Volume 42, Issue 5, Pages 707-718 (May 2005) 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  Journal of Hepatology  Volume 42, Issue 5, Pages 707-718 (May 2005) DOI: 10.1016/j.jhep.2004.12.033 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

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 54kDa), pMAPK/MAPK (signal of 44kDa), pAkt/Akt and PCNA proteins are shown as the bar graphs. Treatment with SB203580 markedly inhibited the increase of pp38 at 24h and 48h after PH. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Immunohistochemistry for Ki-67 after PH of rat livers treated with and without SB203580. Bar 40μm. The corresponding the numbers of Ki-67-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. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

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 40μm (A), 60μm (B), 10μm (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 48h after PH. (B) At 48h after PH, claudin-1 was localized from periportal areas to middle areas of liver lobules, and at 48h 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 48h after PH in livers with or without SB203580 treatment. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Freeze fracture replicas at 48h 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 50nm (E), 100nm (F). Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

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 48h 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, E-cadherin and PCNA proteins are shown as bar graphs. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

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 48h 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, JAM-1, ZO-1, and E-cadherin proteins are shown as bar graphs. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Northern blots for Cx32, claudin-1, -2 (CL-1, -2), occludin (Oc) and JAM-1 at 48h 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. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

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 10μm. (A) In treatment with SB203580 for 4h, 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 1h and expression of Cx32 and claudin-1 proteins was increased from 4h. (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 1h after SB203580 treatment. (D) In Northern blots, mRNA of Cx32, but not claudin-1 was increased from 1h after SB203580 treatment. Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

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 4h, re-expression of lines of Cx32-immunoreactivity co-localized with claudin-1-immunoreactivity at apical regions of some cells. Bars 10μm. [This figure appears in colour on the web.] Journal of Hepatology 2005 42, 707-718DOI: (10.1016/j.jhep.2004.12.033) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions