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GROWTH, DIFFERENTIATION, AND APOPTOSIS
1Central Texas Veterans Health Care System, 2Department of Medicine and 3Systems Biology and Translational Medicine, 4Division of Research and Education, 5Division of Molecular Cardiology, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, Texas; 6Department of Human Anatomy, University of Rome, "La Sapienza," Rome; 7Division of Gastroenterology, Tohoku University Hospital, Aobaku, Sendai, Japan; 8Department of Experimental Medicine, State University of l'Aquila, l'Aquila; and 9Department of Gastroenterology, Università Politecnica delle Marche, Ancona, Italy
Submitted 16 August 2007 ; accepted in final form 21 May 2008
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ABSTRACT |
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heterogeneity; calcium; intrahepatic biliary epithelium; mitosis; transcription factors
In normal states, cholangiocytes are mitotically dormant (8) but proliferate in response to a number of pathological stimuli, including extrahepatic bile duct obstruction (BDL) (4, 8, 26, 52), 
-naphthylisothiocyanate chronic feeding (42), sustained forskolin administration (21) or acute carbon tetrachloride (CCl4) administration (40). The proliferative responses of cholangiocytes to these pathological maneuvers are heterogeneous and size-dependent (35). These findings have significant clinical relevance since human cholangiopathies selectively target different-sized bile ducts (38, 46). In rats, large but not small cholangiocytes selectively proliferate in response to BDL by activation of the cAMP system (3, 8). Although the activation of cAMP-dependent signaling is essential for the proliferation of large rat cholangiocytes (3, 21), it has been suggested (46) that the function of small cholangiocytes (that de novo proliferate following CCl4-induced damage of large cholangiocytes and after chronic bile acid feeding) (6, 40, 42) may depend on the activation of the myo-D-inositol 1,4,5,trisphosphate (IP3)/Ca2+-dependent signaling pathway. However, this is conjectural since direct evidence for the role of this signaling pathway in the regulation of small cholangiocyte proliferation is lacking.
Histamine receptors, which are classified as HRH1, HRH2, HRH3, and HRH4, are expressed by a number of cells (50, 53). HRH1 exert their functions by coupling to Gq mobilizing intracellular Ca2+ concentration ([Ca2+]i) (15), whereas HRH2 is modulated by Gs proteins coupled to adenylyl cyclase (49). Conversely, the HRH3/HRH4 couples to Gi/o resulting in a negative regulation of cAMP (43). Because HRH1 exert their functions by coupling to Gq mobilizing [Ca2+]i (15), we hypothesized that activation of HRH1 modulates the proliferation of small cholangiocytes by a transduction pathway involving the activation of IP3 and Ca2+.
Ca2+/calmodulin-dependent protein kinases or CaMK are serine/threonine-specific protein kinases that are primarily regulated by the Ca2+/calmodulin complex (12, 29). CaMK I, II, and IV require Ca2+/CaM binding and phosphorylation for activation (28). CaMK I and IV can phosphorylate the activating serine-133 (S133) residue of the cAMP-response element binding protein (CREB) (17, 57) and, in turn, stimulate CRE-mediated transcription. In support of the notion that CaMK regulates cholangiocyte functions, we have shown (45) that endogenous opioids modulate the growth of cholangiocytes in the course of cholestasis by an IP3/CaMK II/protein kinase C (PKC)--dependent pathway. Furthermore, Ca2+-dependent activation of Cl– currents in the cholangiocarcinoma cell line Mz-ChA-1 is regulated by a CaMK II-dependent mechanism (48). No data exists regarding the role of CaMK in the modulation of small cholangiocyte proliferation.
We used immortalized cell lines of small and large murine cholangiocytes (61) that have been morphologically, phenotypically, and functionally characterized (61) and retain characteristics similar to that of freshly isolated small and large mouse cholangiocytes (25). Using this model, we evaluated: 1) the expression of histamine receptor subtypes in small and large cholangiocytes and 2) the role and intracellular mechanisms by which the specific HRH1 agonist histamine trifluoromethyl toluidide (HTMT) dimaleate (56) modulates the growth of immortalized small and large cholangiocytes.
