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Leukotriene D₄ induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKC

¹ Experimental Pathology, Department of Laboratory Medicine, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden
² Molecular Medicine, Department of Laboratory Medicine, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden

View larger version (53K): [in a new window]   Fig. 1. Effects of LTD4, CNF-1 and C3 exoenzyme on stress-fibre formation. Int 407 cells were incubated with or without LTD4, C3 exozyme or CNF-1. The cells were subsequently fixed, permeablised, stained for F-actin with Alexa 488 phalloidin and examined by confocal microscopy. The upper two panels show unstimulated (Control) and LTD4-treated cells. The two panels in the middle (C3) illustrate cells incubated with 5 µg/ml C3 exoenzyme and 5 µg/ml lipofectamine for 10 hours in the absence or presence of LTD4. The lower two panels (CNF-1) show cells incubated with 300 ng/ml CNF-1 for 16 hours in the absence or presence of LTD4. The results illustrated in this figure are representative of ten separate experiments.

View larger version (57K): [in a new window]   Fig. 2. Effects of L63-RhoA and N19-RhoA on stress-fibre formation. (A) Cells were transfected with EGFP-L63-RhoA and then fixed, permeablised, stained for F-actin with Alexa 546 phalloidin and examined by confocal microscopy. (B) Cells were transfected with EGFP-N19-RhoA in the absence or presence of 40 nM LTD4 for 5 minutes and then processed as in A. Merged images are shown on the right. The results illustrated in this figure are representative of six separate experiments.

View larger version (87K): [in a new window]   Fig. 3. Effects of LTD4 and TPA on stress-fibre formation. Following stimulation with either 40 nM LTD4 for 5 minutes or 100 nM TPA for 15 minutes, the cells were fixed, permeablised, stained for F-actin with Alexa 488 phalloidin and examined by confocal microscopy. The different panels show cells that were not stimulated (control) or stimulated with LTD4 or TPA and also were or were not exposed to 2µm GF109203X or 2 µm Gö6976 for 15 minutes. The results illustrated in this figure are representative of five separate experiments.

View larger version (59K): [in a new window]   Fig. 4. Effects of different PKC regulatory domains (RD) on stress-fibre formation. Cells were first transfected with vectors encoding EGFP-RD-PKC, —RD-PKCßII, —RD-PKC, or —RD-PKC and then stimulated with 40 nM LTD4 for 5 minutes. Following stimulation, the cells were fixed, permeablised, stained for F-actin with Alexa 546 phalloidin and examined by confocal microscopy. Merged images are shown on the right. The results illustrated in this figure are representative of five separate experiments.

View larger version (19K): [in a new window]   Fig. 5. Effects of C3 exoenzyme on LTD4-induced translocation of PKC, PKC and PKC to the membrane fraction. Cells were or were not pretreated with 5 µg/ml C3 exoenzyme together with 5 µg/ml lipofectamine for 10 hours and then were or were not stimulated with 40 nM LTD4 (30, 60, 90 and 300 minutes). Thereafter, the cells were lysed and centrifuged at 200,000 g for 30 minutes as described in the Materials and Methods. The resulting membrane-rich pellet was separated by SDS-PAGE and immunoblotted with an anti-PKC, anti-PKC or anti-PKC antibody. The illustrated blots are representative of at least three separate experiments.

View larger version (50K): [in a new window]   Fig. 6. Effects of RhoA and PKC on stress-fibre formation. (A) Cells were transfected with the vector encoding EGFP-N19-RhoA and then stimulated with 100 nM TPA for 15 minutes, and they were subsequently fixed, permeablised, stained for F-actin with Alexa 546 phalloidin and examined by confocal microscopy. (B) Cells were transfected with vector encoding EGFP-RD-PKC- and then stimulated with 300 ng/ml CNF-1 for 16 hours and subsequently processed as in A. (C) Cells were stimulated with 300 ng/ml CNF-1 in the absence or presence of 2 µM GF109203X for 16 hours and then processed as in A. Merged images are shown on the right. The results illustrated in this figure are representative of five separate experiments.

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