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Leukotriene D₄ activates MAPK through a Ras-independent but PKC-dependent pathway in intestinal epithelial cells

¹ Division of Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital Malmö, SE-205 02 Malmö
² Department of Cell and Molecular Biology, University of Umeå, SE-901 87 Umeå, Sweden
³ Division of Molecular Medicine, Department of Laboratory Medicine, Lund University, University Hospital Malmö, SE-205 02 Malmö

View larger version (23K): [in a new window]   Fig. 1. LTD4-induced proliferation of Int 407 cells. Cells were pre-incubated in the absence or presence of GF109203X (GFX; 30 µM for 30 minutes), PD98059 (50 µM for 30 minutes), FTI-277 (20 µM for 48 hours), PTX (500 ng/ml for 2 hours) or transfected with N17 Ras. Thereafter, the cells were incubated in the absence or presence of 80 nM LTD4 or 100 ng/ml EGF in the absence or presence of the indicated inhibitor for 48 hours. After 48 hours, the proliferative responses were determined. In (A), the conversion of tetrazolium into formazan was measured by analyzing the absorbance of formazan at 490 nm. The absorbance values are given as percentages relative to untreated cells. (B) outlines the results of the cell count, and the values are given as percentages relative to untreated cells. The data in both panels represent means±s.e.m. of four separate experiments. Statistical significant effects of the inhibitors (compared with untreated cells) were evaluated using the unpaired Student's t-test, *P<0.05 and **P<0.01.

View larger version (28K): [in a new window]   Fig. 2. Characterization of LTD4-induced activation of Erk-1/2. Cells were incubated in the absence or presence of 80 nM LTD4 and the indicated inhibitors. Cell lysates were separated by SDS-PAGE and immunoblotted with antibodies specific for phosphorylated Erk-1/2. Thereafter, the blots were reprobed for total Erk-1/2. (A) outlines a representative blot and the accumulated concentration curve of LTD4-induced Erk-1/2 phosphorylation. Cells were treated with the indicated concentrations of LTD4 for 3 minutes with or without ZM-198,615 (ZM; 50 µM, 15 minutes) after which their degrees of Erk-1/2 phosphorylation were determined. (B) shows a representative blot and graph of the accumulated time course of LTD4-induced Erk-1/2 phosphorylation. Cells were treated with 80 nM LTD4 for the indicated periods of time, after which Erk-1/2 activities were assayed as described in the Materials and Methods. (C) Cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours), GF109203X (GFX; 30 µM for 30 minutes), PP1 (10 µM for 15 minutes) or PD98059 (50 µM for 30 minutes). The cells were then incubated in the absence or presence of 80 nM LTD4 for 3 minutes after which their Erk-1/2 activities were assayed. In (D), cells were pre-incubated in the absence or presence of Rp-cAMPS (50 µM for 30 minutes), wortmannin (100 nM for 10 minutes) or LY294002 (50 µM for 30 minutes) and incubated with or without 80 nM LTD4 for 3 minutes after which their Erk-1/2 activities were assayed. The blots shown are representative of at least five separate experiments. Values obtained from the densitometric analyses are calculated as a percentage of untreated control cells and given as mean±s.e.m. of five separate experiments.

View larger version (21K): [in a new window]   Fig. 3. Characterization of EGF-induced Erk-1/2 activation and LTD4-induced EGF receptor phosphorylation in Int 407 cells. The cells were pre-incubated in the absence or presence of genistein (Gen; 50 µg/ml for 30 minutes), PP1 (10 µM for 15 minutes), PTX (500 ng/ml for 2 hours), PD98059 (50 µM for 30 minutes), GF109203X (GFX; 30 µM for 30 minutes) or TPA (1 µM for 24 hours, i.e. PKC depletion). Then the cells were stimulated with 100 ng/ml EGF (5 minutes), lysed, and the lysates were separated by SDS-PAGE as described in the Materials and Methods. (A) shows a representative immunoblot with a specific anti-phospho-Erk-1/2 antibody that was then reprobed with an anti-total-Erk-1/2 antibody. (B) illustrates results from cells that were stimulated with either 80 nM LTD4 for 3 minutes, 100 ng/ml EGF for 5 minutes or not stimulated at all (control). Lysates of these cells were separated by SDS-PAGE, immunoblotted with an anti-phospho-EGF receptor antibody and then reprobed with an anti-total-EGF receptor antibody. (C) illustrates results from cells pre-incubated with or without 2 µM PD153035 for 30 minutes and thereafter stimulated as above. Lysates of these cells were separated by SDS-PAGE, immunoblotted with a specific anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. The blots shown are representative of five separate experiments.