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MATERIALS AND METHODS |
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, β, 
, and 
) raised against a peptide mapping at the COOH terminus of CaMK I of mouse origin. CaMK II (M-176) is a rabbit polyclonal antibody (that detects all CaMK II subunits) raised against amino acids 303-478 mapping at the COOH terminus of CaMK II- (Ca2+/CaMK II, , β, , and ) of mouse origin. CaMK IV (M-20) is an affinity purified goat polyclonal antibody raised against a peptide mapping at the C-terminus of CaMK IV of mouse origin. All human antibodies cross-reacted with mouse. The HRH1 agonist HTMT dimaleate (56) was purchased from Tocris Bioscience (Ellisville, MO). The intracellular Ca2+ chelator BAPTA/AM (23, 27), and W7 (a calmodulin antagonist that binds to calmodulin and inhibits Ca2+/calmodulin-regulated enzyme activities such as CaMK protein kinase) (59) were obtained from Calbiochem (San Diego, CA). The RIA kits for the determination of intracellular cAMP (cAMP 125I Biotrak Assay System, RPA509) and IP3 (3H Biotrak Assay System, TRK1000) levels were purchased from GE Healthcare (Piscataway, NJ).
Morphological, Phenotypic, and Functional Characterization of Immortalized Small and Large Murine Cholangiocytes Small and large murine cholangiocytes isolated from normal mice (BALB/c) and immortalized by the introduction of the SV40 large-T antigen gene were previously established at the laboratory of Y. Ueno (61). The cell lines were maintained in minimum essential medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated FBS, 1% penicillin and streptomycin, and 2 mmol/l L-glutamine at 37°C in a humidified 5% CO2 incubator. We have previously shown that these cells display morphological (difference in size and transepithelial resistance) (61), phenotypic (expression of cytokeratin-19) (61), and functional features (cAMP response in large but not small cholangiocytes) (61) similar to freshly isolated small and large mouse (25) and rat (5, 7) cholangiocytes. We have performed additional experiments aimed to evaluate: 1) the morphological appearance (by light microscopy) and cell size (by fluorescence-activated cell sorting or FACS) of immortalized small and large murine cholangiocyte lines and 2) if immortalized small and large murine cholangiocytes are positive for cytokeratin-7 (CK-7, a cholangiocyte-specific marker) (26) but differentially express secretin receptor, CFTR, and Cl–/HCO3– AE2, proteins that play important roles in the regulation of proliferative and secretory functions of large bile ducts (2, 3, 5, 7, 40, 41).
For light microscopy, trypsinized immortalized small and large cholangiocytes were smeared on slides, fixed in ice-cold acetone, allowed to air dry, and counterstained with 0.4% trypan blue. Images were obtained with an Olympus BX40 microscope equipped with an Olympus DP12 camera (Tokyo, Japan). Flow cytometry size determinations of cultured small and large cholangiocytes were performed on a BD FACSCalibur system using the CellQuest Pro software package (BD Biosciences, San Jose, CA). Before measurements, small and large cholangiocytes were diluted to 106 cells/ml in cell suspension buffer. Forty thousand events were recorded in each experiment. Cell sizes were estimated by comparing forward scatter (an index of cell size) signals with those of unstained polystyrene reference microspheres (5, 10, 15, and 20 µm; Polysciences, Warrington, PA) diluted in cell buffer at a concentration of 106 beads/ml. All of the bead standards had a relative refractive index (1.59 at 589 nm). Before sampling, the microspheres were uniformly suspended by mixing the suspensions.
Immunofluorescence for secretin receptor, CFTR, Cl–/HCO3– AE2, and CK-7 was performed as follows. Immortalized small and large cholangiocytes were seeded on cover slips and allowed to adhere overnight. Cells were washed in 1x PBS containing 0.2% Triton X-100 (PBST), and blocked in 4% BSA (in PBST) for 1 h. The selected antibody was diluted (1:50) in 1% BSA/PBST, added to the cover slips, and incubated overnight at 4°C. Cells were washed three times for 10 min in PBST, and a 1:50 dilution (in 1% BSA/PBST) of cy3-conjugated anti-rabbit secondary antibody (Jackson Immunochemicals, West Grove, PA) was added for 2 h at room temperature. Cells were washed again and mounted on microscope slides with Antifade gold containing 4,6-diamidino-2-phenylindole (DAPI) as a nuclear stain (Molecular Probes, Eugene, OR). Negative controls were performed by usage of a preimmune serum in the place of the primary antibody. Staining was visualized using an Olympus IX-71 inverted confocal microscope.
