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p38 MAP-kinase regulates function of gap and tight junctions during regeneration of rat hepatocytes
Journal of Hepatology 42 (2005) 707–718 www.elsevier.com/locate/jhep
p38 MAP-kinase regulates function of gap and tight junctions during regeneration of rat hepatocytes Toshinobu Yamamoto, Takashi Kojima*, Masaki Murata, Ken-ichi Takano, Mitsuru Go, Naoko Hatakeyama, Hideki Chiba, Norimasa Sawada Department of Pathology, Sapporo Medical University School of Medicine, S1. W17, Sapporo 060-8556, Japan
Background/Aims: Hepatocyte regeneration is considered to be associated with adaptive changes in expression of gap and tight junctions through multiple signal transduction pathways including p38 MAP-kinase. The role of the stress responsitive MAP-kinase, p38 MAP-kinase, signaling pathway in function of gap and tight junctions was examined during regeneration of rat hepatocytes in vivo and in vitro. Methods: We examined changes in formation, expression and function of gap and tight junctions in rat livers after 70% partial hepatectomy and in primary cultures of rat hepatocytes, by using a p38 MAP-kinase inhibitor, SB203580. Results: When p38 MAP-kinase was activated during partial hepatectomy, down-regulation of Cx32 and up-regulation of claudin-1 were observed. By SB203580 treatment, the down-regulation of Cx32 was inhibited and the up-regulation of claudin-1 was enhanced, well maintaining the structures of gap and tight junctions. SB203580 treatment did not affect the increase of hepatocyte proliferation. In EGF induced proliferative rat hepatocytes treated with SB203580, the expression and function of Cx32 and claudin-1 were increased. Conclusions: Dynamic changes of formation of gap and tight junctions during regeneration of rat hepatocytes in vivo and in vitro are in part controlled via a p38 MAP-kinase signaling pathway, and are independent of cell growth. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: p38 MAPK; Gap junctions; Tight junctions; Liver; Regeneration 1. Introduction Hepatic gap and tight junctions play crucial roles in bile secretion and in the barrier to keep bile in bile canaliculi away from the blood circulation [1–3]. Furthermore, we have hypothesized that the close interaction between gap and tight junctions of hepatocytes also regulates the blood– biliary barrier [4,5]. Gap junction channels, composed of proteins termed connexins, mediate reciprocal exchange of ions and small molecules of less than 1000 Da, including second messengers such as cyclic AMP, IP3 and Ca2C, between adjacent cells [4,6–8]. Gap junctional intercellular communication (GJIC) is thought to play a crucial role in
Received 6 May 2004; received in revised form 2 December 2004; accepted 14 December 2004; available online 16 March 2005 * Corresponding author. Tel.: C81 11 611 2111x2703; fax: C81 11 613 5665. E-mail address: [email protected] (T. Kojima).
development, cell growth and cell differentiation [9–13]. Tight junctions, the apicalmost component of intercellular junctional complexes, separate the apical from the basolateral cell surface domains to establish cell polarity (performing the function of a fence); tight junctions also provide a barrier function, inhibiting solute and water flow through the paracellular space [14,15]. They show a particular net-like meshwork of fibrils formed by the integral membrane proteins, occludin, the claudin family, and JAM [4,15–17]. Several peripheral membrane proteins, ZO-1, -2, -3, 7H6 antigen, cingulin, symplekin, Rab3B, Ras target AF-6, and ASIP, an atypical protein kinase C-interacting protein, were reported [4,15–17]. Communication via gap junctions may be needed for extensive initiation of synchronous DNA synthesis in hepatocytes during liver regeneration. It is well known that the structures and expression of gap junctions in most hepatocytes of all liver lobules disappear in the early stage after PH [18,19]. The construction of the hepatocyte tight
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.12.033
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junction is one of the most important events during regeneration of the liver leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division [20]. In a recent study, protein expression of tight junction components such as claudin-3, ZO-1 and ASIP was found to increase after PH [21]. We previously found that the PI3-kinase pathway rather than the MAPkinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of gap junction protein Cx32, but not tight junction protein claudin-1 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP-kinase [22]. However, the detail mechanisms, including other signal transduction pathways, for regulation of gap and tight junctions during liver regeneration are as yet unclear. Signal transduction pathways, including MAP-kinase, p38 MAP-kinase and PI3-kinase are involved in growth, cell survival and cell death. p38 MAP-kinase is a stressresponsive MAP-kinase and is phosphorylated and activated by proinflammatory cytokines and environmental stress [23,24]. Liver regeneration is also controlled by multiple signaling pathways and partial hepatectomy can activate MAP-kinase, p38 MAP-kinase and c-Jun NH2-terminal kinase (JNK) in the rat liver [25]. Recent evidence indicates that p38 MAP-kinase plays a major role in hepatocyte proliferation, whereas chronic activation of MAP-kinase has been shown to lead to cellular growth arrest [25]. Furthermore, it is thought that the p38 MAP-kinase signaling pathway might be important for hepatic damage after liver injury such as ischemia/reperfusion, hepatitis, and alcoholic liver disease [26–29]. Therefore, we focused on the role of the p38 MAP-kinase signaling pathway for formation, expression and function of gap and tight junctions during regeneration of rat hepatocytes in vivo and in vitro, using the p38 MAP-kinase inhibitor SB203580.
2. Materials and methods 2.1. Animal model Male Fisher 344 rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) weighing about 170–200 g were used for all experiments of animal models. For them, 70% partial hepatectomy (PH) was performed according to the previously described method [30]. Sham operation consisted of laparotomy and exposure of the liver. At 30 min before PH surgery, some rats were injected intraperitoneally with the p38 MAP-kinase inhibitor, SB203580 (Calbiochem-Novabiochem Corp., San Diego, CA), dissolved in 0.9% NaCl at a dose of 20 mg/kg body weight.
2.2. Rat hepatocyte isolation and cell culture Male Sprague–Dawley rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) weighing about 250–300 g were used to isolate hepatocytes by the two-step liver perfusion method of Seglen [31] with some modification [32]. Hepatocytes used in all experiments were maintained in the modified L-15 medium until day 10 after plating. For the experiment on EGF-induced proliferative rat hepatocytes, some cells at
day 10 after plating were cultured with modified DMEM medium (0.2% BSA, 20 mM HEPES, 0.5 mg/l insulin, 10K7 M dexamethasone, 1 g/l galactose, 30 mg/l proline, 20 mM NaHCO3) supplemented with 10 ng/ml EGF and 10 mM nicotinamide for 72 h [32], and then they were treated with 20 mg/ml SB203580 for 1, 4 and 10 h.
2.3. Immunostaining Frozen sections made with a cryostat and cells grown on glass coverslips were fixed with cold absolute acetone for 10 min. Immunostaining with monoclonal anti-Cx32 (Zymed) and polyclonal anti-claudin-1 (Zymed) antibodies was performed. These specimens were visualized using Alexia 488- or 594-conjugated anti-mouse or anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR). The specimens were examined using a laserscanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA). The number of Cx32-positive spots in liver sections was counted using a Scionimage analysis system. Immunostaining for claudin-1 was also performed in deparaffinized liver sections.
2.4. Ki-67 labeling index To examine the Ki-67 labeling index, immunostaining for Ki-67 (DAKO A/S, Glostrup, Denmark) was performed in deparaffinized 3 mm sections of liver. Labeled cells that had nuclei stained for Ki-67 were counted using a microscope (magnification !200). More than 1000 cells were counted per section using a Scion-image analysis system.
2.5. Freeze-fracture analysis For the freeze-fracture technique, the livers were immersed in 40% glycerin solution after fixation in 2.5% glutaraldehyde/0.1 M phosphatebuffered saline (PBS). The specimens were fractured at K160 8C in a JFD7000 freeze-fracture device (JEOL Ltd, Tokyo, Japan) and replicated by deposition of platinum/carbon from an electron beam gun positioned at a 458 angle followed by carbon applied from overhead. Replicas were examined at 100 kV with a JEM transmission electron microscope (JEOL Ltd, Tokyo, Japan).