View larger version (22K): [in a new window]   Fig. 4. Identification of possible PKC isoform(s) involved in LTD4-induced Erk-1/2 activation. In (A), cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours) and then stimulated with or without 80 nM LTD4 for the indicated periods of time. Membrane and cytosolic fractions were isolated, separated on SDS-PAGE and immunoblotted with the indicated specific anti-PKC isoform antibodies as described in the Materials and Methods. In (B), cells were pre-incubated in the absence or presence of 10 µM MAPTAM for 1 hour or 1 µM ionomycin for 5 minutes and thereafter stimulated or not with 80 nM LTD4 for 3 minutes after which they were lysed. The cell lysates were then separated by SDS-PAGE and immunoblotted with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. In (C), cells were pre-incubated in the absence or presence of rottlerin (10 or 30 µM for 30 minutes), stimulated or not with 80 nM LTD4 for 3 minutes and then lysed. The cell lysates were separated by SDS-PAGE and immunoblotted as in (B). In (D), the results in the left panel were obtained from cells treated with 1 µM TPA for the indicated periods of time after which whole-cell lysates were analyzed by immunoblotting for PKC isoforms , and . The right panel of (D) show results from cells pre-incubated with TPA, as in the left panel, but then stimulated or not with 80 nM LTD4 for 3 minutes. After these 3 minutes, whole-cell lysates were prepared and analyzed by immunoblotting with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. (E) shows the results from cells transfected with either an empty vector (lanes 1-2), a RD-PKC-expressing or a RD-PKC-expressing vector that were stimulated or not with 80 nM LTD4 for 3 minutes. After 3 minutes, whole-cell lysates were prepared and analyzed by immunoblotting with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody and finally an anti-GFP antibody. (F) shows results from cells transfected with an empty vector (lanes 1-2) or a vector expressing K-PKC that were stimulated or not with 80 nM LTD4 for 3 minutes. After 3 minutes, whole-cell lysates were prepared and analyzed by immunoblotting for phospho-Erk-1/2, total Erk-1/2 and finally K-PKC. All blots in this figure are representative of at least three separate experiments.

View larger version (26K): [in a new window]   Fig. 5. Effects of LTD4 on the activities of Raf-1, B-Raf and MEK in Int 407 cells. In (A) and (B), the cells were stimulated with 80 nM LTD4 for the indicated periods of time, after which kinase assays were performed with anti-Raf-1 (A) and anti-B-Raf (B) immunoprecipitates (as described in the Materials and Methods). In (C), cells were transfected with an empty vector or a HA-tagged K-Raf-1 expressing vector and then stimulated or not with 80 nM LTD4 for 3 minutes. After 3 minutes, whole cell lysates were prepared and analyzed by immunoblotting with an anti-phospho-Erk-1/2, an anti-total-Erk-1/2 and finally an anti-HA antibody. In (D), cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours), GF109203X (GFX; 30 µM for 30 minutes), TPA (1 µM for 24 hours; PKC deplet) and thereafter stimulated or not with 80 nM LTD4 for 3 minutes. After 3 minutes, the Raf-1 kinase activities were performed as in (A). In (E), cells were pre-incubated in the absence or presence of PTX (500 ng/ml for 2 hours), PP1 (10 µM for 15 minutes) or PD98059 (50 µM for 30 minutes) and thereafter stimulated or not with 80 nM LTD4 for 3 minutes. After 3 minutes, the MEK kinase activities were performed as in (B). Representative control blots of the different immunoprecipitates are shown in panels (A,B,D,E). The kinase activity values are expressed as a percentage of untreated control cells and given as mean±s.e.m. of four separate experiments. Statistically significant effects (compared with untreated cells) were evaluated using an unpaired Student's t-test, *P< 0.05 and **P< 0.01.

View larger version (25K): [in a new window]   Fig. 6. LTD4-induced activation of Ras and its role in Erk-1/2 activation. In (A), cells were stimulated with 80 nM LTD4 for the indicated periods of time or with 100 ng/ml EGF for 5 minutes, after which the cells were lysed. The active form of Ras was then precipitated with the minimal RBD fragment of Raf-1 fused with GST and then separated by SDS-PAGE and immunoblotted with an anti-Ras antibody. A similar analysis is shown in the top panel of (B), but here the cells were pre-incubated in the absence or presence of the Ras inhibitor FTI-277 (20 µM for 48 hours) or transfected with HA tagged N17 Ras as indicated and then stimulated with LTD4 (80 nM) for 1 minute or EGF (100 ng/ml) for 5 minutes. In the second panel of (B), whole lysates from the samples used in the top panel were separated by SDS-PAGE and immunoblotted with an anti-Ras antibody. The blot show a gelshift of Ras derived from FTI-277 treated cells and the two bands of Ras from cells transfected with HA-tagged N17 Ras (the lower endogenous Ras band and the upper HA-tagged Ras band). In the third and fourth panels, the blot was probed with an anti-phospho-Erk-1/2 antibody and then reprobed with an anti-total-Erk-1/2 antibody. The relative densities given in the figure refer to densitometric analysis of LTD4 and EGF-induced activation of Ras and Erk-1/2. The blots shown are representative of four separate experiments.

View larger version (48K): [in a new window]   Fig. 7. Effect of N17 Ras expression on LTD4- and EGF-induced Erk-1/2 activation. The cells were cotransfected with a vector expressing dominant-negative N17 Ras along with an empty EGFP vector and then stimulated with LTD4 (80 nM for 1 minute), EGF (100 ng/ml for 5 minutes) or not at all. The cells were then fixed, permeabilised and stained for phospho-Erk-1/2 as described in the Materials and Methods. The top three panels show from the left to the right: EGFP expression, phospho-Erk-1/2 staining and an overlay image of non-stimulated control cells. The middle three panels and the bottom three panels show the same analysis from cells stimulated with LTD4 and EGF, respectively. The results illustrated are representative of three separate experiments.

View larger version (15K): [in a new window]   Fig. 8. A schematic model of the LTD4-induced signalling pathways involved in the regulation of proliferation in human intestinal epithelial cells.

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