Isolation of Small and Large Normal Mouse Cholangiocytes Because the size of isolated small and large normal mouse cholangiocytes (25) is similar to that of small and large purified rat cholangiocytes (5), we have used similar parameters (5) for the isolation of these cells. Following standard collagenase perfusion, a mixed nonparenchymal cell fraction was separated into two distinct subpopulations of small and large cholangiocytes by centrifugal elutriation (2,500 revolutions/min at the flow rates of 22 and 70 ml/min, respectively) using a Beckman J2–21M centrifuge equipped with a JE-6B rotor (Beckman Instruments, Fullerton, CA) (5). Small and large cholangiocytes were further purified by a monoclonal antibody, rat IgG2a (provided by Dr. R. Faris, Providence, RI), against an antigen expressed by mouse cholangiocytes (26). Cell number and viability were assessed by trypan blue exclusion. The size of the two subpopulations of cholangiocytes was evaluated by FACS analysis.
Expression of Histamine Receptor HRH1 receptor expression was determined by 1) immunohistochemistry in normal mouse liver sections and 2) immunofluorescence, immunohistochemistry, and immunoblots in immortalized small and large cholangiocytes. Real-time PCR was used to further confirm the presence of HRH1 in freshly isolated small and large cholangiocytes and immortalized small and large cholangiocytes. Furthermore, we evaluated the expression of HRH2, HRH3, and HRH4 in 1) liver sections by immunohistochemistry and 2) small and large immortalized cholangiocytes by real time PCR.
Immunohistochemistry Normal mouse liver sections (4 µm) and cell smears of immortalized small and large cholangiocytes (fixed in Bouin solution) were mounted on glass slides coated with acetone aminopropyltriehoxylan (2%) solution. After deparaffination, endogenous peroxidase activity was blocked by incubation (30 min) in methanolic hydrogen peroxide (2.5%). Sections were hydrated in graded ethanol and rinsed in 1x PBS. The endogen biotin was blocked by the Biotin Blocking System (Dako Cytomation, Glostrup, Denmark). Following washes in 1x PBS, sections were incubated overnight at 4°C with antibodies for HRH1, HRH2, HRH3, or HRH4 (1:100 dilution), whereas cell smears were incubated with HRH1 antibody (1:100 dilution). Samples were rinsed with 1x PBS, incubated for 10 min at room temperature with a secondary biotinylated antibody (Dako Cytomation LSAB Plus System-HRP) and then with Dako ABC (Dako Cytomation LSAB Plus System-HRP), developed with 3–3' diaminobenzidine, and counterstained with hematoxylin. For all immunoreactions, negative controls (preimmune serum substituted for primary antibody) were included. Observations and light microscopy photographs of liver sections were taken by Leica Microsystems DM 4500 B Light Microscopy (Weltzlar, Germany) with a Jenoptik Prog Res C10 Plus Videocam (Jena, Germany).
Immunofluorescence Immunofluorescence for HRH1 in immortalized small and large murine cholangiocyte lines was performed as described above for secretin receptor, CFTR, and Cl–/HCO3– AE2 with the exception that an antibody (1:10 dilution) specific for HRH1 was used. Following incubation with HRH1 antibody, cells were washed three times for 10 min in PBST, and a 1:50 dilution (in 1% BSA/PBST) of cy3-conjugated anti-rabbit secondary antibody (Jackson Immunochemicals) was added for 2 h at room temperature. Cells were washed again and mounted on microscope slides with Antifade gold containing DAPI as a nuclear stain. Negative controls were performed by usage of a preimmune serum in the place of the primary antibody. Staining was visualized using an Olympus IX-71 inverted confocal microscope.