2.6. Western blot analysis The livers were minced with a scalpel blade, resuspended in buffer (1 mM NaHCO3 and 2 mM phenylmethyl sulfonyl fluoride (PMSF)) and sonicated for 10 s. The dishes were washed with PBS twice, and 300 ml of the buffer (1 mM NaHCO3 and 2 mM PMSF) was added to 60-mm dishes. The cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 s. Western blots were performed as described previously [32]. The membranes were incubated with polyclonal anti-phospho-p38 (Cell Signaling, Beverly, MA), polyclonal anti-p38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal anti-phospho-JNK (Thr183/ Tyr185; Cell Signaling, Beverly, MA), polyclonal anti-SAPK/JNK (Cell Signaling), polyclonal anti-phospho-p44/p42 MAPK (Thr202/Tyr204; Cell Signaling), polyclonal anti-MAPK (ERK1/2; Promega Corporation, Madison, WI), monoclonal anti-phospho-Akt (Ser473; Cell Signaling), polyclonal anti-Akt (Cell Signaling), monoclonal anti-Cx32 (Zymed Laboratories, Inc., San Francisco, CA), polyclonal anti-Cx26 (Zymed), polyclonal anti-occludin (Zymed), polyclonal anti-claudin-1, -2, -3 (Zymed), polyclonal anti-ZO-1 (Zymed), polyclonal anti-JAM-1 (33; Zymed), monoclonal anti-E-cadherin (Sigma), monoclonal anti-PCNA (Amersham Corp., Buckinghamshire, UK) and polyconal anti-actin (Sigma) antibodies at room temperature (RT) for 1 h.
2.7. Preparation of Triton X-100-soluble and -insoluble protein fractions To isolate Triton X-100-soluble and -insoluble protein fractions, the protein samples were lysed in Triton X-100 buffer (1% Triton X-100, 100 mM NaCl, 10 mM HEPES, pH 7.6, 2 mM EDTA, 1 mM PMSF, 4 mM sodium orthovanadate, 40 mM sodium fluoride), and then passed through a 21-gauge needle 10 times. The lysates were centrifuged at 15,000!g for
T. Yamamoto et al. / Journal of Hepatology 42 (2005) 707–718 30 min at 4 8C. The resulting supernatant was considered the Triton X-100soluble fraction. The pellet was solubilized in Triton X-100 buffer containing 1% SDS using an ultrasonic disintegrator, cleared by centrifugation at 15,000!g for 5 min at 4 8C, and referred to as the Triton X-100-insoluble fraction.
2.8. Northern blot analysis Total RNA was extracted from the cells using TRIzol reagent (Gibco BRL). Northern blots were performed as described previously [32]. For detection of Cx32, claudin-1, occludin and JAM mRNAs, the membranes were hybridized overnight at 42 8C in the same solution with 32P-labeled cDNA probes for Cx32, claudin-1, occludin and JAM, which were amplified from rat liver first-strand DNA by PCR using primers corresponding to the previously reported sequences [33,34]. The membranes were washed twice in 2!SSC buffer containing 0.1% SDS for 5 min at RT and twice in 2!SSC buffer containing 1% SDS for 30 min at 68 8C before exposure to film.
2.9. Measurement of GJIC
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MAP-kinase inhibitor SB203580 could inhibit the activation of p38 MAP-kinase in rat livers during PH, we performed Western blotting of phospho-p38 MAP-kinase (pp38) of rat livers after PH that were treated with or without the inhibitor, compared to those of phospho-JNK (pJNK), phospho-MAP-kinase (pMAPK), and phospho-Akt (pAkt). Expression of pp38 and pAkt were gradient increased from 24 to 72 h after PH (Fig. 1A and B). Expression of pJNK and pMAPK were transiently increased at 24 h after PH (Fig. 1A and B). Treatment with SB203580 markedly inhibited the increase of pp38, but not pAkt at 24 and 48 h after PH (Fig. 1A and B). Treatment with SB203580 slightly inhibited the increase of pJNK and pMAPK at 24 h after PH (Fig. 1A and B). 3.2. Effect of SB203580 on hepatocyte proliferation during PH
For measuring GJIC, we used the scrape loading/dye transfer method [35] with some modification [32]. The hepatocytes on 35-mm dishes were rinsed several times with PBS. Two or three lines were made around the center of the dish using a scalpel and 2 ml of 0.05% lucifer yellow CH (LY; Sigma) in PBS was added. Three minutes after the dye treatment, the cells were rinsed several times with PBS to remove excess dye. We used rhodamine dextran (10 kDa; Sigma), which is known not to move through gap junctions, as a control. We immediately observed the intensity of the dye transfer with an Olympus inverse microscope equipped with appropriate filters (Olympus, Tokyo, Japan) and photographed it.
To examine the effect of SB203580 on hepatocyte proliferation after PH, we performed Western blotting of PCNA (Fig. 1) and counting of the labeling index for Ki-67 (Fig. 2) in rat livers after PH pretreated with or without SB203580. Increases of PCNA expression and the Ki-67 labeling index were observed after PH, but treatment with SB203580 did not affect the hepatocyte proliferation (Figs. 1A, B and 2).
2.10. Fence function assay (diffusion of Bodipy-sphingomyelin)
3.3. Effect of SB203580 on localization of Cx32 and claudin-1 in hepatocytes during PH
This was performed according to Balda et al. [36]. Spingomyelin/ bovine serum albumin (BSA) complex (5 nM/ml) was prepared in P buffer (10 nM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl, and 145 mM NaCl) using Bodipy-FL-sphyngomyelin (Molecular Probes) and defatted BSA. Cells plated on glass-bottomed microwell plates (Mat Tek Corp., Ashland, MA) were loaded with Bodipy-sphyngomyelin/ BSA complex for 1 min on ice, after which they were rinsed with cold DMEM and mounted in DMEM on a glass slide. The samples were analyzed by confocal laser scanning microscopy. All pictures shown were generated within the first 5 min of analysis.
In frozen sections of rat livers, Cx32-positive spots disappeared at cell borders of hepatocytes at 24 and 48 h after PH, while many Cx32-positive spots were observed in most all hepatocytes after sham operation (Fig. 3A). Treatment with SB203580 significantly inhibited reduction of Cx32-positive spots at 24 and 48 h after PH (Fig. 3A). In deparaffinized sections of rat liver, claudin-1 was strongly localized at periportal areas of liver lobules after sham operation (Fig. 3B). At 48 h after PH, claudin-1 was localized from periportal areas to middle areas of liver lobules, and at 48 h after PH of livers treated with SB203580, claudin-1 was diffusely localized from periportal areas to central areas of liver lobules (Fig. 3B). Dilated bile canaliculi in hepatocytes, where were indicated by claudin-1 staining, were observed at 48 h after PH in the livers treated with and without SB203580 (Fig. 3C).
2.11. Statistical analysis Signals were quantified by the Scion-Image Densimetric analysis program (Scion Corporation, Frederick, MA). Each set of results shown is as meanGSEM. Differences between groups were tested by the two-tailed Student’s t-test for unpaired data. Data were expressed as the mean SD of experiments with at least three independent treatments per group.
