alter cellular translation. After 70% partial hepatectomy, biliary miR-181b expression is significantly up-regulated compared with shameoperated on animals.17 Also, miR-125b is
significantly down-regulated in hepatocellular cancer18 and cholangiocarcinoma.19 Another study found that miR-125b inhibits hepatocellular cell migration and invasion,20 suggesting that this miRNA is involved in angiogenesis. On the basis of this background, we proposed to demonstrate that miR-125b regulates large biliary hyperplasia by modulating the HDC/VEGF axis.
Materials and Methods All reagents were obtained from Sigma-Aldrich, Co (St. Louis, MO), unless otherwise indicated. All primers and transfection plasmids were obtained from SA Biosciences (Frederick, MD), unless stated otherwise. Antibodies for immunohistochemistry (IHC) and Western blot analysis were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), unless indicated otherwise. To determine histamine levels, we used an enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI).
Figure 1 Evaluation of HDC expression. A: HDC expression in bile ducts compared with normal mice and after treatment with the HDC inhibitor, a-methylDHis. Original magnification, 20. Inset: Increased resolution of image. B: HDC gene expression in large cholangiocytes compared with BDL mice and after amethyl-DHis treatment. Data are the means SEM of eight experiments from cumulative preparations of large cholangiocytes from at least eight animals from each treatment group. *P < 0.05 versus normal; yP < 0.05 versus BDL. C: After BDL, histamine secretion in serum compared with normal and after treatment with the HDC inhibitor compared with both normal and BDL. D: In isolated large cholangiocyte supernatant, histamine secretion after BDL compared with normal, and after BDL plus a-methyl-DHis treatment compared with normal and BDL. Data are the means SEM of 12 experiments from cholangiocyte supernatants collected from cumulative preparations of large cholangiocytes from at least six animals from each treatment group. *P < 0.05 versus normal; y P < 0.05 versus BDL.
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Animal Models and Isolated Cholangiocytes Mice were subjected to sham surgery (normal) or BDL and treated, immediately after surgery, by daily i.p. injections with vehicle (0.9% NaCl) or a-methyl-DL-histidine (amethyl-DHis; 150 mg/body weight; M8628; SigmaAldrich)10,21; after 3 and 7 days, liver blocks (paraffin and frozen, 4 to 5 mm thick), serum, organs, and isolated large cholangiocytes were collected. Large cholangiocytes were provided by Dr. Gianfranco Alpini (Scott and White, Central Texas Veterans Health Care System and Texas A&M Health Science Center, Temple).4,22
LMCC supernatants were measured by commercially available enzyme immunolink assays (Cayman Chemical).10 Intrahepatic bile duct mass and cholangiocyte proliferation were determined in all animal groups by semiquantitative IHC staining for cytokeratin-19 and proliferating cellular nuclear antigen (PCNA), respectively.11,25 In isolated large cholangiocytes from all animal groups and LMCC, protein lysates were collected and Western blot analysis was performed for PCNA to determine cholangiocyte proliferation. Western by scanning video densitometry using the ImageQuant TL Software version 2003.02.