Real-Time PCR
We evaluated the expression of: 1) HRH1 mRNA in total RNA (1.0 µg) from immortalized small and large murine cholangiocytes and freshly isolated small and large normal mouse cholangiocytes and 2) HRH2, HRH3, and HRH4 in immortalized small and large cholangiocytes by using the RT2 Real-Time assay from SuperArray (Frederick, MD), an approach we have previously used to evaluate the expression of HRH3 in purified rat cholangiocytes (20). cDNA template (1 µl) was added to 12.5 µl of master mix, 10.5 µl of deionized water, and 1 µl of RT2 PCR mouse primers (SuperArray) designed specifically for the messages for HRH1, HRH2, HRH3, and HRH4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping gene (3, 20). A 
CT (delta delta of the threshold cycle) analysis was performed using the normal pooled cholangiocytes as the control sample. Data were expressed as relative mRNA levels ± SE of HRH1 to GAPDH ratio (n = 3).
Immunoblotting Immunoblotting analysis for HRH1 was performed as described (23) in protein (10 µg) from brain (positive control) or whole cell lysate that was harvested from immortalized small and large murine cholangiocytes by sonication (6 times in 30-s bursts) in RIPA buffer [50 mmol/l Tris, pH 7.5, 150 mmol/l NaCl, 1% Nonidet P-40 (NP-40), 0.5% deoxycholic acid, 0.1% SDS, 2 mmol/l EDTA, 10 mmol/l NaF, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mmol/l phenylmethylsulfonyl fluoride]. After stripping, the amount of protein loaded was normalized by immunoblots for β-actin (6). The intensity of the bands was determined by scanning video densitometry using the phospho-imager, Storm 860, (GE Healthcare) and the ImageQuant TL software version 2003.02 (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Effect of HTMT Dimaleate on the Proliferation of Immortalized Small and Large Cholangiocytes The proliferation of immortalized small and large cholangiocytes was evaluated by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) (18) that uses a novel tetrazolium compound, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, in combination with an electron coupling reagent, phenazine ethosulfate, to produce a colorimetric change. Absorbance was measured at 490 nm on a microplate spectrophotometer (Versamax; Molecular Devices, Sunnyvale, CA). Data were expressed as the degree of change of treated cells compared with vehicle-treated controls. After trypsinization, immortalized small and large cholangiocytes were seeded in 96-well plates (10,000/well) in a final volume of 200 µl of medium. Subsequently, cells were stimulated for 24 and 48 h with HTMT dimaleate (10 µM) (56) before evaluation of proliferation by CellTiter Assay. In different sets of experiments, immortalized small cholangiocytes were stimulated for 48 h with: 1) HTMT dimaleate (10 µM) in the absence or presence of preincubation (1 h) with BAPTA/AM (an intracellular Ca2+ chelator, 1 µM) (51), W7 (a calmodulin antagonist, 10 µM), (54), or terfenadine (a specific HRH1 inhibitor, 5 µM) (33); or 2) histamine (1 µM) in the absence or presence of terfenadine (5 µM).
To exclude the role of CaMK II in HTMT dimaleate-induced increase in small cholangiocyte growth, we evaluated the effect of HTMT dimaleate in small cholangiocytes in the absence or presence of the highly-specific CaMK II inhibitor autocamtide-2-related inhibitory peptide (AIP) (30, 31) that at the concentration used (1 µM) completely inhibits CaM kinase II but does not affect PKC, PKA, CaMK I, CaMK IV, and other brain extract kinases (9, 31).
