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. 2009 Oct 9;284(41):28253-28262.
doi: 10.1074/jbc.M109.004101. Epub 2009 Aug 13.

Cytomegalovirus promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors

Beatriz Alvarez-Castelao  1 , Idoia Martín-Guerrero  2 , África García-Orad  2 , José G Castaño  3
Affiliations

Cytomegalovirus promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors

Beatriz Alvarez-Castelao et al. J Biol Chem. .

Retraction in

Abstract

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

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Figures

FIGURE 1.
FIGURE 1.
Dose-dependent accumulation of EYFP-C-Cot in transiently transfected PC12 and NIH3T3 cells treated with proteasome inhibitors. Cells were treated with the indicated dose of proteasome inhibitors for 12 h. a, graphs of the relative change in fluorescence of the cell population in response to different doses of proteasome inhibitors: ○, mean fluorescence; △, percentage of positive cells; ●, product of mean fluorescence multiplied by the percentage of positive cells. b, representative cell fluorescence profiles of the experiments presented in a. c, representative immunoblots with anti-EGFP antibodies of the changes in protein levels at different doses of proteasome inhibitors.
FIGURE 2.
FIGURE 2.
Fluorescence microscopy analysis of EYFP-C-Cot transiently transfected PC12 and NIH3T3 cells treated with different doses of proteasome inhibitors. Cells were transiently transfected with EYFP-C-Cot and treated for 12 h with the indicated doses of proteasome inhibitors. Upper panels for each cell type show EYFP fluorescence, and lower panels show 4′,6-diamidino-2-phenylindole fluorescence (DNA staining). Note that, as presented in a quantitative way in Fig. 1, both the percentage of positive cells and the fluorescence intensity per cell increase in a dose-dependent manner. Low magnification confocal images (×200) are shown. All pictures were taken with the same settings of the confocal microscope.
FIGURE 3.
FIGURE 3.
Effect of proteasome, transcription, and translation inhibitors on cell fluorescence, mRNA, and protein abundance of PC12 cells stably transfected with EGFPd2. a, representative experiment of cell fluorescence profiles of stable EGFPd2 PC12 cells treated with proteasome, transcription, and translation inhibitors. b, upper panel, graph of mean cell fluorescence intensity analyzed by flow cytometry and expressed as mean ± S.E. from three different experiments, each run in triplicate. Lower panels show representative experiments of the levels of mRNA (EGFP and actin, which was used as control) analyzed by 1.5% agarose gel and ethidium bromide staining of RT-PCR products obtained from the respective total cell mRNAs samples and protein (EGFPd2 and tubulin, which was used as control) analyzed by immunoblotting with anti-EGFP and anti-tubulin antibodies. Untransfected PC12 cells (1) or stably transfected cell lines (2–13) were untreated (control; 2) or treated for 12 h with proteasome inhibitors (10 μm MG132, 10 μm lactacystin (Lacta), or 100 nm epoxomycin (Epoxo)) in the absence or in the presence of 500 ng/ml ActD or 20 μg/ml CHX as indicated (3–13).
FIGURE 4.
FIGURE 4.
Fluorescence microscopy analysis of EGFPd2 stably transfected PC12 cells in response to proteasome, transcriptional, and translational inhibitors. Cells stably expressing EGFPd2 were untreated (controls) or treated as indicated for 12 h with MG132, ActD, and CHX or the combinations indicated (see legend to Fig. 3 for doses). Living cells were examined under confocal microscopy with low magnification (×200). Fluorescence images are superimposed over phase-contrast images of the cells. All fluorescent images were captured with the same settings of the confocal microscope.
FIGURE 5.
FIGURE 5.