3. Results 3.1. Effect of SB203580 on expression of phospho-p38 MAPK during PH Partial hepatectomy can activate MAP-kinase, p38 MAP-kinase and JNK in the rat liver and the activation of p38 MAP-kinase has been suggested to play an important role in liver regeneration [25]. To confirm whether the p38
3.4. Effects of SB203580 on formation of gap junction plaques and tight junction strands during PH In the freeze-fracture replicas of rat livers after sham operation, typical large gap junction plaques at the basolateral membrane and well-developed networks formed by continuous lined tight junction strands at the subapical membrane near the bile canalicular microvilli
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Fig. 1. (A) Western blots for phospho-p38 MAPK (pp38), phospho-JNK, phospho-p44/p42 MAPK (pMAPK), phospho-Akt (pAkt) and PCNA after partial hepatectomy (PH) of rat livers treated with and without SB203580 (SB). (B) The corresponding expression levels of pp38/p38, pJNK/JNK (signal of 54 kDa), pMAPK/MAPK (signal of 44 kDa), pAkt/Akt and PCNA proteins are shown as the bar graphs. Treatment with SB203580 markedly inhibited the increase of pp38 at 24 h and 48 h after PH.
were observed (Fig. 4A and B). In untreated rat livers after PH, gap junction plaques were broken up into smaller aggregates and the tight junction strand counts, density and orientation parallel to bile canaliculi were all reduced and the strands were changed from lines to dots (Fig. 4C and D). After PH of rat livers treated with SB203580, large gap junction plaques and continuous tight junction strands were observed as after sham operation (Fig. 4E and F).
3.5. Effect of SB203580 on protein expression of Cx32, claudin-1, -2, -3, occludin, JAM-1, ZO-1, and E-cadherin during PH In Western blots of whole cell lysates at 48 h after PH, expression of Cx32 protein was decreased and expression of claudin-1 and E-cadherin proteins was increased compared to sham operation, whereas no changes of claudin-2, -3, occludin, JAM-1 and ZO-1 proteins were observed (Fig. 5).
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Fig. 2. Immunohistochemistry for Ki-67 after PH of rat livers treated with and without SB203580. Bar 40 mm. The corresponding the numbers of Ki67-positive nuclei are shown as the bar graph in A. In SB203580 treated rat livers, the number of Ki-67-positive nuclei was similar to that after sham operation.
Treatment with SB203580 significantly inhibited the reduction of Cx32 protein and enhanced the increase of claudin-1 protein (Fig. 5). In Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer, Cx32 expression in the soluble and the insoluble fractions was decreased and claudin-1 expression in the insoluble fraction was increased (Fig. 6). Treatment with SB203580 significantly inhibited the reduction of Cx32 in both fractions and enhanced the increase of claudin-1 in the insoluble fraction (Fig. 6). 3.6. Effect of SB203580 on mRNA expression of Cx32, claudin-1, -2, occludin, and JAM-1 during PH In Northern blots at 48 h after PH, Cx32 mRNA was decreased and claudin-1, -2, occludin and JAM-1 mRNAs were increased compared to sham-operation levels (Fig. 7). Treatment with SB203580 significantly inhibited reduction of Cx32 mRNA at 48 h after PH (Fig. 7). 3.7. Effect of SB203580 on expression, function and localization of Cx32 and claudin-1 in EGF-induced proliferative rat hepatocytes In the primary cultured rat hepatocytes at day 10 after plating, Cx32 and claudin-1 were observed at cell borders and the cells had extensive gap junctional intercellular
communication (GJIC) as wide dye-spreading and a wellmaintained fence function as BODIPY-sphingomyelin retained in the apical domain (Fig. 8A). In EGF-induced proliferative rat hepatocytes, Cx32 and claudin-1 disappeared at cell borders (Fig. 8A). GJIC decreased and BODIPY-sphingomyelin diffused and appeared to penetrate the cells (Fig. 8A). After treatment with SB203580 for 4 h, Cx32 and claudin-1 reappeared at cell borders and both functions were recovered (Fig. 8A). In Western blots of whole cell lysates after SB203580 treatment, phospho-p38 MAP-kinase was decreased from 1 h and expression of Cx32 and claudin-1 proteins was increased from 4 h (Fig. 8B). In Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer, expression of Cx32 and claudin-1 in the insoluble fraction was increased from 1 h after SB203580 treatment (Fig. 8C). In Northern blots, mRNA of Cx32, but not claudin-1 was increased from 1 h after SB203580 treatment (Fig. 8D). On the other hand, in primary rat hepatocytes using this culture system, lines of Cx32-immunoreactivity were co-localized with claudin-1-immunoreactivity at apical regions of cell borders, whereas Cx32-positive spots were observed at basolateral regions (Fig. 9A). In EGF-induced proliferative rat hepatocytes treated with SB203580 for 4 h, re-expression of lines of Cx32-immunoreactivity were colocalized with claudin-1-immunoreactivity at apical regions of some cells (Fig. 9B).
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Fig. 3. Immunohistochemistry for Cx32 (A) and claudin-1 (B, C) after partial hepatectomy (PH) of rat livers treated with and without SB203580 (SB). P, periportal area; C, central area. Bars 40 mm (A), 60 mm (B), 10 mm (C). The corresponding numbers of Cx32-positive spots are shown in the bar graph in A. (A) Treatment with SB203580 significantly inhibited reduction of Cx32-positive spots at 24 and 48 h after PH. (B) At 48 h after PH, claudin-1 was localized from periportal areas to middle areas of liver lobules, and at 48 h after PH of livers treated with SB203580, claudin-1 was diffusely localized from periportal areas to central areas of liver lobules. (C) Dilated bile canaliculi in hepatocytes are observed at 48 h after PH in livers with or without SB203580 treatment.
4. Discussion Recent studies indicate that gap and tight junctions may be regulated in some tissues in vivo via a p38 MAP-kinase signaling pathway. In myocardium during ischemia/reperfusion condition, p38 MAP-kinase in part modulates gap junction-mediated communication [37]. TGF-b3 regulates
the blood-testis barrier (BTB) in vivo and in vitro via the p38 MAP-kinase pathway [38]. In the present study, dynamic changes of formation, expression and function of gap and tight junctions during regeneration of rat hepatocytes in vivo may be in part controlled via a p38 MAP-kinase signaling pathway, and are independent of cell growth.
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hepatocytes, SB203580 treatment enhanced expression of Cx32 protein and the mRNA and the GJIC without a change of cell growth. These results suggested that during regeneration of hepatocytes in vivo and in vitro, the formation, expression and function of gap junctions are regulated at the transcriptional level via a p38 MAP-kinase signaling pathway, and also suggest down-regulation in formation and expression of hepatocyte gap junctions is in part independent of hepatocyte proliferation after PH. 4.2. p38 MAP-kinase modulates formation, expression and function of tight junction during regeneration of hepatocytes in vivo and in vitro
Fig. 4. Freeze fracture replicas at 48 h after partial hepatectomy (PH) of rat livers treated with and without SB203580 (SB). In rat livers after sham operation (A, B) and PH of the liver treated with SB203580 (E, F), typical large gap junction plaques and continuous lined tight junction strands are observed. In rat liver after PH (C, D), gap junction plaques broke up into smaller aggregates and tight junction strands changed from lines to dots. Bars 50 nm (E), 100 nm (F).