Evaluation of the Intracellular Mechanisms of HRH1 Modulation of Small Cholangiocyte Proliferation Measurement of IP3, Ca2+, and cAMP levels. The effects of HTMT dimaleate on the intracellular levels of: 1) IP3 in immortalized small and large cholangiocytes and 2) cAMP in immortalized small cholangiocytes were evaluated by commercially available RIA kits (2, 3, 5, 21, 37, 39) according to the vendors' instructions. After trypsinization, immortalized cholangiocytes were washed with 1x Hanks' balanced salt solution (1x HBSS), incubated for 1 h at 37°C (21, 23, 37) to regenerate membrane proteins damaged by trypsinization (32), and stimulated at room temperature with 0.2% BSA (basal) or HTMT dimaleate (10 µM) for 10 min (39) before measurement of IP3 levels and 5 min (2, 3, 5, 21, 37, 39) before cAMP evaluation (2, 3, 5, 21, 37, 39). [Ca2+]i fluorescence measurements in immortalized small cholangiocytes were performed using fluo 3-AM (Molecular Probes) and a Fluoroskan Ascent FL (ThermoLabsystems, Helsinki, Finland) microplate reader equipped with three injectors as described in detail (19, 22, 36, 58). Small cholangiocytes (4 x 104/well) were loaded for 1 h at room temperature with 5 µM of fluo 3-AM in 1x HBSS with 0.1% Pluronic F-127 (Molecular Probes). After washes with 1x HBSS, the loaded cells were added to a 96-well black microplate. The baseline fluorescence was measured 50 times after 1 s at 2-s intervals; 1x HBSS alone or HTMT dimaleate (10 µM) dissolved in buffer was injected sequentially in separate wells, and the fluorescence intensity was measured at 538 nm for 3 min at 1-s intervals. The excitation wavelength was 485 nm. [Ca2+]i was calculated as follows: [Ca2+]i = Kd(F – Fmin)/(Fmax – F). Fmax refers to fluorescence intensity measured after permeabilization of the cells with 1% NP-40, and Fmin is the minimum fluorescence. Next, 0.1 M EGTA (pH 8.0) was added to chelate Ca2+, and Fmin was obtained. Ionomycin (a Ca2+ ionophore, 10 µM) (58) was used at the end of each Ca2+ determination to ensure cholangiocyte responsiveness.
Role of CaMK Isoforms in HTMT Dimaleate Modulation of Cholangiocyte Proliferation We first evaluated: 1) the protein expression of CaMK I, CaMK II, and CaMK IV in normal mouse liver sections by immunohistochemistry and 2) protein and message expression of CaMK I and CaMK II (the two isoforms expressed by bile ducts in liver sections) in immortalized small and large cholangiocytes by immunoblots and real-time PCR, respectively (23).
We evaluated, by semiquantitative immunohistochemistry of paraffin-embedded serial liver sections (4 µm), the number of cholangiocytes positive for CaMK I, CaMK II, and CaMK IV. Immunohistochemistry was performed as described above for staining of histamine receptors with the exception that the antibodies specific for the CaMK isoforms were used. The data were obtained from the cumulative evaluation of 10 different fields from three different sections.
Immortalized small and large cholangiocytes were lysed as described above, and 10 µg of protein were used to determine the expression of CaMK I and CaMK II by immunoblotting using antibodies against the two isoforms. Following stripping, β-actin was used for normalization of the amount of proteins used (6). The intensity of the bands was quantified as described above.
Real-time PCR for CaMK I-, -, and - and CaMK II-, -, and - in immortalized small and large murine cholangiocytes was performed as described above for histamine receptors using primers from SuperArray.
We next evaluated the effect of HTMT dimaleate on the phosphorylation of CaMK I in immortalized small cholangiocytes. Briefly, 1 x 106 cholangiocytes were seeded in six-well plates and allowed to grow overnight to 
80% confluence. Subsequently, cells were stimulated with 0.2% BSA (basal) or HTMT dimaleate (10 µM) for 24 h. The relative levels of phosphorylated CaMK I were determined in protein (10 µg) from whole cell lysate by immunoblotting. After being stripped, immunoblots were normalized with the protein for total CaMK. The intensity of the bands was quantified as described above.
