Intercellular communication via gap junctions in activated rat hepatic stellate cells
2005; Elsevier BV; Volume: 128; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2004.11.065
ISSN1528-0012
AutoresRichard Fischer, Roland Reinehr, Thuy Phung Lu, Alexandra Schönicke, Ulrich Warskulat, Hans Peter Dienes, Dieter Häussinger,
Tópico(s)Biochemical effects in animals
ResumoBackground & Aims: Gap junctional communication was studied in quiescent and activated hepatic stellate cells. Methods: Connexin expression and intercellular dye transfer were studied in rat hepatic stellate cells in culture and in vivo. Results: Protein expression of connexin 43 was up-regulated in activated hepatic stellate cells in vivo and in vitro and was mainly localized on the cell surface, whereas connexin 26 was found intracellularly. In contrast to hepatocytes, hepatic stellate cells do not express connexin 32. Confluent hepatic stellate cells in culture communicate via gap junctions, resulting in lucifer yellow transfer and propagation of intracellular calcium signals. Phorbol ester induces a protein kinase C–dependent hyperphosphorylation and degradation of connexin 43 and inhibits intercellular communication on a short-term time scale. At the long-term level, vitamin D3, lipopolysaccharide, thyroid hormone T3, dexamethasone, platelet-derived growth factor, endothelin 1, and interleukin 1β up-regulate connexin 43 protein and messenger RNA expression and enhance intercellular communication. Slight down-regulation of connexin 43 is observed in response to vitamin A. Connexin 43 induction by endothelin 1 is inhibited by both endothelin A and endothelin B receptor antagonists. In coculture systems, hepatic stellate cells communicate with each other, which is suggestive of a syncytial organization, but no communication was found between hepatic stellate cells and other liver cell types. As shown by immunohistochemistry and electron microscopy, gap junctions are formed between activated hepatic stellate cells in vivo. Conclusions: Gap junctional communication occurs between hepatic stellate cells, is enhanced after activation, and underlies complex regulation by cytokines, hormones, and vitamins. Background & Aims: Gap junctional communication was studied in quiescent and activated hepatic stellate cells. Methods: Connexin expression and intercellular dye transfer were studied in rat hepatic stellate cells in culture and in vivo. Results: Protein expression of connexin 43 was up-regulated in activated hepatic stellate cells in vivo and in vitro and was mainly localized on the cell surface, whereas connexin 26 was found intracellularly. In contrast to hepatocytes, hepatic stellate cells do not express connexin 32. Confluent hepatic stellate cells in culture communicate via gap junctions, resulting in lucifer yellow transfer and propagation of intracellular calcium signals. Phorbol ester induces a protein kinase C–dependent hyperphosphorylation and degradation of connexin 43 and inhibits intercellular communication on a short-term time scale. At the long-term level, vitamin D3, lipopolysaccharide, thyroid hormone T3, dexamethasone, platelet-derived growth factor, endothelin 1, and interleukin 1β up-regulate connexin 43 protein and messenger RNA expression and enhance intercellular communication. Slight down-regulation of connexin 43 is observed in response to vitamin A. Connexin 43 induction by endothelin 1 is inhibited by both endothelin A and endothelin B receptor antagonists. In coculture systems, hepatic stellate cells communicate with each other, which is suggestive of a syncytial organization, but no communication was found between hepatic stellate cells and other liver cell types. As shown by immunohistochemistry and electron microscopy, gap junctions are formed between activated hepatic stellate cells in vivo. Conclusions: Gap junctional communication occurs between hepatic stellate cells, is enhanced after activation, and underlies complex regulation by cytokines, hormones, and vitamins. 