In Vitro Cell Lines In vitro studies were performed in SV-40etransformed large mouse cholangiocyte lines (LMCC)23 and human intrahepatic epithelial cells (HiBECs). HiBECs were purchased from Sciencell Research Laboratories (Carlsbad, CA).10 Cells were cultured according to previous studies.10,12,23
Evaluation of HDC Expression and Histamine Secretion To determine the expression of HDC in the liver, we performed IHC in liver sections (4 to 5 mm thick) and real-time and standard RT-PCR in isolated large cholangiocytes, hepatocytes, and LMCC. IHC was performed in paraffinembedded liver sections. The antibody for HDC (sc-34458; Santa Cruz Biotechnology) was used at 1:50. Negative controls were performed in parallel using nonimmune serum as the primary antibody. Light microscopy (Leica Microsystems, Wetzlar, Germany) was performed to obtain and quantify images. Positive cells were counted in six nonoverlapping fields (20 magnification) for each slide, and the data expressed as percentage of positive cells. Light microscopy and IHC observation were performed in a blinded manner by two independent morphologists. Real-time PCR was performed in total RNA extracted from isolated large cholangiocytes from all animal groups to determine gene expression of HDC. For all PCRs, mouse primers (SA Biosciences) were used and a DDCT analysis was performed. Total cellular RNA (1 mg) was used for PCRs, and cytokeratin-19 (a cholangiocyte-specific marker)14 or glyceraldehyde-3-phosphate dehydrogenase14 was used as the control. Standard RT-PCR was performed in LMCC to determine mRNA levels of HDC, as described.24 Western blot analysis for HDC expression was performed on protein from whole cell lysates from purified large cholangiocytes, hepatocytes, and LMCC. Cells were trypsinized and lysed for immunoblots and SDS-PAGE, as described.10 Western blot analysis was performed with 10 to 20 mg of protein, and band intensity was determined ImageQuant TL Software version 2003.02 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Histamine levels in serum, isolated large cholangiocyte supernatants (after incubation for 6 hours at 37 C), and
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Figure 2 Evaluation of intrahepatic bile duct mass after a-methyl-DHis treatment. Ductal mass after BDL compared with normal shown by IHC (A). Quantification of IHC (B). Original magnifications: 20; 40 (inset). *P < 0.05 versus normal; yP < 0.05 versus BDL.
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Determination of VEGF Expression VEGF cholangiocyte expression was evaluated by IHC in paraffin-embedded liver sections (4 mm thick), and realtime and standard RT-PCR in purified large cholangiocytes and LMCC, respectively. Sections were incubated overnight at 4 C with the following antibodies from Santa Cruz Biotechnology: VEGF-A (sc-152; 1:1000) and VEGF-C (sc-9047; 1:200). The immunoreactivity of VEGF-A/C in cholangiocytes was automatically quantified by ImageScope (Aperio Scanscope CS System, Vista, CA). Bile ducts were randomly selected by the operator, and an image analysis algorithm that automatically analyzes the selected areas was used to quantify the number of positive pixels in cholangiocytes. The algorithm was applied to the entire section of at least three different sections from each liver fragment. The percentage of positive pixels and the average intensity of positivity were obtained from the software, and quantification of IHC was obtained by multiplying the percentage of positive pixels by the average intensity of positivity. Immunofluorescence and Western blot analysis were performed in LMCC to detect VEGF-A/C protein levels, as
described.11,15 Cells were seeded on coverslips in a six-well plate (500,000 cells per well) and allowed to adhere overnight. Immunofluorescence was performed, as described, using a specific antibody for VEGF-A and VEGF-C (diluted 1:50 in 1% bovine serum albumin/PBS with Tween or nonimmune serum for negative controls).10,12 Images were obtained using an inverted Olympus IX-71 confocal microscope (Olympus, Tokyo, Japan). Quantification of immunofluorescent staining was performed manually and expressed as the percentage of VEGF-A/Cepositive cholangiocytes per area (six areas were counted by a blinded operator). Primary antibodies for Western blot analysis were diluted at 1:50.
Evaluation of miR-125b To determine whether HDC is a target of the predicted miR125b, we extracted the mRNA sequence of HDC and scanned potential binding sites for miRNAs using both Targetscan (Whitehead Institute for Biomedical Research, Cambridge, MA) and PicTar 4-way software (Center for Comparative Functional Genomics, New York, New York) and performed miRNA profiling by microarray analysis.
Evaluation of PCNA expression after a-methyl-DHis treatment. A: The number of PCNA-positive cholangiocytes after BDL and after a-methyl-DHis treatment shown by IHC. Original magnifications: 20; 40 (inset). Quantification of IHC (B). PCNA protein expression after BDL and after treatment with amethyl-DHis compared with BDL mice (C). Data are the means SEM of at least four experiments from cumulative preparations of large cholangiocytes from at least eight animals from each treatment group. *P < 0.05 versus normal; yP < 0.05 versus BDL.