Role of CaMK I in HRH1 Modulation of Small Cholangiocyte Proliferation
To further confirm the role of CaMK I in HRH1 modulation of small cholangiocyte proliferation, we used short hairpin RNA (shRNA) to develop a stable transfected cell line. Knockdown of the CaMK I gene was performed using a SureSilencing shRNA (SuperArray) plasmid for mouse CaMK I containing the gene for neomycin (geneticin; Invitrogen) resistance for the selection of transfected cells, according to the instructions provided by the vendor. Briefly, immortalized small cholangiocytes were transfected with either CaMK I shRNA or the empty shRNA vector in a six-well plate with Mirus TransIT-LT1 Transfection Reagent. Transfected cells were selected by the addition of 500 µg/ml of neomycin in the media, and the selection process was allowed to continue for 3–4 days. Surviving cells were then split to a lower density in a 12-well plate so that each clone stems from only 5–10 cells. A total of 24 clones was then assessed for the relative knockdown of CaMK I using real-time PCR (as described above), and a single clone with the greatest degree of knockdown was selected for subsequent experiments. After establishing knockdown efficiency (70%), small cholangiocyte proliferation was evaluated by CellTiter Assay after stimulation with 0.2% BSA or HTMT dimaleate (10 µM) for 48 h.
Pharmacologic and Genetic Manipulation of CREB Activation We evaluated the role of HRH1 and CaMK I on CREB activation by using the TransAM pCREB Transcription Factor Assay Kit from Active Motif. The kit was performed according the directions provided by the vendor. Nuclear extracts were obtained using the Nuclear Extraction Kit from Active Motif. In pharmacological experiments, nuclear extracts were prepared from immortalized small cholangiocytes stimulated for 48 h at 37°C with 0.2% BSA (basal) or HTMT dimaleate (10 µM) in the absence or presence of preincubation with W7 (10 µM) in serum-free conditions. For genetic manipulation of CaMK I-dependent CREB activity, we used the CaMK I knockdown cell line (described above) to obtain nuclear extracts. Knockdown and empty vector cells were plated and stimulated with 0.2% BSA (basal) or HTMT dimaleate (10 µM) for 48 h. The assay was performed as follows. Nuclear extracts were plated on a precoated 96-well plate with 30 µl of complete binding buffer. Nuclear extracts were incubated for 3 h at room temperature on a rocking platform. After careful washing, 100 µl of phospho-CREB antibody (1:500 dilution in 1x antibody binding buffer) were added to all wells. The plate was incubated for 1 h at room temperature. Wells were washed three times, and 100 µl of diluted HRP antibody (1:100 dilution in 1x antibody binding buffer) were added to each well and incubated for 1 h at room temperature. After washing (4 times), 100 µl of developing solution was added to all wells, and the plate was incubated for 10 min at room temperature protected from light. Stop solution (100 µl) was added, and the plate was read within 5 min at 450 nm. Data were determined as a degree of increase over basal values.
Statistical Analysis All data are expressed as means ± SE. The differences between groups were analyzed by Student's t-test when two groups were analyzed or ANOVA if more than two groups were analyzed. A P value <0.05 was used to indicate statistically significant differences.
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40 and 50%, respectively, after HRH1 stimulation for 24 and 48 h (Fig. 3A). The stimulation of small cholangiocyte proliferation by HTMT dimaleate was blocked by the HRH1 antagonist terfenadine (33), the intracellular Ca2+ chelator BAPTA/AM (51), and the calmodulin antagonist W7 (59), but not by the highly-specific CaMK II inhibitor AIP (30, 31) (Fig. 3B). Terfenadine, BAPTA/AM, W7, or AIP alone did not change the proliferation of small cholangiocytes (not shown). Histamine stimulation of small cholangiocyte proliferation was prevented by preincubation with terfenadine (Fig. 3B).
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, , and ) and CAMK II (, , and ) (Fig. 5C). Although CaMK I βI mRNA has been shown to be expressed by rat liver (63), we did not evaluate the expression of this isoform in mouse cholangiocytes, since the gene has not been cloned in mice yet. Although immortalized small and large murine cholangiocytes express CaMK I and CaMK II, we only evaluated the effect of HTMT dimaleate on the phosphorylation of CaMK I in immortalized small cholangiocytes. The rationale for this experiment is based on the fact that: 1) the HRH1 agonist HTMT dimaleate did not increase the proliferation of immortalized large cholangiocytes, thus excluding the need for evaluating the phosphorylation of CaMK I in large cholangiocytes, and 2) the CaMK II isoform does not play a role in the regulation of small cholangiocyte proliferation, since the specific CaMK II inhibitor, AIP (30, 31), did not prevent the HTMT dimaleate-induced increase in small cholangiocyte proliferation (Fig. 3). The data show that HTMT dimaleate increased the phosphorylation of CaMK I in immortalized small cholangiocytes compared with small cholangiocytes treated with 0.2% BSA (basal) (Fig. 5D). Protein expression of total CaMK I was similar in immortalized small cholangiocytes treated with HTMT dimaleate or 0.2% BSA (basal) (Fig. 5D).