4.1. p38 MAP-kinase modulates formation, expression and function of gap junctions during regeneration of hepatocytes in vivo and in vitro During liver regeneration, dynamic changes in formation and expression of gap junctions in most hepatocytes of all liver lobules are well known [18,19]. Furthermore, gap junction proteins Cx32 and Cx26 of cultured hepatocytes are down-regulated in the S-phase of the cell cycle [19,32]. The extent of synchronous initiation and termination of DNA synthesis in regenerating liver are altered in Cx32-deficient mice [39]. In Cx32 dominantnegative mice, hepatocyte proliferation during liver regeneration after PH is retarded [40]. In the present study, treatment with a p38 MAP-kinase inhibitor, SB203580, inhibited down-regulation of Cx32 protein and the mRNA and the change into smaller aggregates of gap junction plaques during liver regeneration after PH, whereas the inhibitor did not affect hepatocyte proliferation after PH. Furthermore, in EGF-induced proliferative rat
It is thought that hepatocyte tight junctional permeability is increased by architectural remodeling in the liver in which loosening of tight junction strands has been indicated during liver regeneration in vivo [20]. Although a recent study reported that protein expression of tight junction components such as claudin-3, ZO-1 and ASIP was increased during liver regeneration after PH [21], the changes of the tight junction components are not fully clarified. In the present study, we also examined changes of hepatocyte tight junction components such as occludin, JAM-1, ZO-1, and claudin-1, -2, -3 during liver regeneration after PH of livers treated with or without the p38 MAP-kinase inhibitor SB203580. In Western blots and in immunostaining of rat livers after PH, protein expression and localization of claudin-1, but not occludin, JAM-1 and claudin-2, -3, were increased, and in rat livers after PH of rat livers treated with SB203580, the increase of claudin-1 protein was enhanced. In Northern blots after PH of rat livers treated with and without SB203580, mRNA expression of claudin-1, -2, occludin and JAM-1 was increased to the same level as after sham operation. Furthermore, in EGF-induced proliferative rat hepatocytes, SB203580 treatment enhanced expression of claudin-1 protein and the fence function, but not the mRNA without affecting of cell growth. These results suggested that during regeneration of hepatocytes in vivo and in vitro, the formation, expression and function of the tight junction component claudin-1 are regulated at the posttranscriptional level via a p38 MAP-kinase signaling pathway. More interestingly, regardless of the increase of tight junction component proteins, including claudin-1 protein, which formed hepatocyte tight junction strands after PH, the tight junctional strand counts, density and orientation parallel to canaliculi were reduced (Fig. 3B and 4C). Furthermore, in EGF induced rat hepatocytes, expression of claudin-1 and formation of tight junction strands were down-regulated (Fig. 8; [32]). Reciprocal changes in expression of claudin-1 during regeneration of hepatocytes in vivo and in vitro were observed. Accordingly, it is thought that expression of tight junction proteins is regulated by other factors as well as p38 MAP-kinase during liver regeneration in vivo.
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Fig. 5. Western blots of whole cell lysates for Cx32, claudin-1, -2, -3 (CL-1, -2, -3), occludin (Oc), JAM-1, ZO-1, E-cadherin and PCNA at 48 h after partial hepatectomy (PH) of livers with and without SB203580 (SB) treatment. (A) Treatment with SB203580 significantly inhibited the reduction of Cx32 protein and enhanced the increase of claudin-1 protein. (B) The corresponding expression levels of Cx32, CL-1, -2, -3, Oc, JAM-1, ZO-1, Ecadherin and PCNA proteins are shown as bar graphs.
4.3. The interaction between gap and tight junctions and the p38 MAP-kinase signaling pathway during regeneration of hepatocytes In a recent study, gap junctions were found to form along the tight junctions by immunogold-electron microscopy using SDS-digested freeze-fracture replicas of regenerating mouse hepatocytes [41]. On the other hand,
in the Cx32-deficient mouse liver, dilation of bile canaliculi that were sealed by tight junctions was observed [42]. In primary cultures of rat hepatocytes, Cx32, but not Cx26, might be closely coordinated with the expression of tight junction proteins (Fig. 9A; [43]). Furthermore, Cx32 formation and/or Cx32-mediated intercellular communication induces expression and function of tight junctions in two mouse hepatocytic cell lines [34,44]. In the present
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Furthermore, there is progress towards clinical development of p38 MAP-kinase inhibitors for the treatment of inflammatory diseases is beginning [46]. In the rat liver, the p38 MAP-kinase plays a major role in hepatocyte proliferation during its normal development [24]. Inhibition of hepatocyte proliferation by chronic ethanol administration is reported to be due to inhibition of p38 MAP-kinase activation [47]. Inhibiting the activation of p38 MAPK is also reported to attenuate warm ischemia-reperfusion injury of the liver [48]. The hepatitis C virus core inhibits the Fasmediated p38 MAP-kinase signaling pathway, which results
Fig. 6. Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer for Cx32, claudin-1, -2, -3 (CL-1, -2, -3), occludin (Oc), JAM-1, ZO-1, and E-cadherin at 48 h after partial hepatectomy (PH) in rat livers treated with and without SB203580 (SB). (A) Treatment with SB203580 significantly inhibited the reduction of Cx32 in soluble and insoluble fractions and enhanced the increase of claudin-1 in the insoluble fraction. (B) The corresponding expression levels of Cx32, CL-1, -2, -3, Oc, JAM1, ZO-1, and E-cadherin proteins are shown as bar graphs.
study, re-expression of lined Cx32-immunoreactivity was co-localized with claudin-1-immunoreactivity at apical regions of some EGF-induced proliferative rat hepatocytes treated with SB203580 (Fig. 9B). This suggests that during regeneration of hepatocytes, Cx32 is also coordinated with claudin-1, which can form tight junction strands via a p38 MAP-kinase signal pathway. Although gap and tight junctions perform very different functions, we show herein that there is common regulation by a p38 MAP-kinase signaling pathway during liver regeneration.
4.4. Potential of p38 MAP-kinase inhibitors in the treatment of regenerating hepatocytes after liver injury p38 MAP-kinase inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury [45].
Fig. 7. Northern blots for Cx32, claudin-1, -2 (CL-1, -2), occludin (Oc) and JAM-1 at 48 h after partial hepatectomy (PH) in rat livers treated with and without SB203580 (SB). (A) Cx32 mRNA was decreased and claudin-1, -2, occludin and JAM-1 mRNAs were increased compared to sham operation levels. Treatment with SB203580 inhibited reduction of Cx32 mRNA. (B) The corresponding expression levels of Cx32, CL-1, -2, Oc, and JAM-1 mRNAs are shown as bar graphs.
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Fig. 8. Immunocytochemistry (A) for Cx32 and claudin-1, functions of gap and tight junctions, GJIC and fence (A), Western blot (B) for phospho-p38 MAP-kinase (pp38), Cx32 and claudin-1 (CL-1) and Northern blot (C) for Cx32 and claudin-1 (CL-1) in EGF-induced proliferative rat hepatocytes treated with SB203580 (SB). Bars 10 mm. (A) In treatment with SB203580 for 4 h, Cx32 and claudin-1 reappeared at cell borders, and GJIC and fence function were recovered. (B) In Western blots of whole cell lysates after SB203580 treatment, phospho-p38 MAP-kinase was decreased from 1 h and expression of Cx32 and claudin-1 proteins was increased from 4 h. (C) In Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer, expression of Cx32 and claudin-1 in the insoluble fraction was increased from 1 h after SB203580 treatment. (D) In Northern blots, mRNA of Cx32, but not claudin-1 was increased from 1 h after SB203580 treatment.
in accelerated Fas-mediated cell death in hepatocytes [27]. In the present study, during liver regeneration after PH in vivo, the p38 MAPK inhibitor SB203580 could inhibit the marked changes in formation and expression of gap and tight junctions. During hepatocyte regeneration after liver injury, maintenance of the functions of hepatic gap and tight junctions may be very important for preventing jaundice as well as hepatic damage, because the interaction between gap
and tight junctions of hepatocytes may regulate the ‘blood– biliary barrier’ [3,4,5]. Further studies are needed to examine changes of gap and tight junctions via various signaling pathways during liver regeneration in vivo. Thus, it is possible that the p38 MAP-kinase inhibitors could be used as therapeutic drugs for regenerating hepatocytes after chronic or severe liver injury, interms of prevention of jaundice.
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Fig. 9. Double immunocytochemistry for Cx32 and claudin-1 in EGF-induced proliferative rat hepatocytes treated with SB203580. (A) In primary cultures of rat hepatocytes at day 10 after plating, lines of Cx32-immunoreactivity were co-localized with claudin-1-immunoreactivity at apical regions of cell borders, whereas Cx32-positive spots were observed at basolateral regions. (B) In EGF-induced proliferative rat hepatocytes treated with SB203580 for 4 h, re-expression of lines of Cx32-immunoreactivity co-localized with claudin-1-immunoreactivity at apical regions of some cells. Bars 10 mm. [This figure appears in colour on the web.]
Acknowledgements We are grateful to Dr T. Kita (Kyoto University) for the JAM-1 antibody. We thank Ms E. Suzuki (Sapporo Medical University) for technical support. This work was supported in by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science, and the Ministry of Health, Labour and Welfare of Japan and by the Smoking Research Foundation, and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.