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The gap junction communication channel.Cell. 1999; 84: 381-388Google Scholar are also involved. Cxs play important roles in cell differentiation, growth control, apoptosis,25Lin J.H. Weigel H. Cotrina M.L. Liu S. Bueno E. Hansen A.J. Hansen T.W. Goldman S. Nedergaard M. Gap-junction-mediated propagation and amplification of cell injury.Nat Neurosci. 1998; 1: 494-500Google Scholar and cell migration.26Lin J.H.C. Takano T. Cotrina M.L. Arcuino G. Kang J. Liu S. Gao Q. Li J. Li F. et al.Connexin43 enhances the adhesivity and mediates the invasion of malignant glioma cells.J Neurosci. 2002; 22: 4302-4311Crossref PubMed Google Scholar Cx-43 influences cytokine expression27Oviedo-Orta E. Gasque P. Evans W.H. Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication.FASEB J. 2001; 15: 768-774Google Scholar and modulates fibroblast28Ehrlich H.P. Gabbiani G. Meda P. Cell coupling modulates the contraction of fibroblast-populated collagen lattices.J Cell Physiol. 2000; 184: 86-92Google Scholar and cardiac muscle29Zhang Y.M. Miura M. Keurs H.E.D.J. Triggered propagated contractions in rat cardiac trabeculae.Circ Res. 1996; 79: 1077-1085Google Scholar contraction. In rat liver, the propagation of nerve signals requires intact gap junctions, and Cx-32–deficient mice show impaired metabolism and increased tumorigenesis.12Nelles E. Butzler C. Jung D. Temme A. Gabriel H.D. Dahl U. Traub O. et al.Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin-32-deficient mice.Proc Natl Acad Sci U S A. 1996; 93: 9565-9570Google Scholar, 30Saez J.C. Intercellular gap junctional communication is required for an optimal metabolic response of the functional units of the liver.Hepatology. 1997; 25: 775-776Google Scholar Whereas the expression of Cx-26 and Cx-32 in PC is well established,10Willecke K. Temme A. Ott T. Functions of hepatic gap junctions analysed in connexin 32-deficient mice.in: Häussinger D. Jungermann K. Liver and nervous system. Falk Symposium 103. German Falk Foundation, Freiburg, Germany1997Google Scholar, 12Nelles E. Butzler C. Jung D. Temme A. Gabriel H.D. Dahl U. Traub O. et al.Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin-32-deficient mice.Proc Natl Acad Sci U S A. 1996; 93: 9565-9570Google Scholar, 14Zhang M. Thorgeirsson S.S. Modulation of connexins during differentiation of oval cells into hepatocytes.Exp Cell Res. 1994; 213: 37-42Google Scholar, 16Kumar N.M. Gilula N.B. The gap junction communication channel.Cell. 1999; 84: 381-388Google Scholar, 30Saez J.C. Intercellular gap junctional communication is required for an optimal metabolic response of the functional units of the liver.Hepatology. 1997; 25: 775-776Google Scholar, 31Greenwel P. Rubin J. Schwartz M. Hertzberg E.L. Rojkind M. Liver fat-storing cell clones obtained from a CCl4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43.Lab Invest. 1993; 69: 210-216Google Scholar little is known about Cxs in primary HSCs. Recently Cx-43 expression in rat cholangiocytes was described.32Bode H.P. Wang L. Cassio D. Leite M.F. St-Pierre M.V. Kirata K. Okazaki K. Sears M.L. Meda P. Nathanson M.H. Dufour J.F. Expression and regulation of gap junctions in rat cholangiocytes.Hepatology. 2002; 36: 631-640Google Scholar We therefore studied the regulation and functional relevance of Cxs and intercellular communication in quiescent and activated HSCs. Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum were obtained from PAA Laboratories (Linz, Austria). Penicillin/streptomycin was from Biochrom (Berlin, Germany), Nycodenz from Nycomed (Oslo, Norway), and pronase from Merck (Darmstadt, Germany). Deoxyribonuclease I and collagenase were from Roche (Mannheim, Germany). Antibodies against α-SMA, bovine serum albumin, lipopolysaccharide (LPS), dexamethasone, vitamin D3 (1,25-dihydroxy-cholecalciferol), triiodo-l-thyronine (thyroid hormone T3), interleukin (IL)-1β, vitamin A (all-trans retinoic acid), PMA (phorbol 12-myristate 13-acetate), lucifer yellow dilithium (LY), the lysosome inhibitor 3-methyladenine, and mouse monoclonal anti–α-SMA-CY3 were obtained from Sigma (Deisenhofen, Germany). The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling detection assay and platelet-derived growth factor (PDGF) were from R&D (Wiesbaden, Germany). All fluorochrome-conjugated secondary antibodies were from Jackson Corp. (West Grove, PA). Horseradish peroxidase–conjugated anti-mouse immunoglobulin (Ig)G, anti-rabbit IgG, and the Bio-Rad protein assay were from Bio-Rad (Hercules, CA); the enhanced chemiluminescence detection kit was from Amersham (Braunschweig, Germany). The PKC inhibitor Gö6850, the proteasome inhibitor MG132, the ETA receptor antagonist BQ123, the ETB receptor antagonist IRL1038, and the ETB receptor agonist sarafotoxin were from Calbiochem (Bad Soden, Germany). Formaldehyde was from Sigma-Aldrich (Steinheim, Germany). Mouse anti-rat ED-1/fluorescein isothiocyanate (FITC) was from Serotec (Oxford, UK). Antibodies against Cx-26 were from Zymed (rabbit and mouse; San Francisco, CA), Chemicon (AB1717; Temecula, CA), and Alpha Diagnostic (Cologne, Germany). Antibodies against Cx-32 were from Zymed (rabbit and mouse), Chemicon (MAB3069), and Alpha Diagnostic (Cx-32B12-A). Antibodies against Cx-43 were from Zymed (rabbit and mouse), Santa Cruz (C-20; Santa Cruz, CA), and Chemicon (MAB3068). Antibodies against phosphorylated serine 255, serine 279/282, and tyrosine 265 Cx-43 were from Santa Cruz. Antibodies against Cx-31, -33, -37, -40, and -46 were from Alpha Diagnostic. Antibodies against Cx-46 were from Chemicon (MAB3101); antibodies against Cx-50 were from Zymed. Phosphorylation-specific antibodies against MAPKs Erk-1/-2 and p38 were from Biosource (Mannheim, Germany). Antibodies against glial fibrillary acidic protein (GFAP) were from Sigma (monoclonal and polyclonal). The Cx-26, -32, and -43 complementary DNA (cDNA) fragments used for detection of the corresponding Cx messenger RNA (mRNA) were provided by Dr. H. Sies (University of Düsseldorf, Germany). The 1.0-kilobase cDNA fragment for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used for standardization, was from Clontech (Heidelberg, Germany); the oligonucleotide-labeling kit was from Pharmacia (Freiburg, Germany). [α-32P]Deoxycytidine triphosphate was from Amersham. HSCs from 1–3-year-old male Wistar rats were prepared as previously described33Fischer R. Schmitt M. Bode J.G. Häussinger D. Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells.Gastroenterology. 2001; 120: 1212-1226Abstract Full Text Full Text PDF Scopus (100) Google Scholar by collagenase/pronase perfusion and isolated by Nycodenz gradient. The cells were seeded at a density of 0.15 × 106/cm2 on glass coverslips in a 24-well culture plate (Falcon, Heidelberg, Germany) or a 60-mm well (Falcon) and maintained in DMEM containing 10% (vol/vol) heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Culture was performed in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The culture medium was changed first after 24 hours and then every 2 days. The purity of HSCs was >95%, as assessed 24 hours after seeding by their typical light-microscopic appearance and vitamin A–specific autofluorescence, their inability to phagocytose fluorescent 1.1-μm latex particles, and their positive immunofluorescent staining for desmin, GFAP, and, at the seventh day of culture, α1-SMA. If not indicated otherwise, all experiments were performed with HSCs at the seventh day of culture. Isolated PCs were prepared from livers of 5–8-week-old male Wistar rats by a collagenase perfusion technique as previously described.33Fischer R. Schmitt M. Bode J.G. Häussinger D. Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells.Gastroenterology. 2001; 120: 1212-1226Abstract Full Text Full Text PDF Scopus (100) Google Scholar Cells were seeded on collagen-coated culture dishes or glass coverslips at a density of 0.7 × 106/cm2 and were cultured for 24 hours in DMEM containing 10% (vol/vol) heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 nmol/L insulin, 100 nmol/L dexamethasone, 30 nmol/L sodium selenite, and 1 μg/mL aprotinin in a humidified atmosphere of 5% CO2 and 95% air at 37°C. For coculture experiments, PCs were plated on HSCs and maintained in the same cell culture medium as described for PC culture. Experiments were performed 24 hours after seeding. Kupffer cells (KCs) and sinusoidal endothelial cells (SECs) were prepared by collagenase/pronase perfusion and separated by a single Nycodenz gradient and centrifugal elutriation as described previously.9Fischer R. Cariers A. Reinehr R. Häussinger D. Caspase 9-dependent killing of hepatic stellate cells by activated Kupffer cells.Gastroenterology. 