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First published online 30 March 2004
doi: 10.1242/jcs.01056

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Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway

¹ Department of Radiobiology and Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan
² National Food Safety Toxicology Center, Department of Pediatrics/Human Development, Michigan State University, East Lansing, MI, USA
³ Department of Histology and Cell Biology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
⁴ Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

View larger version (53K): [in a new window]   Fig. 1. Typical digitized fluorescence images and plots of fluorescence recovery after photobleaching. With (anisomycin) or without (control) anisomycin treatment for 60 minutes, cells were labelled with 5,6-carboxyfluorescein diacetate. Suitable fields of cells were identified using a 40x objective lens. Such fields contained many cells that were in contact with each other but not too confluent. Each field was scanned to generate a digital image of fluorescence (Prebleach). After the initial scan, selected cells were photobleached (0 minute, numbers 1-6). Sequential scans were then carried out at 30 second intervals to detect recovery of fluorescence in the bleached cells (4 minute, numbers 1-6). Images were digitally recorded for analysis. Several unbleached cells were also monitored to provide control data (number 7). Typical plots of fluorescence recovery after photobleaching are shown (proportion of prebleaching against time). A rising slope indicates the recovery of fluorescence. The percentage recovery of fluorescence over time was determined for each selected cell and the data were corrected for the background loss of fluorescence in one area (number 7).

View larger version (30K): [in a new window]   Fig. 2. Time (A) and dose (B) course analyses of the effect of anisomycin on MAP kinase activity. After treatment with anisomycin, cell lysates were prepared and 20 µg protein was separated on 12.5% gel and transferred onto a PVDF membrane. The same membrane was probed and reprobed after stripping with antibodies against doubly phosphorylated p38 MAP kinase, phosphorylated p46/p54 and ß-actin as control in protein loading. These results were representative of three experiments, each performed with a different preparation of cells, and are expressed as the fold activity (means±s.e.m.) of band density relative to that of untreated control by densitometric analysis.

View larger version (11K): [in a new window]   Fig. 3. Time (A) and dose (B) course analyses of the effect of anisomycin on GJIC. GJIC was estimated by FRAP in cells treated with anisomycin. Results are expressed as the percentage (mean±s.e.m.) of RR relative to that of control cells (set to 100%) treated with distilled water. These results are representative of at least three experiments. **P<0.01 versus cells incubated with control. (C) To examine the effect of anisomycin as protein synthesis inhibitor, we used a [35S]-methionine metabolic labelling assay. At each concentration (0.01 µg ml-1, 0.1 µg ml-1 and 10 µg ml-1) of anisomycin, [35S]-labelled protein level was shown as radioactivity by liquid scintillation spectrometry. For comparison, the radioactivity of control was arbitrarily set at 1. Results were expressed as means±s.e.m. of three separate experiments.

View larger version (19K): [in a new window]   Fig. 4. Phosphorylation of Cx43 protein in anisomycin-treated cells. (A) Typical western blots of the Cx43 protein, with ZO-1, occludin, E-cadherin and ß-catenin as reference proteins and ß-actin as a control for protein loading. Numbers indicate the time from the addition of anisomycin. After incubation with anisomycin (10 µg ml-1) for 5, 30, 60, 90, 120 and 180 minutes, whole-cell extracts (20 µg per lane) were probed with antibodies. All samples show multiple Cx43 protein bands (P0, P1 and P2) depending on the phosphorylation level, and equal levels of reference proteins. (B) Densitometric analysis of the most important bands of Cx43 protein. The relative band intensities are shown. For assessment of each protein, the total amounts of control were arbitrarily set at 1. (C) Each column displays the ratio of (P1+P2):P0. Values are the means±s.e.m. of three separate experiments.

View larger version (25K): [in a new window]   Fig. 5. SB203580 interferes with the effects of anisomycin p38 MAP kinase activity on GJIC. Each treatment group was exposed to 0.02% dimethyl sulfoxide as vehicle alone for 90 minutes (control), anisomycin at 10 µg ml-1 for 60 minutes, SB203580 plus anisomycin (pretreatment of SB203580 for 30 minutes, then co-treatment with anisomycin for an additional 60 minutes) or SB203580 alone for 90 minutes. Cells with or without pretreatment with SB203580 at 10 µM were exposed to 10 µg ml-1 of anisomycin for 60 minutes. After treatment, samples were prepared as described in Fig. 2B. (A) Typical immunoblot analyses using specific antibodies against phosphorylated forms of p38 MAP kinase, JNK and ß-actin as protein-loading control. (B) This result is representative of three experiments, each performed with a different preparation of cells and plotted as the fold activity (mean±s.e.m.) of band density relative to that of control by densitometric analysis. (C) Results are expressed as the percentage (mean±s.e.m.) of RR relative to that of control cells (100%) of at least three separate experiments. *P<0.05 versus cells incubated with control; **P<0.01 versus cells incubated with control. (D) To examine the effect of anisomycin as protein synthesis inhibitor, we used [35S]-methionine metabolic labelling. The [35S]-methionine-incorporated protein levels were shown as radioactivity by liquid scintillation spectrometry. For comparison, the radioactivity of control was arbitrarily set at 1. Results were expressed as means±s.e.m. of three separate experiments.

View larger version (88K): [in a new window]   Fig. 6. Anisomycin introduced redistribution of Cx43 and gap-junction plaques. Cx43 was visualized as green spots by indirect immunofluorescence using FITC-labelled secondary antibody. Two sets of quadruple photographs are shown containing immunofluorescence images (IF) and Nomarski differential interference contrast images (DIC) of the same fields. These are typical images of each treatment group. Each treatment group was exposed to 0.02% dimethyl sulfoxide as vehicle alone for 90 minutes (control), anisomycin at 10 µg ml-1 for 60 minutes, SB203580 plus anisomycin (pretreatment of SB203580 for 30 minutes, then co-treatment with anisomycin for an additional 60 minutes) or SB203580 alone for 90 minutes. Cytoplasmic staining for Cx43 was also observed (indicated by white arrows). All images in each panel are of the same magnification. Scale bar, 20 µm.

View larger version (31K): [in a new window]   Fig. 7. Western blot analysis of Cx43 protein expression in cells treated with anisomycin and/or SB203580. Each treatment group was exposed to 0.02% dimethyl sulfoxide as vehicle alone for 90 minutes (control), anisomycin at 10 µg ml-1 for 60 minutes, SB203580 plus anisomycin (pretreatment of SB203580 for 30 minutes, then co-treatment with anisomycin for an additional 60 minutes) or SB203580 alone for 90 minutes. (A) A typical western blot. Whole-cell extracts (20 µg per lane) were probed with an antibody against Cx43 or against occludin, ß-catenin as reference proteins, and against ß-actin as a control for protein loading. (B) The relative band intensities of P1, P2 and P0 of Cx43. (C) Autoradiograph of [32P]-orthophosphate labelled Cx43 and its densitometric analysis on an SDS-PAGE gel. Results are from one representative experiment of two separate experiments. To assess each sample, the total value of control radiation was arbitrarily set at 1.

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