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p38 MAP-kinase regulates function of gap and tight junctions during regeneration of rat hepatocytes Toshinobu Yamamoto, Takashi Kojima*, Masaki Murata, Ken-ichi Takano, Mitsuru Go, Naoko Hatakeyama, Hideki Chiba, Norimasa Sawada Department of Pathology, Sapporo Medical University School of Medicine, S1. W17, Sapporo 060-8556, Japan
Background/Aims: Hepatocyte regeneration is considered to be associated with adaptive changes in expression of gap and tight junctions through multiple signal transduction pathways including p38 MAP-kinase. The role of the stress responsitive MAP-kinase, p38 MAP-kinase, signaling pathway in function of gap and tight junctions was examined during regeneration of rat hepatocytes in vivo and in vitro. Methods: We examined changes in formation, expression and function of gap and tight junctions in rat livers after 70% partial hepatectomy and in primary cultures of rat hepatocytes, by using a p38 MAP-kinase inhibitor, SB203580. Results: When p38 MAP-kinase was activated during partial hepatectomy, down-regulation of Cx32 and up-regulation of claudin-1 were observed. By SB203580 treatment, the down-regulation of Cx32 was inhibited and the up-regulation of claudin-1 was enhanced, well maintaining the structures of gap and tight junctions. SB203580 treatment did not affect the increase of hepatocyte proliferation. In EGF induced proliferative rat hepatocytes treated with SB203580, the expression and function of Cx32 and claudin-1 were increased. Conclusions: Dynamic changes of formation of gap and tight junctions during regeneration of rat hepatocytes in vivo and in vitro are in part controlled via a p38 MAP-kinase signaling pathway, and are independent of cell growth. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: p38 MAPK; Gap junctions; Tight junctions; Liver; Regeneration 1. Introduction Hepatic gap and tight junctions play crucial roles in bile secretion and in the barrier to keep bile in bile canaliculi away from the blood circulation [1–3]. Furthermore, we have hypothesized that the close interaction between gap and tight junctions of hepatocytes also regulates the blood– biliary barrier [4,5]. Gap junction channels, composed of proteins termed connexins, mediate reciprocal exchange of ions and small molecules of less than 1000 Da, including second messengers such as cyclic AMP, IP3 and Ca2C, between adjacent cells [4,6–8]. Gap junctional intercellular communication (GJIC) is thought to play a crucial role in
Received 6 May 2004; received in revised form 2 December 2004; accepted 14 December 2004; available online 16 March 2005 * Corresponding author. Tel.: C81 11 611 2111x2703; fax: C81 11 613 5665. E-mail address: [email protected] (T. Kojima).
development, cell growth and cell differentiation [9–13]. Tight junctions, the apicalmost component of intercellular junctional complexes, separate the apical from the basolateral cell surface domains to establish cell polarity (performing the function of a fence); tight junctions also provide a barrier function, inhibiting solute and water flow through the paracellular space [14,15]. They show a particular net-like meshwork of fibrils formed by the integral membrane proteins, occludin, the claudin family, and JAM [4,15–17]. Several peripheral membrane proteins, ZO-1, -2, -3, 7H6 antigen, cingulin, symplekin, Rab3B, Ras target AF-6, and ASIP, an atypical protein kinase C-interacting protein, were reported [4,15–17]. Communication via gap junctions may be needed for extensive initiation of synchronous DNA synthesis in hepatocytes during liver regeneration. It is well known that the structures and expression of gap junctions in most hepatocytes of all liver lobules disappear in the early stage after PH [18,19]. The construction of the hepatocyte tight
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.12.033
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junction is one of the most important events during regeneration of the liver leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division [20]. In a recent study, protein expression of tight junction components such as claudin-3, ZO-1 and ASIP was found to increase after PH [21]. We previously found that the PI3-kinase pathway rather than the MAPkinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of gap junction protein Cx32, but not tight junction protein claudin-1 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP-kinase [22]. However, the detail mechanisms, including other signal transduction pathways, for regulation of gap and tight junctions during liver regeneration are as yet unclear. Signal transduction pathways, including MAP-kinase, p38 MAP-kinase and PI3-kinase are involved in growth, cell survival and cell death. p38 MAP-kinase is a stressresponsive MAP-kinase and is phosphorylated and activated by proinflammatory cytokines and environmental stress [23,24]. Liver regeneration is also controlled by multiple signaling pathways and partial hepatectomy can activate MAP-kinase, p38 MAP-kinase and c-Jun NH2-terminal kinase (JNK) in the rat liver [25]. Recent evidence indicates that p38 MAP-kinase plays a major role in hepatocyte proliferation, whereas chronic activation of MAP-kinase has been shown to lead to cellular growth arrest [25]. Furthermore, it is thought that the p38 MAP-kinase signaling pathway might be important for hepatic damage after liver injury such as ischemia/reperfusion, hepatitis, and alcoholic liver disease [26–29]. Therefore, we focused on the role of the p38 MAP-kinase signaling pathway for formation, expression and function of gap and tight junctions during regeneration of rat hepatocytes in vivo and in vitro, using the p38 MAP-kinase inhibitor SB203580.
2. Materials and methods 2.1. Animal model Male Fisher 344 rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) weighing about 170–200 g were used for all experiments of animal models. For them, 70% partial hepatectomy (PH) was performed according to the previously described method [30]. Sham operation consisted of laparotomy and exposure of the liver. At 30 min before PH surgery, some rats were injected intraperitoneally with the p38 MAP-kinase inhibitor, SB203580 (Calbiochem-Novabiochem Corp., San Diego, CA), dissolved in 0.9% NaCl at a dose of 20 mg/kg body weight.
2.2. Rat hepatocyte isolation and cell culture Male Sprague–Dawley rats (Shizuoka Laboratory Animal Center, Hamamatsu, Japan) weighing about 250–300 g were used to isolate hepatocytes by the two-step liver perfusion method of Seglen [31] with some modification [32]. Hepatocytes used in all experiments were maintained in the modified L-15 medium until day 10 after plating. For the experiment on EGF-induced proliferative rat hepatocytes, some cells at
day 10 after plating were cultured with modified DMEM medium (0.2% BSA, 20 mM HEPES, 0.5 mg/l insulin, 10K7 M dexamethasone, 1 g/l galactose, 30 mg/l proline, 20 mM NaHCO3) supplemented with 10 ng/ml EGF and 10 mM nicotinamide for 72 h [32], and then they were treated with 20 mg/ml SB203580 for 1, 4 and 10 h.
2.3. Immunostaining Frozen sections made with a cryostat and cells grown on glass coverslips were fixed with cold absolute acetone for 10 min. Immunostaining with monoclonal anti-Cx32 (Zymed) and polyclonal anti-claudin-1 (Zymed) antibodies was performed. These specimens were visualized using Alexia 488- or 594-conjugated anti-mouse or anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR). The specimens were examined using a laserscanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA). The number of Cx32-positive spots in liver sections was counted using a Scionimage analysis system. Immunostaining for claudin-1 was also performed in deparaffinized liver sections.
2.4. Ki-67 labeling index To examine the Ki-67 labeling index, immunostaining for Ki-67 (DAKO A/S, Glostrup, Denmark) was performed in deparaffinized 3 mm sections of liver. Labeled cells that had nuclei stained for Ki-67 were counted using a microscope (magnification !200). More than 1000 cells were counted per section using a Scion-image analysis system.
2.5. Freeze-fracture analysis For the freeze-fracture technique, the livers were immersed in 40% glycerin solution after fixation in 2.5% glutaraldehyde/0.1 M phosphatebuffered saline (PBS). The specimens were fractured at K160 8C in a JFD7000 freeze-fracture device (JEOL Ltd, Tokyo, Japan) and replicated by deposition of platinum/carbon from an electron beam gun positioned at a 458 angle followed by carbon applied from overhead. Replicas were examined at 100 kV with a JEM transmission electron microscope (JEOL Ltd, Tokyo, Japan).