Figure 3
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Meng et al After identification of the target miR-125b, we measured miR-125b expression in isolated cholangiocytes from normal and BDL mice by microarray analysis performed by the Center for Targeted Therapy, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center (Houston) and verified by real-time PCR analysis, as previously described, using a specific TaqMan miRNA assay (Life Technologies, Carlsbad, CA).
Luciferase Reporter Assay An intact putative miR-125b recognition sequence from the 30 euntranslated region (UTR) of HDC/VEGF (pMIRe HDCewte30 -UTR or pGL3eVEGFe30 -UTR) or with random mutations (pMIReHDCemute30 -UTR) was cloned downstream of the firefly luciferase reporter gene. For the miR-125beHDC target relationship, luciferase assays were performed at 72 hours after transfection with control and miR-125b precursor or inhibitor (50 nmol/L) in HiBEC cells using the Dual Luciferase Reporter Assay system (Promega, Madison, WI). To determine whether miR-125b directly targets VEGF, HiBEC cells were plated (2 106 cells per well) in 6-well plates. The pGL3eVEGFe30 -UTR firefly luciferase expression construct (1 mg) and a Renilla luciferase expression construct, pRL-TK (1 mg; Promega), were cotransfected with the miR-125b or control precursor or
inhibitor. Luciferase assays were performed 48 hours after transfection using the dual Luciferase Reporter Assay System (Promega).
Genetic Modification of HDC and miR-125b in LMCC To demonstrate that the effects of HDC and miR-125b are specific to cholangiocyte proliferation, we performed in vitro independent experiments to genetically knock down HDC or overexpress miR-125b in LMCC. By using an HDC shRNA plasmid, we generated a stably transfected HDC LMCC knockdown cell line (LMCC-HDC), along with a negative (LMCC-neg) cell line, as described.10 Overexpression of miR125b (preemiR-125b) was performed in standard media containing 10% fetal bovine serum and using preemiR-125b (50 nmol/L) from Ambion Inc. (Austin, TX), along with appropriate controls (pre-miRNA), and miR-125b expression was measured by TaqMan real-time PCR assay (Applied Biosystem Inc., Grand Island, NY). In LMCC-HDC or miR-125b overexpressed cells (and respective controls), HDC, VEGF-A, and VEGF-C gene and protein levels were determined. In LMCC-HDC and respective control cells, we also evaluated the expression of the mRNA for H1 to H4 histamine receptors by real-time PCR. Histamine secretion was measured in conditioned media from genetically modified cells using EIA, and cholangiocyte
Figure 4 In vivo expression of VEGF-A and VEGF-C in liver sections. VEGF-A and VEGF-C expression after BDL compared with normal, and after treatment with the HDC inhibitor, a-methyl-DHis, compared with BDL. Original magnifications: 20; 40 (inset). Data are the means SEM from at least four animals per group. *P < 0.05 versus normal; yP < 0.05 versus BDL.
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Figure 5
In vivo evaluation of miR-125b expression and verification by PCR. miR-125b levels after BDL compared with normal by microarray and confirmed by real-time PCR (A) and in isolated large cholangiocytes with total RNA extracted from control and BDL mice (B). Data are the means SEM of six experiments. *P < 0.05 versus normal. C: Targeting of HDC 30 -UTR by miR-125b. The sequences of the mutated target site of HDC with mutations to disrupt base pairing between the specific binding sites and miR-125b are displayed. D: Luciferase reporter constructs containing the miR-125b recognition sequence from the 30 -UTR of HDC inserted downstream of the luciferase gene were generated in HiBEC cholangiocytes. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample transfected with control and miR-125b precursor (50 nmol/L) for 72 hours. Data are the means SEM of six experiments. *P < 0.05 versus control.
proliferation was determined by MTT assays12 and Western blot analyses12 for PCNA. Apical and basolateral membranes were collected from LMCCs, according to our previous studies.26,27 Specifically, cells were placed in transwell chambers and brought to confluency, and transepithelial resistance was measured before experimental analysis of histamine levels by EIA.
otherwise. Differences between groups were analyzed by the Student’s unpaired t-test when two groups were analyzed, and by analysis of variance when more than two groups were analyzed, followed by an appropriate post hoc test. The null hypothesis was rejected at P < 0.05.
