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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01696

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Proteasome inhibition activates the transport and the ectodomain shedding of TNF- receptors in human endothelial cells

INSERM UMR626, IFR125 IPHM, Faculté de Médecine, 27 Bd Jean Moulin, Marseilles 13385 Cedex 5, France

View larger version (25K): [in a new window]   Fig. 1. In HUVECs, the ectodomain-shedding of TNFRs involves metalloprotease activity and is increased by proteasome inhibition. Ro 32-7315 (5 µM) was added or not to HUVECs 15 minutes before the cells were incubated for 4 hours with MG-262 (600 nM) or lactacystin (40 mM) as indicated. (A) TNFR1 and TNFR2 that had accumulated in the culture media was measured by ELISA. (B) Cell surface expression of TNFR1 and TNFR2 was analyzed by flow cytometry. Cont, control; Lac, lactacystin; Ro, Ro 32-7315. Flowcytometry experiments were performed in duplicate and repeated three times. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.

View larger version (27K): [in a new window]   Fig. 2. In ECV-304 cells, the ectodomain-shedding of ectopically expressed TNF- is under the control of a TACE-like activity and is stimulated by proteasome inhibition. (A) Ro 32-7315 (5 µM) was added or not to ECV-304 cells stably expressing TNF-, 15 minutes before the cells were incubated for 4 hours with PMA (20 nM) or MG-262 (600 nM) as indicated. TNF- that had accumulated in the medium (black bars) and was present in the cell lysates at the end of the experiment (white bars) was measured by ELISA. Inset shows the mRNA levels of TNF- and eukaryotic elongation factor (EF1) analyzed by RT-PCR in (lane 1) ECV-304 cells and (lanes 2-4) ECV-304 cells expressing TNF- under basal conditions (lanes 1, 2), and after 4 hours of (lane 3) MG-262 or (lane 4) PMA treatment. (B) ECV-304 cells stably expressing TNF- were treated for 4 hours with MG-262 (600 nM), lactacystin (40 µM) or E-64d (10 µM), and TNF- that had accumulated in the medium was measured by ELISA. Cont: control; Lac: lactacystin; Ro: Ro 32-7315. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.

View larger version (32K): [in a new window]   Fig. 3. Proteasome inhibition triggers TACE-dependent release of TNF- without altering the amount of TACE antigen. (A) ECV-304 cells stably expressing TNF- were incubated for 20 minutes without (white bars) or with 300 nM of TIMP-3 (black bars) or TIMP-1 (striped bars) before MG-262 (100 nM) was added or not. TNF- that had accumulated in the culture medium over a period of 4 hours was measured by ELISA. (B) ECV-304 cells were co-transfected with TNF- and a dominant-negative form of TACE (DN), or a control vector (Cont). Fourty-eight hours after the transfection, cells were treated or not with MG-262 as described for Fig. 2. TNF- that had accumulated in the medium over a period of 4 hours (black bars) and was present in the cell lysates at the end of the experiment (white bars) was measured by ELISA. (C) The total cellular amount of TACE was immunoblotted as described in Materials and Methods. The immature form of TACE is indicated by `i', the mature form by `m'. (D) The cell surface expression of TACE was measured by flow cytometry. Experiments were performed in duplicate and repeated three times. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.

View larger version (44K): [in a new window]   Fig. 4. Proteasome inhibition specifically stimulates TACE-dependent ectodomain shedding. (A) ECV-304 cells were transiently transfected with TNFR2. Fourty-eight hours post transfection cells were treated as described for Fig. 2. Ectopically expressed TNFR2 (black bars) and endogenous TNFR1 from untransfected cells (white bars) that had accumulated in the medium over a period of 4 hours were measured by ELISA. (B) Cells ectopically expressing TNFR2 at their surface were counted by flow cytometry. (C) Endogenous ICAM-1, released in the culture media of ECV-304 cells, was measured by ELISA and its cell surface expression was measured by flow cytometry. (D) Epifluorescence microscopy of the intracellular distribution of ICAM-1 in untreated and MG-262-treated ECV-304 cells. Flowcytometry experiments were performed in duplicate and repeated three times. Significance of difference between treated and control cells: *, P<0.05 and **, P<0.001.

View larger version (16K): [in a new window]   Fig. 5. Proteasome inhibition increases the phosphorylation of MAP kinase p42/p44. HUVECs and ECV-304 cells were serum-starved overnight and treated with MG-262 (600 nM). Phosphorylation of MAP kinase p42/p44 was analyzed by immunoblot after 0, 90 and 180 minutes of treatment (upper panels). Corresponding amounts of total p42 are shown in lower panels.

View larger version (31K): [in a new window]   Fig. 6. Proteasome inhibition increases the amount of TACE substrates at the surface of TACE-inhibited cells. Native cells and ECV-304 cells stably expressing TNF- were treated with Ro 32-7315 (5 µM) 15 minutes before the addition of vehicle alone (Cont), MG-262 (600 nM) or PMA (20 nM) as indicated. Four hours later, cell surface expression of ectopic TNF- (black bars) and endogenous TNFR1 on native ECV-304 cells (white bars) were measured by flow cytometry. Experiments were performed in duplicate and repeated three times. Significance of difference between treated and control cells: **, P<0.001.

View larger version (49K): [in a new window]   Fig. 7. In ECV-304 cells, ectopically expressed TNF- is localized in the Golgi complex. Immunofluorescence analyses by confocal microscopy of ectopic TNF- and endogenous Golgin-97 in ECV-304 cells that stably express TNF-. TNF, localization of ectopic TNF-; Golgin: localization of endogenous Golgin-97; TNF + Golgin: superposition of the two previous images.

View larger version (69K): [in a new window]   Fig. 8. Proteasome inhibition triggers the intracellular dispersion of TNF- and TNFR2 from the Golgi-complex pool. ECV-304 cells that stably express TNF- (A-F) or transiently express TNFR2 (G, H), or native ECV-304 cells (I,J) were treated with TACE inhibitor Ro 32-7315 (5 µM) 15 minutes before the addition of vehicle alone (A,G,I), MG-262 (B,H,J), lactacystin (C), PMA (D), PD98054 (E) or PD98054+MG-262 (F). Four hours later, cellular expressions of TNF- (A-F), TNFR2 (G,H) and Golgin-97 (I,J) were analyzed by immunocytochemistry and epifluorescence microscopy as described in Materials and Methods.

View larger version (50K): [in a new window]   Fig. 9. The association of TACE with proteasome inhibitors increases the cell susceptibility to the proapoptotic effect of TNF-. ECV-304 cells were incubated overnight in the absence of serum with Ro 32-7315 (5 mM) and/or MG-262 (30 nM) in presence of TNF- (60 U/ml) as indicated. Surface binding of annexin V-FITC and propidium iodide labeling were measured by flow cytometry. Apoptotic cells are in square `a', necrotic cells in square `n' and secondary necrotic cells in square `a/n'. The percentage of apoptotic cells (a and a/n) is indicated. Ro, Ro32-7315. Experiments were performed in triplicate and repeated twice.

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