2.6. Western blot analysis The livers were minced with a scalpel blade, resuspended in buffer (1 mM NaHCO3 and 2 mM phenylmethyl sulfonyl fluoride (PMSF)) and sonicated for 10 s. The dishes were washed with PBS twice, and 300 ml of the buffer (1 mM NaHCO3 and 2 mM PMSF) was added to 60-mm dishes. The cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 s. Western blots were performed as described previously [32]. The membranes were incubated with polyclonal anti-phospho-p38 (Cell Signaling, Beverly, MA), polyclonal anti-p38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal anti-phospho-JNK (Thr183/ Tyr185; Cell Signaling, Beverly, MA), polyclonal anti-SAPK/JNK (Cell Signaling), polyclonal anti-phospho-p44/p42 MAPK (Thr202/Tyr204; Cell Signaling), polyclonal anti-MAPK (ERK1/2; Promega Corporation, Madison, WI), monoclonal anti-phospho-Akt (Ser473; Cell Signaling), polyclonal anti-Akt (Cell Signaling), monoclonal anti-Cx32 (Zymed Laboratories, Inc., San Francisco, CA), polyclonal anti-Cx26 (Zymed), polyclonal anti-occludin (Zymed), polyclonal anti-claudin-1, -2, -3 (Zymed), polyclonal anti-ZO-1 (Zymed), polyclonal anti-JAM-1 (33; Zymed), monoclonal anti-E-cadherin (Sigma), monoclonal anti-PCNA (Amersham Corp., Buckinghamshire, UK) and polyconal anti-actin (Sigma) antibodies at room temperature (RT) for 1 h.
2.7. Preparation of Triton X-100-soluble and -insoluble protein fractions To isolate Triton X-100-soluble and -insoluble protein fractions, the protein samples were lysed in Triton X-100 buffer (1% Triton X-100, 100 mM NaCl, 10 mM HEPES, pH 7.6, 2 mM EDTA, 1 mM PMSF, 4 mM sodium orthovanadate, 40 mM sodium fluoride), and then passed through a 21-gauge needle 10 times. The lysates were centrifuged at 15,000!g for
T. Yamamoto et al. / Journal of Hepatology 42 (2005) 707–718 30 min at 4 8C. The resulting supernatant was considered the Triton X-100soluble fraction. The pellet was solubilized in Triton X-100 buffer containing 1% SDS using an ultrasonic disintegrator, cleared by centrifugation at 15,000!g for 5 min at 4 8C, and referred to as the Triton X-100-insoluble fraction.
2.8. Northern blot analysis Total RNA was extracted from the cells using TRIzol reagent (Gibco BRL). Northern blots were performed as described previously [32]. For detection of Cx32, claudin-1, occludin and JAM mRNAs, the membranes were hybridized overnight at 42 8C in the same solution with 32P-labeled cDNA probes for Cx32, claudin-1, occludin and JAM, which were amplified from rat liver first-strand DNA by PCR using primers corresponding to the previously reported sequences [33,34]. The membranes were washed twice in 2!SSC buffer containing 0.1% SDS for 5 min at RT and twice in 2!SSC buffer containing 1% SDS for 30 min at 68 8C before exposure to film.
2.9. Measurement of GJIC
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MAP-kinase inhibitor SB203580 could inhibit the activation of p38 MAP-kinase in rat livers during PH, we performed Western blotting of phospho-p38 MAP-kinase (pp38) of rat livers after PH that were treated with or without the inhibitor, compared to those of phospho-JNK (pJNK), phospho-MAP-kinase (pMAPK), and phospho-Akt (pAkt). Expression of pp38 and pAkt were gradient increased from 24 to 72 h after PH (Fig. 1A and B). Expression of pJNK and pMAPK were transiently increased at 24 h after PH (Fig. 1A and B). Treatment with SB203580 markedly inhibited the increase of pp38, but not pAkt at 24 and 48 h after PH (Fig. 1A and B). Treatment with SB203580 slightly inhibited the increase of pJNK and pMAPK at 24 h after PH (Fig. 1A and B). 3.2. Effect of SB203580 on hepatocyte proliferation during PH
For measuring GJIC, we used the scrape loading/dye transfer method [35] with some modification [32]. The hepatocytes on 35-mm dishes were rinsed several times with PBS. Two or three lines were made around the center of the dish using a scalpel and 2 ml of 0.05% lucifer yellow CH (LY; Sigma) in PBS was added. Three minutes after the dye treatment, the cells were rinsed several times with PBS to remove excess dye. We used rhodamine dextran (10 kDa; Sigma), which is known not to move through gap junctions, as a control. We immediately observed the intensity of the dye transfer with an Olympus inverse microscope equipped with appropriate filters (Olympus, Tokyo, Japan) and photographed it.
To examine the effect of SB203580 on hepatocyte proliferation after PH, we performed Western blotting of PCNA (Fig. 1) and counting of the labeling index for Ki-67 (Fig. 2) in rat livers after PH pretreated with or without SB203580. Increases of PCNA expression and the Ki-67 labeling index were observed after PH, but treatment with SB203580 did not affect the hepatocyte proliferation (Figs. 1A, B and 2).
2.10. Fence function assay (diffusion of Bodipy-sphingomyelin)
3.3. Effect of SB203580 on localization of Cx32 and claudin-1 in hepatocytes during PH
This was performed according to Balda et al. [36]. Spingomyelin/ bovine serum albumin (BSA) complex (5 nM/ml) was prepared in P buffer (10 nM HEPES, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl, and 145 mM NaCl) using Bodipy-FL-sphyngomyelin (Molecular Probes) and defatted BSA. Cells plated on glass-bottomed microwell plates (Mat Tek Corp., Ashland, MA) were loaded with Bodipy-sphyngomyelin/ BSA complex for 1 min on ice, after which they were rinsed with cold DMEM and mounted in DMEM on a glass slide. The samples were analyzed by confocal laser scanning microscopy. All pictures shown were generated within the first 5 min of analysis.
In frozen sections of rat livers, Cx32-positive spots disappeared at cell borders of hepatocytes at 24 and 48 h after PH, while many Cx32-positive spots were observed in most all hepatocytes after sham operation (Fig. 3A). Treatment with SB203580 significantly inhibited reduction of Cx32-positive spots at 24 and 48 h after PH (Fig. 3A). In deparaffinized sections of rat liver, claudin-1 was strongly localized at periportal areas of liver lobules after sham operation (Fig. 3B). At 48 h after PH, claudin-1 was localized from periportal areas to middle areas of liver lobules, and at 48 h after PH of livers treated with SB203580, claudin-1 was diffusely localized from periportal areas to central areas of liver lobules (Fig. 3B). Dilated bile canaliculi in hepatocytes, where were indicated by claudin-1 staining, were observed at 48 h after PH in the livers treated with and without SB203580 (Fig. 3C).
2.11. Statistical analysis Signals were quantified by the Scion-Image Densimetric analysis program (Scion Corporation, Frederick, MA). Each set of results shown is as meanGSEM. Differences between groups were tested by the two-tailed Student’s t-test for unpaired data. Data were expressed as the mean SD of experiments with at least three independent treatments per group.
3. Results 3.1. Effect of SB203580 on expression of phospho-p38 MAPK during PH Partial hepatectomy can activate MAP-kinase, p38 MAP-kinase and JNK in the rat liver and the activation of p38 MAP-kinase has been suggested to play an important role in liver regeneration [25]. To confirm whether the p38
3.4. Effects of SB203580 on formation of gap junction plaques and tight junction strands during PH In the freeze-fracture replicas of rat livers after sham operation, typical large gap junction plaques at the basolateral membrane and well-developed networks formed by continuous lined tight junction strands at the subapical membrane near the bile canalicular microvilli
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Fig. 1. (A) Western blots for phospho-p38 MAPK (pp38), phospho-JNK, phospho-p44/p42 MAPK (pMAPK), phospho-Akt (pAkt) and PCNA after partial hepatectomy (PH) of rat livers treated with and without SB203580 (SB). (B) The corresponding expression levels of pp38/p38, pJNK/JNK (signal of 54 kDa), pMAPK/MAPK (signal of 44 kDa), pAkt/Akt and PCNA proteins are shown as the bar graphs. Treatment with SB203580 markedly inhibited the increase of pp38 at 24 h and 48 h after PH.
were observed (Fig. 4A and B). In untreated rat livers after PH, gap junction plaques were broken up into smaller aggregates and the tight junction strand counts, density and orientation parallel to bile canaliculi were all reduced and the strands were changed from lines to dots (Fig. 4C and D). After PH of rat livers treated with SB203580, large gap junction plaques and continuous tight junction strands were observed as after sham operation (Fig. 4E and F).