Statistical Analysis
HDC Expression and Histamine Release in Vivo
Data are expressed as means SEM from at least three separate experiments performed in triplicate, unless noted
Prolonged administration (3 and 7 days) of a-methyl-DHis to normal mice induced no changes in intrahepatic bile duct
Results
Figure 6
Standard RT-PCR evaluation of HDC, VEGF-A, and VEGF-C expression in LMCCs. LMCCs express HDC, VEGF-A, and VEGF-C. Total RNA from liver was used as a positive control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 140 bp; HDC, 162 bp; VEGF-A, 62 bp; VEGF-C, 118 bp.
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Meng et al mass, liver weight, body weight, and liver/body weight ratio (data not shown). Therefore, we performed all studies in normal and BDL mice treated for 7 days with vehicle or a-methyl-DHis. HDC was expressed mainly in large cholangiocytes (Figure 1A) and increased after BDL. Realtime PCR (Figure 1B) for HDC expression demonstrates that HDC gene expression is increased in large cholangiocytes after BDL and reduced in BDL þ a-methylDHisetreated mice. By immunoblotting, a slight expression of HDC was found in mouse hepatocytes, whereas a strong expression was found in isolated mouse cholangiocytes, thus strengthening the autocrine role of HDC in biliary proliferation (Supplemental Figure S1). Histamine levels increase in serum and cholangiocyte supernatant obtained from BDL mice when compared with normal mice, whereas treatment with a-methyl-DHis significantly decreased histamine release in serum and cholangiocyte supernatant (Figure 1, C and D).
Evaluation of Intrahepatic Bile Duct Mass and Cholangiocyte Proliferation After BDL, intrahepatic bile duct mass increases compared with normal mice; treatment with a-methyl-DHis significantly decreases intrahepatic bile duct mass when compared with BDL alone (Figure 2). The number of PCNA-positive cholangiocytes increased after BDL and was significantly reduced when animals were treated with a-methyl-DHis (Figure 3A). There was a significant decrease in PCNA protein expression in large cholangiocytes from a-methylDHisetreated BDL mice compared with BDL mice (Figure 3B). We evaluated lobular damage, necrosis, and inflammation in the liver and other major organs (heart, lung, intestine, spleen, and stomach) in all groups and found
that a-methyl-DHis has no pathological effects in our animal model, and no significant morphological changes were noted (data not shown). These data demonstrate that HDC is an important regulator of cholangiocyte proliferation.
VEGF Expression in Vivo The expression of VEGF-A and VEGF-C significantly increased in cholangiocytes from mouse liver after BDL when compared with normal (Figure 4). Furthermore, after HDC inhibition, cholangiocyte VEGF-A/C expression decreased, suggesting that HDC regulates the expression of VEGF during biliary proliferation.
In Vivo Evaluation of miR-125b and Its Target Proteins in BDL Mice The potential miRNAs predicted to target HDC were selected from the list of miRNAs in which expression levels were reduced in BDL mouse liver (<0.05-fold, P < 0.01). miR-125b is significantly decreased in BDL liver versus normal mice (Figure 5A) (ArrayExpress database; http:// www.ebi.ac.uk/arrayexpress, accession number E-MTAB2267). Real-time PCR analysis revealed that, in cholangiocytes isolated from BDL and normal mouse liver, miR-125b gene expression was significantly downregulated after BDL compared with normal (Figure 5B). HDC is a predicted target of miR-125b, as shown by bioinformatic analysis (Figure 5C). To verify that HDC is a direct target of miR-125b, we used luciferase reporter constructs containing the miR-125b recognition sequence from the 30 -UTR of HDC inserted downstream of the luciferase gene (Figure 5C). Transfection with precursors or inhibitors of miR-125b significantly modulated reporter activity of
Figure 7
Effects of overexpression of miR-125b in LMCCs. A: miR-125b expression compared with control pre-miRNA (top panel). HDC expression after overexpression of miR-125b (bottom panel). B: By realtime PCR, VEGF-A, VEGF-C, and H1 to H4 histamine receptor gene expression levels were measured after miR-125b overexpression compared with control premiRNA. Data are the means SEM of six experiments. *P < 0.05 versus control transfected cells.