Knockdown of the CaMK I Gene Ablates the Stimulatory Effects of HTMT Dimaleate on Small Cholangiocyte Proliferation
Genetic knockdown of CaMK I was performed and confirmed by real-time PCR for CaMK I in all the clones. Clone 22 (representative of all clones that we have evaluated for CaMK I message expression) had the highest degree of knockdown efficiency (70%, Fig. 6A). Relative mRNA expression is shown in Fig. 6A. Following knockdown of the CaMK I mouse gene, HTMT dimaleate (10 µM) failed to increase the proliferation (evaluated by CellTiter Assay) (18) of immortalized small cholangiocytes compared with BSA-treated, transfected cells (Fig. 6B). HTMT dimaleate significantly increased the proliferation of small, empty vector-transfected cells compared with basal-treated empty vector-transfected cells (Fig. 6B). These data provide direct evidence for the role of CaMK I-dependent signaling in HRH1 modulation of small cholangiocyte growth.
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DISCUSSION |
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Small and large rat cholangiocytes differentially proliferate in a number of experimental models of cholestasis (6, 24, 35, 40–42, 46). Following BDL, large (but not small) cholangiocytes proliferate by activation of the cAMP-dependent signaling pathway (24, 35, 46). Small cholangiocytes (which are constitutively mitotically dormant) (24, 35, 46), de novo proliferate in pathological conditions associated with damage of large cholangiocytes (e.g., after CCl4 administration) (40, 41). Feeding of taurocholic and taurolithocholic acid to rats increased proliferation of large cholangiocytes and induced the proliferation of small cholangiocytes (6). A number of theories have proposed regarding the function of small cholangiocytes (16, 35, 47, 60). Three-dimensional analysis of the architecture of Herring canals in normal and diseased human livers demonstrated that small cholangiocytes lining these canals constitute a functional stem cell population (60). We have also proposed that small cholangiocytes may be poorly differentiated primordial cells (35) in contrast to the more differentiated large cholangiocytes, which secrete water and electrolytes and proliferate in response to pathophysiological stimuli (3–5, 35, 41). Along this view, Masyuk et al. (47) have suggested that the differential proliferative capacity of different-sized cholangiocytes in response to various stimuli may be related not only to the nature of the stimulus but also to the heterogeneity of distributed proteins along the length of the biliary tree. However, despite these findings, the function of small cholangiocytes is undefined.
We provide further evidence that our immortalized small and large cholangiocytes (61) display features of freshly isolated cholangiocytes since: 1) the size of small and large immortalized cholangiocytes was similar to that of freshly isolated small and large mouse cholangiocytes, and 2) immortalized large cholangiocytes expressed the protein for secretin receptor, CFTR, and Cl–/HCO3– AE2. Our findings, demonstrating that HRH1 are expressed by small and large bile ducts in normal mouse liver sections, immortalized small and large cholangiocyte lines, and freshly isolated small and large mouse cholangiocytes, further support the concept that immortalized small and large cholangiocytes retain phenotypes similar to freshly isolated cholangiocytes.
We next evaluated the effect of HTMT dimaleate on cholangiocyte proliferation and demonstrated that: 1) HTMT dimaleate increases small but not large cholangiocyte proliferation, and 2) the HTMT dimaleate-induced increase in small cholangiocyte proliferation was blocked by terfenadine, BAPTA/AM, and W7. Our studies, which provide the first evidence that small cholangiocyte proliferation (following activation of the HRH1) is induced by activation of the IP3/CaMK I/CREB-dependent signaling pathway, have important clinical implications. Indeed, in patients with cystic fibrosis, it has been suggested that the IP3/Ca2+-dependent functional activity of small ducts (35) may compensate for the loss of large, cAMP-dependent cholangiocytes (2, 3, 5, 7, 24, 35, 40–42).
To explain why we have used different concentrations of HTMT dimaleate and histamine, we refer to a study that demonstrates that histamine has a much higher binding affinity compared with HTMT dimaleate (64). In this study (64), the authors used COS-1 cells (transiently transfected with AXOR35, a G protein-coupled receptor) to demonstrate competitive binding between histamine and numerous histamine receptor agonists and antagonists. The authors showed, with competitive [3H]histamine binding, that histamine had a much greater affinity [inhibitory constant (Ki) = 17 ± 4 nM] compared with HTMT dimaleate (Ki = 1,229 ± 4 nM) to AXOR35 receptor-transfected membranes. This represents an 100-fold difference between histamine and the HRH1 agonist, thus supporting our reasons and results obtained with our concentrations of 1 and 10 µM of histamine and HTMT dimaleate, respectively, on small cholangiocyte proliferation.
A possible explanation as to why there is a differential proliferative response of small and large cholangiocytes to HTMT dimaleate may be because of the selective increase in intracellular IP3/Ca2+ by this cell subtype, and a subsequent activation of CAMK I. In agreement with this notion, we demonstrated that HTMT dimaleate did not increase intracellular cAMP levels in small cholangiocytes. Upregulation of HRH1 expression in small compared with large cholangiocytes may also explain why HTMT dimaleate stimulates only small cholangiocyte proliferation.
We next performed experiments aimed to pinpoint the specific CaMK isoform(s) involved in HTMT dimaleate stimulation of small cholangiocyte proliferation. We first demonstrated that both small and large cholangiocytes express all the isoforms of CaMK I (, , and ) and CaMK II (, β, , and ) but not CaMK IV. Although CaMK I β- I mRNA is present in rat liver (63), we did not evaluate the expression of this isoform in mouse cholangiocytes, since the gene has not been cloned in mice yet. We demonstrated the key role of CaMK I in HRH1 modulation of small cholangiocyte growth, since HTMT dimaleate stimulation was associated with phosphorylation of CaMK I and blocked by gene silencing for this CaMK isoform. Although both small and large cholangiocytes express CaMK I and CaMK II, we evaluated the role of CaMK I based on the following observations. First, we excluded the role of CaMK II by demonstrating that the highly specific CaMK II inhibitor AIP (30, 31) did not block the HTMT dimaleate-induced increase of small cholangiocyte proliferation. Second, we did not evaluate the role of CaMK I in large murine cholangiocyte proliferation, since these cells do not respond to HTMT dimaleate. We did not evaluate the role of CaMK IV in the regulation of cholangiocyte proliferation, since this isoform was not expressed by these cells. Taken together, these findings demonstrated that HTMT dimaleate stimulation of small cholangiocyte proliferation depends on the phosphorylation of CaMK I.
We next performed experiments to evaluate if HTMT dimaleate stimulation of small cholangiocyte proliferation depends on CaMK I-dependent activation of CREB and demonstrated that HTMT dimaleate activation of small mouse cholangiocytes was associated with phosphorylation of CREB, which was blocked by W7 and CaMK I silencing. The ability of Ca2+ to regulate CREB activity involves phosphorylation of CREB by Ca2+/CaMK (55, 62). CaMK I, II, and IV have been shown to phosphorylate CREB in vitro, providing evidence that each of these protein kinases may be a candidate for mediating the effects of Ca2+ on CREB activity (13, 14, 57).
Our current studies have important clinical implications, since a number of human liver diseases (e.g., primary biliary cirrhosis) are characterized by selective rather than diffuse proliferation or loss restricted to certain-sized ducts (4, 35). Our findings further support the concept that small and large cholangiocytes differentially respond to different stimuli (3, 35, 40–42). Indeed, depending on the need of the liver, either growth-promoting receptor is activated (like HRH1 or HRH2), or growth-inhibiting (such as HRH3 or HRH4) receptors are "switched on." This fine balance exists to regulate the function(s) of cholangiocytes in the modulation of liver diseases. The regulation of the biogenic amine histamine (via 4 independent receptors) may be important in this balance of heterogeneous proliferation and loss in liver disease management.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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