Volume 128, Issue 2, Pages 433-448 (February 2005) Intercellular communication via gap junctions in activated rat hepatic stellate cells  Richard Fischer, Roland Reinehr, Thuy Phung Lu, Alexandra Schönicke, Ulrich Warskulat, Hans Peter Dienes, Dieter Häussinger  Gastroenterology  Volume 128, Issue 2, Pages 433-448 (February 2005) DOI: 10.1053/j.gastro.2004.11.065 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 The mRNA and protein expression of Cx-43 in quiescent and activated HSCs in culture. (A) Quiescent and activated HSCs in culture express Cx-43 mRNA, but no Cx-32 mRNA. Faint expression of Cx-26 mRNA becomes detectable in transformed HSCs. For mRNA analysis, cells were harvested after the time periods indicated and subjected to Northern blot analysis for Cx-43, -26, and -32 and GAPDH as described in Materials and Methods. (B) Cx-43 protein expression increases with culture time. Cultured PCs, KCs, and SECs express only small amounts of Cx-43. Phosphorylation of Cx-43 at serine 279/282 in HSCs increases during culture. Also, in KCs, PCs, and SECs, Cx-43 is phosphorylated. Cx-26 is strongly up-regulated in cultured HSCs and is also expressed in PCs and SECs, whereas Cx-32 is expressed only in PCs and SECs. Protein levels are determined by Western blot analysis by using specific antibodies against the proteins indicated (Cx-43: Zymed rabbit anti–Cx-43; same result obtained with antibody Cx-43 C-20 [Santa Cruz] and mouse anti–Cx-43 MAB3068 [Chemicon]; Cx-26: AB1717 [Chemicon]; same result obtained with Cx-26-A [Alpha Diagnostic]; Cx-32: MAB3069 [Chemicon]; same result obtained with rabbit and mouse anti–Cx-32 [Zymed] and Cx-32B12-A [Alpha Diagnostic]). GAPDH expression served as a control for protein loading. Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Expression of Cx-43 and Cx-26 in activated HSCs. (A) The level of Cx-43 (♦) over HSC culture time was quantified by determination of optical density (Western blotting; rabbit Cx-43 [Zymed]). The level of protein expression is given in relation to GAPDH protein. Intercellular communication in confluent HSC monoculture was determined by lucifer yellow microinjection, and the number of dye-coupled cells was counted 3 minutes after microinjection (•). (B) Immunocytochemical staining of HSCs (seventh day of culture) indicates a membrane localization of Cx-43, whereas Cx-26 is intracellular (mainly perinuclear). Antibodies used were as follows: Cx-43—Zymed rabbit anti–Cx-43, same result obtained with antibody Cx-43 C-20 (Santa Cruz) and mouse anti–Cx-43 MAB3068 (Chemicon); Cx-26—AB1717 (Chemicon), same result obtained with Cx-26-A (Alpha Diagnostic). Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 In vivo expression of Cxs in HSCs. Cx expression was studied in normal rat liver (A–C and G–L) and CCl4-induced liver fibrosis (D–F and M). Cx-43 is expressed in HSCs, as GFAP (C) (green) colocalizes (B) with Cx-43 (red) (A). Expression of Cx-43 is increased (D–F) in activated HSCs in CCl4-induced liver fibrosis. Quiescent HSCs do not express Cx-32 (red) (G) or Cx-26 (red) (J), and no colocalization (H and K) is found with HSC-specific GFAP (green) (I and L). The antibodies used were from Zymed (rabbit); monoclonal antibodies from Zymed (mouse) yielded similar results. (M) Magnification of 2 neighboring HSCs from (E), showing Cx-43 expression at HSC cell–cell contacts. (N) Electron micrograph of a CCl4-treated rat liver in situ: activated HSCs are seen beside a hepatocyte (HC). Although the HSC located at the upper right corner of the electron micrograph still shows intracellular lipid droplets, indicating an early stage of HSC transdifferentiation, the HSC located in the left lower corner represents a late stage of HSC transdifferentiation with respect to organelle content and loss of intracellular lipid droplets. The abundance of mitochondria identifies the cell located in the lower right corner as a hepatocyte. As indicated by the arrow, a gap junction is visible between the 2 HSCs (original magnification, 24,000-fold). Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Determination of intercellular HSC communication by microinjection of lucifer yellow (A and B) and intercellular calcium transfer (C). (A) After microinjection of lucifer yellow into a single HSC (first figure), a time-dependent dye transfer to neighboring HSCs occurs (microscopic fluorescence images and the corresponding phase contrast are given). (B) HSCs from at least 3 different preparations were microinjected with lucifer yellow, and the time course of the consecutive dye transfer to neighboring HSCs was visualized by fluorescence microscopy. Dye transfer was maximal 2–3 minutes after microinjection. (C) Intracellular calcium was measured in a single HSC (circle) at a distance of 2 cells from an HSC microinjected (arrow) with 1 mmol/L CaCl2/140 mmol/L KCl (120 hPa; 30 seconds; 37°C). Twenty of 26 HSCs responded with an intracellular calcium signal seconds after microinjection of the distant cell, thus indicating intercellular calcium transfer. No calcium signal was observed after inhibition of gap junctional communication by 1-octanol (1 mmol/L; n = 12; not shown). Intercellular calcium transfer was preserved in 13 of 17 cells in the absence of extracellular calcium. For positive control, all cells were exposed to adenosine triphosphate (10 μmol/L) that evoked a typical intracellular calcium response. All experiments were performed with HSCs cultured for 7 days and are representative of at least 3 independent cell preparations. ATP, adenosine triphosphate. Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 PMA-induced phosphorylation and degradation of Cx-43 in activated HSCs. (A) HSCs (seventh day of culture) were incubated with PMA (1 μmol/L) for the time periods indicated. The PKC inhibitor Gö6850 (10 μmol/L) was incubated 30 minutes before PMA stimulation; the proteasome inhibitor MG132 (10 μmol/L) and the inhibitor of the lysosomal pathway 3-methyladenine (10 mmol/L) were added 18 hours before PMA stimulation. PMA induces hyperphosphorylation and degradation of Cx-43 in a Gö6850-sensitive way, but it has no effect on Cx-26 expression. Also, MG132 or methyladenine diminished the PMA-induced Cx-43 disappearance. GAPDH served as a loading control. (B) The level of Cx-43 in the presence of MG132 (♦ 18 hours of preincubation) or 3-methyladenine (▴; 18 hours of preincubation) or under control (•) conditions was quantified by optical densitometry of Western blottings. The level of protein expression is given in relation to GAPDH protein. MG132 and 3-methyladenine prevent PMA-induced degradation of Cx-43. (C) HSCs (seventh day of culture) were incubated for 30 minutes with the compounds indicated. The cytokines had no effect on Cx-43 expression or Cx-43 phosphorylation. Epidermal growth factor (50 ng/mL), ET-1 (40 nmol/L), and PDGF (50 ng/mL) induced phosphorylation of the MAPKs Erk-1/-2 and p38. Phosphorylation of the MAPKs Erk-1/-2 and p38 was detected by using phosphospecific antibodies in Western blotting. Cx-43 phosphorylation and expression were analyzed with Western blotting. GAPDH expression showed equal protein loading in the latter samples. Representative experiments from at least 3 independent cell preparations are shown. Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Long-term effects of cytokines and hormones on Cx-43 protein and mRNA expression. (A) HSCs were incubated with the effectors indicated from the third to the seventh day of culture for determination of Cx-43 protein. Medium containing effectors was changed daily. For determination of Cx-43 mRNA, HSCs (seventh day of culture) were incubated with the substances indicated for 8 hours. Vitamin D3 (100 nmol/L), dexamethasone (1 μmol/L), LPS (1 μg/mL), IL-1β (100 U/mL), ET-1 (10 nmol/L), PDGF (50 ng/mL), and triiodothyronine (T3; 100 nmol/L) increased Cx-43 expression, whereas vitamin A (100 nmol/L) slightly decreased Cx-43 expression. The antibodies used were from Zymed (rabbit Cx-43). Cx protein expression was analyzed by Western blotting. GAPDH served as a loading control. Messenger RNA expression was analyzed by Northern blot analysis with specific cDNA probes for GAPDH and Cx-43, as described in Materials and Methods. (B) Statistical analysis: exposure of HSCs for 4 days to dexamethasone, vitamin D3, triiodothyronine (T3), IL-1β, LPS, ET-1, and PDGF significantly increased, whereas vitamin A decreased, Cx-43 protein expression. Incubation of HSCs (seventh day of culture) for 8 hours with the same substances showed a similar Cx-43 mRNA regulation. Messenger RNA and protein expression were analyzed by densitometry of Western and Northern blots, as described in Materials and Methods. Relative Cx-43 mRNA levels are given in relation to corresponding GAPDH mRNA. The level of protein expression is given in relation to GAPDH protein. Cx-43 protein and mRNA expression are given relative to control values (arbitrarily set to 1). *Statistically significant compared with control by using the Student t test. Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 ET-1–induced Cx-43 expression in activated HSCs involves ETA and ETB receptors. (A) In activated HSCs (seventh day of culture), ET-1 induced a dose-dependent Cx-43 protein expression (stimulation for 8 hours at concentrations as indicated) as determined by Western blot analysis. GAPDH expression was used as a control and showed equal protein loading. ET-1 20 nmol/L rapidly induced Cx-43 mRNA in activated HSCs (seventh day of culture) as determined by Northern blot analysis. For control, the corresponding GAPDH mRNA levels are given. (B) ET-1–induced (20 nmol/L) expression of Cx-43 protein in HSCs (seventh day of culture) is diminished by both the ETA receptor antagonist BQ123 (5 μmol/L; 30 minutes’ preincubation) and the ETB receptor antagonist IRL1038 (5 μmol/L; 30 minutes’ preincubation). The ETB receptor agonist sarafotoxin (20 nmol/L) induced Cx-43 protein expression. Chelation of intracellular calcium by BAPTA-AM (50 μmol/L) reduced ET-1–induced Cx-43 expression, whereas 8-CPT-cAMP (200 μmol/L) induced Cx-43 expression. Omission of extracellular calcium and chelation with EDTA (2.5 mmol/L) did not prevent ET-1–induced Cx-43 expression. Inhibition of the Erk-1/-2 MAPK pathway with PD98059 (10 μmol/L), inhibition of the MAPK p38 with SB203580 (20 μmol/L), and inhibition of PI3 kinase with LY294002 (10 μmol/L) or wortmannin (100 nmol/L) did not significantly alter ET-1–induced Cx-43 expression. GAPDH expression showed equal protein loading in the latter samples. HSCs were stimulated for 8 hours, and Cx-43 protein expression was determined by using Western blot analysis. All experiments are representative of at least 3 independent cell preparations. (C) Statistical analysis of the data shown in (B) for Cx-43 protein expression, which was analyzed by densitometry of Western blots as described in Materials and Methods. The level of protein expression is given in relation to GAPDH protein. Statistically significant compared with *ET-1 or +control with the Student t test. Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Cx-dependent single cell contraction of activated HSCs. Microinjection of calcium (1 mmol/L CaCl2) with lucifer yellow dilithium (0.33 mol/L; 120 hPa; 30 seconds; 37°C) in activated HSCs (seventh day of culture) induced visible contraction not only of the injected cell, but also of a neighboring HSC, in 48% (n = 27; control). Neighbor cell contraction was prevented after inhibition of gap junctional communication by 1-octanol (1 mmol/L 30 minutes before stimulation; n = 26). Black arrows indicate the microinjected HSCs, and white arrows indicate neighboring HSCs. Cell contraction is shown by phase-contrast microscopy 5 minutes after microinjection (right). Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 HSCs in coculture do not communicate with PCs or KCs. HSCs and PCs or HSCs and KCs were cocultured as described in Materials and Methods. (A) A PC (white arrows) in direct contact with an HSC (SMA positive) was microinjected with lucifer yellow (blue arrow). Dye transfer occurred only to a second PC (green fluorescent cells; 3 minutes after microinjection), but not to the neighboring HSC, which stained positive for SMA. (B) An HSC in direct contact with a PC (white arrows) was microinjected with lucifer yellow (blue arrow). Dye transfer was observed to a neighboring HSC (green fluorescent cells; 3 minutes after microinjection), but not to PCs. After microinjection, HSCs were specifically stained with antibody against SMA (red) (A and B). (C) In HSC/KC coculture, a microinjected HSC does not communicate with KCs (white arrows). After microinjection, KCs were specifically stained with antibody against ED-1 (right). (D) A microinjected KC (white arrows) does not show any dye transfer to neighboring HSCs. After microinjection, HSCs were specifically stained with antibody against SMA (right). Gastroenterology 2005 128, 433-448DOI: (10.1053/j.gastro.2004.11.065) Copyright © 2005 American Gastroenterological Association Terms and Conditions