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miR-125b Regulation of Biliary Growth HDC in human cholangiocytes (Figure 5D). Studies were repeated with random mutations in the shared recognition sequence (Figure 5D), which resulted in abolition/ enhancement of the HDC reporter activation by miR-125b overexpression or inhibition (Figure 5D). To determine that miR-125b also targets VEGF, we performed a comparison of bp between mature human miR-125b, human VEGF-A, mouse mmu-miR-125b, and mouse VEGF-A, and found a sequence conservation between the species (Supplemental Figure S2). Furthermore, by using luciferase assays, we found that both overexpression (miR-125b precursor) and inhibition (antiemiR-125b) of miR-125b induced a significant change of VEGF-A luciferase activity (Supplemental Figure S2), which demonstrated that VEGF-A is also a direct target of miR-125b in biliary epithelial cells.
(Figure 5B), we overexpressed miR-125b in LMCCs. Overexpression of miR-125b decreased HDC, H1 to H4 histamine receptors, and VEGF-A/C gene expression when compared with pre-miRNA control-transfected LMCCs (Figure 7). HDC and VEGF-A/C protein expression and histamine release were significantly reduced after miR-125b overexpression compared with miRNA control precursor (Figure 8, AeC). Cholangiocyte proliferation was reduced after miR-125b overexpression (Figure 8, D and E) compared with control precursor-transfected cells. To further confirm that miR-125b is an important regulator of HDC (and downstream targets), we silenced miR-125b (Supplemental Figure S3) and performed real-time PCR for HDC. We found that, after knockdown of miR-125b, there is an increase in HDC expression compared with control-transfected cells (Supplemental Figure S3). These data suggest that miR-125b is an important target and regulator of the HDC/VEGF axis.
In Vitro Evaluation of miR-125b Effects on HDC and VEGF Expression and Cholangiocyte Proliferation
Validation of HDC Effects in Vitro
The LMCCs23 express HDC and VEGF-A/C (Figure 6). Because miR-125b expression is greatly reduced after BDL
To validate that the effects of miR-125b can be mimicked by silencing HDC, we generated a stably transfected HDC
Figure 8
Effects of miR-125b in vitro. Effects of miR125b overexpression in LMCCs on HDC, VEGF-A, and VEGF-C protein expression, histamine secretion, and cholangiocyte proliferation. HDC (A) and histamine secretion in conditioned medium from miR-125b overexpressed cells compared with control pre-miRNA transfected cells (B) C: Protein expression of VEGF-A/C after miR-125b overexpression compared with control pre-miRNA transfected cells. D and E: PCNA protein expression (D) and MTT assay (E) after miR-125b overexpression compared with control pre-miRNAe transfected cells. Data are the means SEM of 12 experiments for secretion, the means SEM of 8 experiments for Western blot analysis, and the means SEM of 16 experiments for MTT assay (24-hour time point). *P < 0.05 versus control pre-miRNAetransfected cholangiocytes.
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Meng et al knockdown LMCC cell line (LMCC-HDC) lacking HDC and both shRNA control-transfected LMCC (LMCC-neg) and nontransfected LMCC (LMCC basal) cells as controls and measured the following: (i) HDC and VEGF-A/C expression, (ii) histamine secretion, and (iii) cholangiocyte proliferation. Loss of HDC (Figure 9, A and B) induced a decrease in these components. VEGF-A/C gene and protein expression was significantly decreased in LMCC-HDC (cells deficient in HDC expression) compared with controltransfected LMCC (LMCC-neg) (Figure 10) and nontransfected LMCC (data not shown) cells. LMCC secretes histamine at both the apical and basolateral membrane (Figure 9C), and histamine release (Figure 9D) and cholangiocyte proliferation (Figure 9, E and F) were diminished after knockdown of HDC when compared with controls. To determine whether histamine rescues the effects of genetic manipulation of HDC inhibition and further demonstrate an autocrine regulation by cholangiocytes, histamine was added back into the LMCC-HDC cells, secretion was measured by EIA, and the expression of H1 to H4 histamine receptors was
evaluated by real-time PCR. Addition of histamine to LMCC-HDC increased histamine secretion (Figure 9D). Finally, we found that after knockdown of HDC (LMCCHDC), the expression of all histamine receptors (H1 to H4) decreased compared with LMCC-neg (Figure 11), and when histamine was added to LMCC-HDC, H1 to H4 gene expression was significantly increased (Figure 11). These data demonstrate a role for HDC in regulating cholangiocyte proliferation and VEGF expression, potentially via one of the four histamine receptors.