2002; 123: 845-861Abstract Full Text Full Text PDF Scopus (85) Google Scholar The phagocytotic activity of KCs was measured by their ability to ingest latex particles. The purity of KC was >95%, as assessed 24 hours after seeding by their positive immunofluorescent staining for ED-1 and anti-macrophage antibodies. KC and SEC culture conditions were identical to those for HSC. For coculture experiments, KCs or SECs were plated on HSCs and maintained in DMEM containing 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin 48 hours before experiments. Male Wistar rats (200–250 g) received an initial dose of CCl4 (150 μL/100 g body weight in paraffin oil 1:1) intraperitoneally followed by 2 intraperitoneal injections per week of CCl4 (100 μL/100 g), as described previously.5Niki T. De Bleser P.J. Su G. Van Den Berg K. Wisse E. Geerts A. Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers.Hepatology. 1996; 23: 1538-1545Google Scholar, 33Fischer R. Schmitt M. Bode J.G. Häussinger D. Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells.Gastroenterology. 2001; 120: 1212-1226Abstract Full Text Full Text PDF Scopus (100) Google Scholar Control was performed by injecting the vehicle (paraffin oil). Groups of 3 CCl4-treated animals and single controls were killed after 3 weeks of treatment, a time period after which fibrosis is observed in the CCl4-treated animals.5Niki T. De Bleser P.J. Su G. Van Den Berg K. Wisse E. Geerts A. Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers.Hepatology. 1996; 23: 1538-1545Google Scholar All rats were fed ad libitum on stock diet and held according to the local ethical guidelines. Cells were harvested in a buffer containing 63 mmol/L Tris/HCl (pH 6.8), 1% sodium dodecyl sulfate (SDS), and a protease/phosphatase inhibitor cocktail (Boehringer Corp., Mannheim, Germany). For determination of the membrane and the cytosolic protein fraction, cells were lysed in a buffer containing 10 mmol/L Tris, 30 mmol/L mannitol, and 10 mmol/L CaCl2 (pH 7.5). After centrifugation of the samples (5 minutes at 1200 × g), the supernatants were subjected to ultracentrifugation (35 minutes at 40,000 × g) to separate cytosolic from membrane fractions. The protein content of all probes was determined as previously described.33Fischer R. Schmitt M. Bode J.G. Häussinger D. Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells.Gastroenterology. 2001; 120: 1212-1226Abstract Full Text Full Text PDF Scopus (100) Google Scholar The cell lysate was mixed 2:1 (vol/vol) with gel loading buffer containing 300 mmol/L dithiothreitol (pH 6.8). After boiling for 5 minutes, the proteins were separated by SDS-polyacrylamide gel electrophoresis (40 μg of protein per lane; 12% gel). After electrophoresis, gels were equilibrated with transfer buffer (39 mmol/L glycine, 48 mmol/L Tris base, 0.03% SDS, and 20% [vol/vol] methanol). Proteins were transferred to nitrocellulose membranes by using a semidry transfer apparatus (Pharmacia) according to the manufacturer's instructions. Blots were blocked in 5% (wt/vol) bovine serum albumin containing 20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, and 0.1% Tween 20 (TBST) and then incubated at 4°C overnight with the primary antibody (dilution 1:2000). After washing with TBST and incubating with horseradish peroxidase–coupled anti–mouse IgG, anti–goat IgG, or anti–rabbit IgG antibody (dilution 1:5000) at room temperature for 2 hours, the blot was washed extensively and developed by using enhanced chemiluminescent detection (Amersham). Blots were exposed to Kodak X-OMAT AR-5 film (Eastman Kodak Co., Rochester, NY) for 1–30 minutes. Suitably exposed films were then