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First published online 30 March 2004
doi: 10.1242/jcs.01071

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The specific fates of tight junction proteins in apoptotic epithelial cells

¹ Institute of Clinical Chemistry and Pathobiochemistry, Charité – Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
² Institute of Biochemistry, Deutscher Platz 6, 04103 Leipzig, Germany
³ Institute of Biochemistry, Fabeckstrasse 36A, 14195 Berlin, Germany
⁴ Department of Gastroenterology, Charité – Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
⁵ Institute of Clinical Physiology, Charité – Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany

View larger version (34K): [in a new window]   Fig. 1. Occludin is cleaved during apoptosis of epithelial cells. (A) H184A1 cell lysates (35 µg) were analyzed by western blotting with the monoclonal anti-occludin (clone 19) antibody at different times after induction of apoptosis with staurosporine. Two specific occludin fragments (fragment 1 and 2) were generated with time. The middle panel shows a shorter exposure of the above western blot to show the decrease in full-length occludin more clearly. The lower panel shows the degradation of PARP in the same cell lysates that were used to analyze occludin cleavage. (B) The generation of fragment 1 and decrease of full-length occludin were quantified by chemiluminescence imaging on a FujiFilm LAS-1000 system. The signal for occludin at 0 hours and the strongest signal for fragment 1 at 12 hours were set to 100%. Data represent mean values of five independent experiments with A as a representative blot. (C) Apoptosis was induced by addition of TNF- for 24 hours. Cell lysates (15 µg) of control (lane 1), adherent (lane 2) and floating apoptotic MDCK cells (lane 3) were analyzed using western blots with the monoclonal anti-occludin (clone 19) antibody. (D) After 6 hours pretreatment of cells with IFN-, apoptosis was induced by addition of anti-CD95/Fas (clone CH11) antibody for 24 hours and western blot analysis of lysates (20 µg) of control (lane 1), adherent (lane 2) and floating H184A1 cells (lane 3) was performed with monoclonal anti-occludin (clone 19) antibody. Western blots in C and D are representatives of at least four independent experiments. (E) Measurement of transepithelial resistance Rt in HT-29/B6 cells seeded on Millicell filters and incubated with 104 U/ml TNF- alone and TNF- in the presence of 50 µM Z-VAD-FMK. Rt was monitored over a period of 12 hours and specified as a percentage of initial Rt values. The results are representative of at least three independent experiments (Student's t-test was used to compare results, **P<0.01).

View larger version (24K): [in a new window]   Fig. 2. Fragment 1 and 2 are generated by caspase and metalloproteinase cleavage, respectively. Western blots in A, B and C are representative of at least four independent experiments. (A) MDCK cell lysates (10 µg) were analyzed 6 hours after induction of apoptosis by staurosporine in the presence or absence of Z-DEVD-FMK with the monoclonal anti-occludin (clone 19) antibody. (B) Apoptosis was induced by 24 hours TNF- treatment and equal amounts of protein from lysates (15 µg) of MDCK cells were analyzed in the presence of caspase and/or metalloproteinases inhibitors (TAPI-2, 50 µM; MMPI-1, 100 µM) using the monoclonal anti-occludin (clone 19) antibody. (C) In vitro cleavage of GST-occludin264-522 by recombinant caspase-3 generates a cleavage fragment co-migrating with fragment 1 from H184A1 cell lysates. In vivo cleavage of occludin in H184A1 cells at 0 hours (lane 1) and 12 hours (lane 2) after induction of apoptosis. In vitro cleaved GST-occludin264-522 (lane 3). Western blots were analyzed with the polyclonal anti-occludin antibody. (D) Schematic model of occludin and the apoptotic cleavage sites.

View larger version (17K): [in a new window]   Fig. 3. Mutagenesis of the caspase cleavage site prevents cleavage of the occludin cytoplasmic domain. Ltk- cells were transiently transfected with either full-length occludin-myc6 or with occludin (D320A)-myc6 with the mutated caspase cleavage site. Apoptosis was induced with 1 µM staurosporine for 3 hours. Western blots of cell lysates (25 µg) were analyzed with monoclonal anti-occludin (clone 19) antibody. Lanes 1-4 control cells, lanes 5-7, cells 3 hours after induction of apoptosis. Lane 1, untransfected Ltk- cells; lanes 2 and 5, Ltk- cells transfected with empty vector; lanes 3 and 6, Ltk- cells transfected with wild-type occludin; lane 4 and 7, Ltk- cells transfected with mutant occludin (D320A). Analysis with an anti-myc antibody gives comparable results (not shown). This western blot is a representative of at least five independent experiments.