Discussion In the present study, we demonstrated the autocrine role of HDC on the modulation of cholangiocyte hyperplasia during extrahepatic bile duct obstruction, and that these effects are specific to cholangiocytes and regulated by miR-125b. We demonstrated that HDC expression is primarily expressed by cholangiocytes and increases after BDL that is coupled with a significant reduction in miR-125b expression. Traditionally,
Figure 9
Overall effects of HDC knockdown on biliary HDC expression, histamine secretion, and proliferation. By real-time PCR (A) and Western blot analysis (B), HDC gene and protein expression after stable knockdown transfection of HDC shRNA (LMCC-HDC) compared with control-transfected, LMCC-neg. Data are the means SEM of three experiments for real-time PCR and eight experiments for Western blot analysis. Histamine at the apical and basolateral membrane of LMCC (nontransfected, basal treatment) (C); histamine release in LMCC lacking HDC (LMCC-HDC); effects of addition of histamine (10 mmol/L) in LMCC-HDC histamine secretion (gray bar) compared with LMCC-neg and LMCC-HDC (D). By MTT assay (E) and PCNA protein expression (F), cholangiocyte proliferation after loss of HDC (LMCC-HDC) compared with cells with normal amounts of HDC (LMCC-neg). Data are the means SEM of 12 experiments. *P < 0.05 versus LMCC-neg, control-transfected cholangiocytes; yP < 0.05 versus LMCC-HDC.
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Figure 11
Evaluation of loss of histamine receptors in LMCCs. After stable transfection and knockdown of HDC, histamine receptor (H1 to H4) expression compared with LMCC-neg cells. Effects of addition of histamine on expression of H1 to H4 compared with LMCC-HDCetransfected cells. Data are the means SEM of six experiments for real-time PCR. *P < 0.05 versus LMCC-neg (control)etransfected cholangiocytes; yP < 0.05 versus LMCC-HDCetransfected cholangiocytes.
this concept, demonstrating that histamine is a trophic, growth-promoting factor.11 Our study shows that cholangiocytes contain the machinery to produce histamine and secrete increased levels of histamine after BDL, likely to sustain the increased ductal mass. Also, preliminary data from BDL rats show that histamine levels are significantly increased in bile [0.756 0.1307 (normal) versus 47.85 1.116 (BDL) nmol/L], which supports our autocrine hypothesis for histamine regulation of cholangiocyte proliferation. In genetically modified, HDC knockout mice, wound-healing time is slower when compared with mice
Figure 10
Evaluation of loss of HDC in LMCCs. After stable transfection and knockdown of HDC, VEGF-A, and VEGF-C gene (left panel) and protein (right panels) expression compared with LMCC-neg cells. Original magnification, 20. Inset: Increased resolution of image (arrows). Data are the means SEM of three experiments for real-time PCR. *P < 0.05 versus LMCC-neg (control)etransfected cholangiocytes.
HDC is expressed primarily by mast cells.28,29 However, in support of our findings, previous studies have shown that HDC expression and histamine synthesis are also found in gastric enterochromaffin-like cells.30 Histamine synthesis plays a role in neuroregulation, and HDC expression regulates day-night rhythms by the changes in the fluctuation of HDC expression in the hypothalamus.31 Numerous factors regulate cholangiocyte proliferation via an autocrine loop, including histamine, melatonin, and VEGF.5,32e34 We have shown that HDC is critical in regulating cholangiocyte hyperplasia, as shown by the loss of intrahepatic bile duct mass and cholangiocyte proliferation after inhibition of HDC. Previous work also supports
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Figure 12
Proposed overall working model of the effects of miR125beregulated HDC expression in BDL mice treated with the HDC inhibitor, a-methyl-DL-histidine.