analyzed by densitometry scanning (Kodak digital image station 440CF) and normalized for their respective GAPDH expression. Total RNA from culture plates of HSCs was isolated by using the RNeasy Total RNA kit (Qiagen, Hilden, Germany). RNA samples (5 μg per lane) were electrophoresed in 0.8% agarose containing 3% formaldehyde and then blotted onto Duralon-UV nylon membranes (Stratagene, Heidelberg, Germany) with 20× standard saline citrate (3 mmol/L NaCl/0.3 mmol/L sodium citrate). After brief rinsing with water and UV cross-linking (Hoefer UV-Crosslinker 500; Hoefer, San Francisco, CA), the membranes were observed under UV illumination to determine RNA integrity and the location of the 28S and 18S ribosomal RNA bands. The blots were then subjected to prehybridization for 3 hours at 43°C in 50% deionized formamide in sodium phosphate buffer (0.25 mmol/L; pH 7.2) containing 0.25 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 100 mg/mL salmon sperm DNA, and 7% (wt/vol) SDS. Hybridization was performed in the same solution with approximately 106 counts per minute per milliliter of [α-32P]deoxycytidine triphosphate–labeled random-primed Cx-26, -32, -43, and GAPDH cDNA probes. Membranes were washed 3 times in 2× standard saline citrate/0.1% SDS for 10 minutes, twice in sodium phosphate buffer (25 mmol/L; pH 7.2)/EDTA (1 mmol/L)/0.1% SDS, and twice in sodium phosphate buffer (25 mmol/L; pH 7.2)/EDTA (1 mmol/L)/1% SDS. Blots were then exposed to Kodak X-OMAT AR-5 films at −70°C with intensifying screens. Suitably exposed films were then analyzed by densitometry scanning (Kodak digital image station 440CF). Gap junctional intercellular communication was assessed by the efficacy of diffusion of LY from a single microinjected cell to its neighboring cells. Microinjection of lucifer yellow (0.33 mol/L) was performed by using microinjector 5246 and micromanipulator 5171 (Eppendorf, Hamburg, Germany). To detect lucifer yellow diffusion, cells were microinjected under constant pressure (120 hPa) at 37°C until LY fluorescence became broadly visible in the injected cell. If not stated otherwise, dye-coupled cells were counted 3 minutes after injection under a fluorescence microscope. In cocultures of HSCs with other cell types, the microinjected cell was in direct contact with the other cell type. For measurement of intercellular calcium transfer, calcium (1 mmol/L in 0.33 mol/L LY or 140 mmol/L KCl) was microinjected (30 seconds; 120 hPa; 37°C), and intracellular calcium was measured in the neighboring cell as previously described.6Reinehr R.M. Kubitz R. Peters-Regehr T. Bode J.G. Häussinger D. Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin-1.Hepatology. 1998; 28: 1566-1577Google Scholar Cells were washed with Krebs–Henseleit buffer (KHB) and loaded with 5 μmol/L Fura-2-acetoxymethylester (AM) for 30 minutes. After the loading period, the coverslips were mounted in a perfusion chamber on an inverted fluorescence microscope (Zeiss, Oberkochen, Germany) and continuously superfused with KHB at 10 mL/min. The KHB was equilibrated with oxygen/CO2 (95/5; vol/vol), resulting in a pH of 7.3. The temperature was kept at 37°C. One cell per coverslip was investigated. Cells loaded with Fura-2 were alternately excited at 340 and 380 nm by use of a high-speed filter wheel (10 turns per second; time resolution, 10 Hz). Emission was measured at 480–520 nm with a photo counting tube (H3460-04; Hamamatsu, Herrsching, Germany). After correction for autofluorescence, the intracellular calcium concentration was calculated from the fluorescence ratio 380:340 nm as previously described.6Reinehr R.M. Kubitz R. Peters-Regehr T. Bode J.G. Häussinger D. Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin-1.Hepatology. 1998; 28: 1566-1577Google Scholar For determination of intercellular calcium transfer via gap junctions between heterogeneous liver cell types, intracellular calcium was measured over at least 5 minutes after microinjection of 1 mmol/L CaCl2 (in 140 mmol/L KCl; 120 hPa, 30 seconds, and 37°C) in a neigh
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