View larger version (144K): [in a new window]   Fig. 4. Localization of occludin in apoptotic cells. MDCK cells were stained with polyclonal anti-occludin antibody. (A) 0 hours; (B) 3 hours STS (staurosporine); (C) 6 hours STS; (D) 6 hours STS + Z-DEVD-FMK; (E) 6 hours STS + TAPI-2; (F) 6 hours STS + Z-DEVD-FMK + TAPI-2. STS, staurosporine. Scale bar: 20 µm.

View larger version (23K): [in a new window]   Fig. 5. Fate of claudin-1 and claudin-2 during apoptosis of epithelial cells. (A) MDCK and H184A1 cell lysates (5 µg) were analyzed at different time points after induction of apoptosis with staurosporine by western blotting with polyclonal rabbit anti-claudin-1 antibody. (B) Apoptosis was induced in MDCK cells by treatment with TNF- for 24 hours (lane 1, 0 hours; lane 2, adherent cells 24 hours; lane 3, floating cells 24 hours). (C) HT-29/B6 cell lysates (5 µg) were analyzed at different time points after induction of apoptosis with staurosporine by western blotting with polyclonal anti-claudin-1 or anti-claudin-2 antibodies, respectively. On both western blots identical amounts of protein from the same cell lysate were analyzed. Western blots show representative data from four independent experiments. (D) Ltk- cells were transiently transfected with full-length claudin-2-FLAG3. Apoptosis was induced with 1 µM staurosporine for 6 hours. Western blots of cell lysates (40 µg) were analyzed with monoclonal anti-FLAG M2 antibody. Lane 1, Ltk- cells transfected with empty vector; lane 2, control Ltk- cells transfected with claudin-2-FLAG3; lane 3, Ltk- cells transfected with claudin-2-FLAG3 6 hours after induction of apoptosis with staurosporine. Western blots are representatives of at least three independent experiments.

View larger version (46K): [in a new window]   Fig. 6. Cleavage of ZO-1 and ZO-2 in apoptotic H184A1 cells. (A) Apoptosis was induced either by staurosporine (lanes 2 and 3) for 12 hours or anti-CD95/Fas (clone CH11) antibody (lanes 4 and 5) for 24 hours. Lysates (10 µg) of adherent (lanes 2 and 4) and floating (lanes 3 and 5) cells were analyzed by western blotting with a polyclonal anti-ZO-1 antibody. Lane 1, control at 0 hours. (B) Western blot analysis of ZO-2 24 hours after induction of apoptosis with the anti-CD95/Fas (clone CH11) antibody. Lane 1, control at 0 hours; lane 2, adherent cells; lane 3, floating cells; lane 4, lysate of pooled adherent cells and remaining floating cells in the presence of inhibiting anti-CD95/Fas (clone ZB4) antibody. Both western blots are representative of at least four independent experiments. (C,D) Apoptosis was induced by staurosporine and 10 µg of lysates of adherent (lane 2) and floating (lane 3) cells were analyzed by western blotting with anti-ZO-1 (C) or anti-ZO-2 (D) antibody. In the presence of Z-DEVD-FMK cells remain attached and fragmentation of ZO-1 and ZO-2 is inhibited (lane 4). Lane 1, control at 0 hours. Arrows in A-D indicate ZO-1 and ZO-2 cleavage products.

View larger version (124K): [in a new window]   Fig. 7. Localization of ZO-1 in apoptotic cells. MDCK cells were stained with polyclonal anti-ZO-1 antibody. (A) 0 hours; (B) 3 hours; (C) 6 hours STS; (D) 6 hours STS + Z-DEVD-FMK; (E) 6 hours STS + TAPI-2; (F) 6 hours STS + Z-DEVD-FMK + TAPI-2. STS, staurosporine. Scale bar: 20 µm.