3.5. Effect of SB203580 on protein expression of Cx32, claudin-1, -2, -3, occludin, JAM-1, ZO-1, and E-cadherin during PH In Western blots of whole cell lysates at 48 h after PH, expression of Cx32 protein was decreased and expression of claudin-1 and E-cadherin proteins was increased compared to sham operation, whereas no changes of claudin-2, -3, occludin, JAM-1 and ZO-1 proteins were observed (Fig. 5).
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Fig. 2. Immunohistochemistry for Ki-67 after PH of rat livers treated with and without SB203580. Bar 40 mm. The corresponding the numbers of Ki67-positive nuclei are shown as the bar graph in A. In SB203580 treated rat livers, the number of Ki-67-positive nuclei was similar to that after sham operation.
Treatment with SB203580 significantly inhibited the reduction of Cx32 protein and enhanced the increase of claudin-1 protein (Fig. 5). In Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer, Cx32 expression in the soluble and the insoluble fractions was decreased and claudin-1 expression in the insoluble fraction was increased (Fig. 6). Treatment with SB203580 significantly inhibited the reduction of Cx32 in both fractions and enhanced the increase of claudin-1 in the insoluble fraction (Fig. 6). 3.6. Effect of SB203580 on mRNA expression of Cx32, claudin-1, -2, occludin, and JAM-1 during PH In Northern blots at 48 h after PH, Cx32 mRNA was decreased and claudin-1, -2, occludin and JAM-1 mRNAs were increased compared to sham-operation levels (Fig. 7). Treatment with SB203580 significantly inhibited reduction of Cx32 mRNA at 48 h after PH (Fig. 7). 3.7. Effect of SB203580 on expression, function and localization of Cx32 and claudin-1 in EGF-induced proliferative rat hepatocytes In the primary cultured rat hepatocytes at day 10 after plating, Cx32 and claudin-1 were observed at cell borders and the cells had extensive gap junctional intercellular
communication (GJIC) as wide dye-spreading and a wellmaintained fence function as BODIPY-sphingomyelin retained in the apical domain (Fig. 8A). In EGF-induced proliferative rat hepatocytes, Cx32 and claudin-1 disappeared at cell borders (Fig. 8A). GJIC decreased and BODIPY-sphingomyelin diffused and appeared to penetrate the cells (Fig. 8A). After treatment with SB203580 for 4 h, Cx32 and claudin-1 reappeared at cell borders and both functions were recovered (Fig. 8A). In Western blots of whole cell lysates after SB203580 treatment, phospho-p38 MAP-kinase was decreased from 1 h and expression of Cx32 and claudin-1 proteins was increased from 4 h (Fig. 8B). In Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer, expression of Cx32 and claudin-1 in the insoluble fraction was increased from 1 h after SB203580 treatment (Fig. 8C). In Northern blots, mRNA of Cx32, but not claudin-1 was increased from 1 h after SB203580 treatment (Fig. 8D). On the other hand, in primary rat hepatocytes using this culture system, lines of Cx32-immunoreactivity were co-localized with claudin-1-immunoreactivity at apical regions of cell borders, whereas Cx32-positive spots were observed at basolateral regions (Fig. 9A). In EGF-induced proliferative rat hepatocytes treated with SB203580 for 4 h, re-expression of lines of Cx32-immunoreactivity were colocalized with claudin-1-immunoreactivity at apical regions of some cells (Fig. 9B).
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Fig. 3. Immunohistochemistry for Cx32 (A) and claudin-1 (B, C) after partial hepatectomy (PH) of rat livers treated with and without SB203580 (SB). P, periportal area; C, central area. Bars 40 mm (A), 60 mm (B), 10 mm (C). The corresponding numbers of Cx32-positive spots are shown in the bar graph in A. (A) Treatment with SB203580 significantly inhibited reduction of Cx32-positive spots at 24 and 48 h after PH. (B) At 48 h after PH, claudin-1 was localized from periportal areas to middle areas of liver lobules, and at 48 h after PH of livers treated with SB203580, claudin-1 was diffusely localized from periportal areas to central areas of liver lobules. (C) Dilated bile canaliculi in hepatocytes are observed at 48 h after PH in livers with or without SB203580 treatment.
4. Discussion Recent studies indicate that gap and tight junctions may be regulated in some tissues in vivo via a p38 MAP-kinase signaling pathway. In myocardium during ischemia/reperfusion condition, p38 MAP-kinase in part modulates gap junction-mediated communication [37]. TGF-b3 regulates
the blood-testis barrier (BTB) in vivo and in vitro via the p38 MAP-kinase pathway [38]. In the present study, dynamic changes of formation, expression and function of gap and tight junctions during regeneration of rat hepatocytes in vivo may be in part controlled via a p38 MAP-kinase signaling pathway, and are independent of cell growth.
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hepatocytes, SB203580 treatment enhanced expression of Cx32 protein and the mRNA and the GJIC without a change of cell growth. These results suggested that during regeneration of hepatocytes in vivo and in vitro, the formation, expression and function of gap junctions are regulated at the transcriptional level via a p38 MAP-kinase signaling pathway, and also suggest down-regulation in formation and expression of hepatocyte gap junctions is in part independent of hepatocyte proliferation after PH. 4.2. p38 MAP-kinase modulates formation, expression and function of tight junction during regeneration of hepatocytes in vivo and in vitro
Fig. 4. Freeze fracture replicas at 48 h after partial hepatectomy (PH) of rat livers treated with and without SB203580 (SB). In rat livers after sham operation (A, B) and PH of the liver treated with SB203580 (E, F), typical large gap junction plaques and continuous lined tight junction strands are observed. In rat liver after PH (C, D), gap junction plaques broke up into smaller aggregates and tight junction strands changed from lines to dots. Bars 50 nm (E), 100 nm (F).
4.1. p38 MAP-kinase modulates formation, expression and function of gap junctions during regeneration of hepatocytes in vivo and in vitro During liver regeneration, dynamic changes in formation and expression of gap junctions in most hepatocytes of all liver lobules are well known [18,19]. Furthermore, gap junction proteins Cx32 and Cx26 of cultured hepatocytes are down-regulated in the S-phase of the cell cycle [19,32]. The extent of synchronous initiation and termination of DNA synthesis in regenerating liver are altered in Cx32-deficient mice [39]. In Cx32 dominantnegative mice, hepatocyte proliferation during liver regeneration after PH is retarded [40]. In the present study, treatment with a p38 MAP-kinase inhibitor, SB203580, inhibited down-regulation of Cx32 protein and the mRNA and the change into smaller aggregates of gap junction plaques during liver regeneration after PH, whereas the inhibitor did not affect hepatocyte proliferation after PH. Furthermore, in EGF-induced proliferative rat
It is thought that hepatocyte tight junctional permeability is increased by architectural remodeling in the liver in which loosening of tight junction strands has been indicated during liver regeneration in vivo [20]. Although a recent study reported that protein expression of tight junction components such as claudin-3, ZO-1 and ASIP was increased during liver regeneration after PH [21], the changes of the tight junction components are not fully clarified. In the present study, we also examined changes of hepatocyte tight junction components such as occludin, JAM-1, ZO-1, and claudin-1, -2, -3 during liver regeneration after PH of livers treated with or without the p38 MAP-kinase inhibitor SB203580. In Western blots and in immunostaining of rat livers after PH, protein expression and localization of claudin-1, but not occludin, JAM-1 and claudin-2, -3, were increased, and in rat livers after PH of rat livers treated with SB203580, the increase of claudin-1 protein was enhanced. In Northern blots after PH of rat livers treated with and without SB203580, mRNA expression of claudin-1, -2, occludin and JAM-1 was increased to the same level as after sham operation. Furthermore, in EGF-induced proliferative rat hepatocytes, SB203580 treatment enhanced expression of claudin-1 protein and the fence function, but not the mRNA without affecting of cell growth. These results suggested that during regeneration of hepatocytes in vivo and in vitro, the formation, expression and function of the tight junction component claudin-1 are regulated at the posttranscriptional level via a p38 MAP-kinase signaling pathway. More interestingly, regardless of the increase of tight junction component proteins, including claudin-1 protein, which formed hepatocyte tight junction strands after PH, the tight junctional strand counts, density and orientation parallel to canaliculi were reduced (Fig. 3B and 4C). Furthermore, in EGF induced rat hepatocytes, expression of claudin-1 and formation of tight junction strands were down-regulated (Fig. 8; [32]). Reciprocal changes in expression of claudin-1 during regeneration of hepatocytes in vivo and in vitro were observed. Accordingly, it is thought that expression of tight junction proteins is regulated by other factors as well as p38 MAP-kinase during liver regeneration in vivo.