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Meng et al with normal histamine levels.35,36 In our current and previously published studies, we have demonstrated that the HDC/histamine axis is a trophic pathway increasing cholangiocyte proliferation. In data presented herein and in preliminary data using HDC/ mice (data not shown), we found that inhibition of HDC (by pharmacological treatment or genetic manipulation) decreases cholangiocyte proliferation via decreased VEGF expression. In contrast, a recent study has shown that, in HDC/ mice, there is an increased rate of colon cancer and skin carcinogenesis.37 This study focused on the paracrine contribution of immature myeloid cell production of HDC and proposes that the balance of histamine synthesis is dysregulated in the tumor microenvironment of skin and colon cancer. Our study focuses on the autocrine release of HDC from the biliary epithelium, which is an unusual tissue. For example, gastrin (which has trophic effects in gastric, colonic, and pancreatic cancer cell lines) inhibits both biliary hyperplasia in BDL rats and cholangiocarcinoma growth of several human cell lines.38,39 We speculate that targeting HDC locally (by nanoparticles containing HDC inhibitors) may be an effective therapeutic approach for modulating HDC directly in the liver. After BDL, the intrahepatic biliary epithelium experiences rapid cholangiocyte proliferation (that occurs before the growth of the peribiliary vascular plexus) that is characterized by increased proliferative activities, including the increased release of histamine and VEGF.5,40 In our study, systemic treatment of a-methyl-DHis decreased cholangiocyte VEGF-A/C expression after biliary injury, presumably to compensate for the decrease in biliary demand. To support our findings, VEGF and angiogenesis are decreased in HDC/ mice granulation tissue when compared with normal controls.16 When HDC/ mice are treated with an H2 histamine receptor agonist, wound healing is recovered and angiogenesis is restored.16 Furthermore, our data demonstrate that, after loss of HDC (or in miR-125beoverexpressed cells), H1 to H4 histamine receptor expression decreases in a similar manner. Addition of histamine to cells with diminished HDC expression resulted in increased histamine secretion and increased receptor expression. We have shown, in our previous work, that there is a differential cholangiocyte response that results from specific receptor stimulation or inhibition, whereas in this study, it appears that knockdown of HDC affects all receptors, leading to a decrease in cholangiocyte proliferation. The exact role of the individual receptors will need to be further investigated. Consistent with our previous findings in human cholangiocytes,10 our present study demonstrates that HDC regulates cholangiocyte VEGF-A/C expression; to expand this, we evaluated the effects of the specific miRNA miR-125b, on HDC. miR-125b expression is decreased in hepatocellular carcinoma, as well as cholangiocarcinoma tissues and cells, whereas overexpression of miR-125b decreases tumor growth.19 After BDL, miR-125b was significantly decreased, which may lead to up-regulation of HDC. After
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overexpression of miR-125b, the HDC/VEGF axis and cholangiocyte proliferation are all down-regulated. Further studies will focus on evaluating other targets (in addition to HDC) for miR-125b that may affect liver functions. In summary, we have provided evidence that HDC plays a critical role in cholangiocyte proliferation. The HDC/ VEGF axis that mediates cholangiocyte proliferation loses function when histamine is no longer being synthesized after the loss of HDC. Furthermore, we found that miR125b regulates the HDC/VEGF axis and may be a potential target for future studies. The current study, coupled with our previous work regarding the role of histamine and VEGF on cholangiocyte proliferation, provides substantial evidence that histamine and HDC (regulated by miR-125b) are key mediators of cholangiocyte response during cholestatic liver injury (Figure 12 depicts an overall working model).
Acknowledgment We thank Dr. Gianfranco Alpini for supplying freshly isolated large mouse cholangiocytes and generously contributing his time in the editing of this work.
Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2013.11.008.
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