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Fig. 5. Western blots of whole cell lysates for Cx32, claudin-1, -2, -3 (CL-1, -2, -3), occludin (Oc), JAM-1, ZO-1, E-cadherin and PCNA at 48 h after partial hepatectomy (PH) of livers with and without SB203580 (SB) treatment. (A) Treatment with SB203580 significantly inhibited the reduction of Cx32 protein and enhanced the increase of claudin-1 protein. (B) The corresponding expression levels of Cx32, CL-1, -2, -3, Oc, JAM-1, ZO-1, Ecadherin and PCNA proteins are shown as bar graphs.
4.3. The interaction between gap and tight junctions and the p38 MAP-kinase signaling pathway during regeneration of hepatocytes In a recent study, gap junctions were found to form along the tight junctions by immunogold-electron microscopy using SDS-digested freeze-fracture replicas of regenerating mouse hepatocytes [41]. On the other hand,
in the Cx32-deficient mouse liver, dilation of bile canaliculi that were sealed by tight junctions was observed [42]. In primary cultures of rat hepatocytes, Cx32, but not Cx26, might be closely coordinated with the expression of tight junction proteins (Fig. 9A; [43]). Furthermore, Cx32 formation and/or Cx32-mediated intercellular communication induces expression and function of tight junctions in two mouse hepatocytic cell lines [34,44]. In the present
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Furthermore, there is progress towards clinical development of p38 MAP-kinase inhibitors for the treatment of inflammatory diseases is beginning [46]. In the rat liver, the p38 MAP-kinase plays a major role in hepatocyte proliferation during its normal development [24]. Inhibition of hepatocyte proliferation by chronic ethanol administration is reported to be due to inhibition of p38 MAP-kinase activation [47]. Inhibiting the activation of p38 MAPK is also reported to attenuate warm ischemia-reperfusion injury of the liver [48]. The hepatitis C virus core inhibits the Fasmediated p38 MAP-kinase signaling pathway, which results
Fig. 6. Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer for Cx32, claudin-1, -2, -3 (CL-1, -2, -3), occludin (Oc), JAM-1, ZO-1, and E-cadherin at 48 h after partial hepatectomy (PH) in rat livers treated with and without SB203580 (SB). (A) Treatment with SB203580 significantly inhibited the reduction of Cx32 in soluble and insoluble fractions and enhanced the increase of claudin-1 in the insoluble fraction. (B) The corresponding expression levels of Cx32, CL-1, -2, -3, Oc, JAM1, ZO-1, and E-cadherin proteins are shown as bar graphs.
study, re-expression of lined Cx32-immunoreactivity was co-localized with claudin-1-immunoreactivity at apical regions of some EGF-induced proliferative rat hepatocytes treated with SB203580 (Fig. 9B). This suggests that during regeneration of hepatocytes, Cx32 is also coordinated with claudin-1, which can form tight junction strands via a p38 MAP-kinase signal pathway. Although gap and tight junctions perform very different functions, we show herein that there is common regulation by a p38 MAP-kinase signaling pathway during liver regeneration.
4.4. Potential of p38 MAP-kinase inhibitors in the treatment of regenerating hepatocytes after liver injury p38 MAP-kinase inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury [45].
Fig. 7. Northern blots for Cx32, claudin-1, -2 (CL-1, -2), occludin (Oc) and JAM-1 at 48 h after partial hepatectomy (PH) in rat livers treated with and without SB203580 (SB). (A) Cx32 mRNA was decreased and claudin-1, -2, occludin and JAM-1 mRNAs were increased compared to sham operation levels. Treatment with SB203580 inhibited reduction of Cx32 mRNA. (B) The corresponding expression levels of Cx32, CL-1, -2, Oc, and JAM-1 mRNAs are shown as bar graphs.
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Fig. 8. Immunocytochemistry (A) for Cx32 and claudin-1, functions of gap and tight junctions, GJIC and fence (A), Western blot (B) for phospho-p38 MAP-kinase (pp38), Cx32 and claudin-1 (CL-1) and Northern blot (C) for Cx32 and claudin-1 (CL-1) in EGF-induced proliferative rat hepatocytes treated with SB203580 (SB). Bars 10 mm. (A) In treatment with SB203580 for 4 h, Cx32 and claudin-1 reappeared at cell borders, and GJIC and fence function were recovered. (B) In Western blots of whole cell lysates after SB203580 treatment, phospho-p38 MAP-kinase was decreased from 1 h and expression of Cx32 and claudin-1 proteins was increased from 4 h. (C) In Western blots of soluble and insoluble fractions using 1% Triton X-100 buffer, expression of Cx32 and claudin-1 in the insoluble fraction was increased from 1 h after SB203580 treatment. (D) In Northern blots, mRNA of Cx32, but not claudin-1 was increased from 1 h after SB203580 treatment.
in accelerated Fas-mediated cell death in hepatocytes [27]. In the present study, during liver regeneration after PH in vivo, the p38 MAPK inhibitor SB203580 could inhibit the marked changes in formation and expression of gap and tight junctions. During hepatocyte regeneration after liver injury, maintenance of the functions of hepatic gap and tight junctions may be very important for preventing jaundice as well as hepatic damage, because the interaction between gap
and tight junctions of hepatocytes may regulate the ‘blood– biliary barrier’ [3,4,5]. Further studies are needed to examine changes of gap and tight junctions via various signaling pathways during liver regeneration in vivo. Thus, it is possible that the p38 MAP-kinase inhibitors could be used as therapeutic drugs for regenerating hepatocytes after chronic or severe liver injury, interms of prevention of jaundice.
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Fig. 9. Double immunocytochemistry for Cx32 and claudin-1 in EGF-induced proliferative rat hepatocytes treated with SB203580. (A) In primary cultures of rat hepatocytes at day 10 after plating, lines of Cx32-immunoreactivity were co-localized with claudin-1-immunoreactivity at apical regions of cell borders, whereas Cx32-positive spots were observed at basolateral regions. (B) In EGF-induced proliferative rat hepatocytes treated with SB203580 for 4 h, re-expression of lines of Cx32-immunoreactivity co-localized with claudin-1-immunoreactivity at apical regions of some cells. Bars 10 mm. [This figure appears in colour on the web.]
Acknowledgements We are grateful to Dr T. Kita (Kyoto University) for the JAM-1 antibody. We thank Ms E. Suzuki (Sapporo Medical University) for technical support. This work was supported in by Grants-in-Aid from the Ministry of Education, Culture, Sports and Science, and the Ministry of Health, Labour and Welfare of Japan and by the Smoking Research Foundation, and the Long-Range Research Initiative Project of the Japan Chemical Industry Association.
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