Effect of proteasome, transcription, and translation inhibitors on cell fluorescence, mRNA, and protein abundance of PC12 cells stably transfected with EGFP. a, representative experiment of cell fluorescence profiles of stable EGFP PC12 cells treated with proteasome, transcription, and translation inhibitors. b, upper panel, graph of mean cell fluorescence intensity analyzed by flow cytometry and expressed as mean ± S.E. from three different experiments, each done by triplicate. Lower panels show representative experiments of the levels of mRNA (EGFP and actin, which was used as control) analyzed by 1.5% agarose gel and ethidium bromide staining of RT-PCR products obtained from the respective total cell mRNAs samples and protein (EGFP and tubulin, which was used as control) analyzed by immunoblotting with anti-EGFP and anti-tubulin antibodies. Untransfected PC12 cells (1) or stably transfected cell lines (2–13) were untreated (control; 2) or treated for 12 h with proteasome inhibitors (10 μm MG132, 10 μm lactacystin (Lacta), or 100 nm epoxomycin (Epoxo)) in the absence or in the presence of 500 ng/ml ActD or 20 μg/ml CHX as indicated (3–13).
FIGURE 6.
FIGURE 6.
Fluorescence microscopy analysis of EYFP stably transfected PC12 cells in response to proteasome, transcriptional, and translational inhibitors. Cells stably expressing EYFP were untreated (controls) or treated as indicated for 12 h with MG132, ActD, and CHX or the combinations indicated (see legend to Fig. 3 for doses). Living cells were examined under confocal microscopy with low magnification (×200). Fluorescence images are superimposed over phase-contrast images of the cells. All fluorescent images were captured with the same settings of the confocal microscope.
FIGURE 7.
FIGURE 7.
Northern blot analysis of the effect of proteasome, transcription, and translation inhibitors on the mRNA expression of stable EYFP. Experimental conditions are identical to those described in the legend to Fig. 3. Total RNA was isolated from cells and analyzed by Northern blot. Membranes with transferred RNAs were stained with methylene blue (28 and 18 S RNAs) and successively hybridized with 32P-labeled probes for EYFP, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and protein-disulfide isomerase (PDI). A representative experiment is presented. The graph shows the relative changes in the amount of mRNA for EYFP quantitated using either glyceraldehyde-3-phosphate dehydrogenase (white bars) or protein-disulfide isomerase (gray bars) mRNAs as controls. Data are expressed as mean ± S.E. from three experiments. Epoxo, epoxomycin; Lacta, lactacystin.
FIGURE 8.
FIGURE 8.
Transcription factors implicated in the activation of CMV fluorescent reporters by proteasome inhibitors. HeLa cells were transiently transfected with 50 ng of the corresponding pCMV GFPu (a) and pCMV EYFP (b) together with 950 ng of the indicated plasmids: empty pCDNA3.1, empty pEF-BOS, and AP1, CREB, or NFκB luciferase reporters. Transfected cells were untreated (control) or treated with MG132 (10 μm) for 12 h. a and b show -fold changes in total cell fluorescence intensity analyzed by flow cytometry. c shows a representative Western immunoblot of the expression of the fluorescent proteins. d, HeLa cells were transiently transfected with a mixture of DNAs containing 100 ng of the indicated firefly luciferase reporters, 50 ng of basal Renilla luciferase plasmid, and 850 ng of empty pcDNA3.1 vector. Transfected cells were untreated (control) or treated with MG132 (10 μm) for 12 h, and luciferase was assayed as described under “Experimental Procedures.” The graph shows -fold changes in the quotient of firefly/Renilla luciferase. Data are expressed as mean ± S.E. from three different experiments. Tub, tubulin; CRE, cAMP-responsive element.
FIGURE 9.
FIGURE 9.
Effect of MG132 treatment of HeLa and PC12 cells on c-Jun and c-Fos expression. HeLa or PC12 cells were untreated (−) or treated (+) with 10 μm MG132 for 12 h. Total cells extracts were analyzed by immunoblots with the indicated antibodies to detect total c-Fos and c-Jun and the phosphorylation of c-Jun (P-c-Jun) as described under “Experimental Procedures.”
FIGURE 10.
FIGURE 10.
Studying protein degradation by the proteasome under controlled cellular conditions. PC12 stably transfected with EGFPd2 were treated with MG132 (5 μm) for 4 h, then washed four times with complete medium to remove the excess proteasome inhibitor, and plated again. 1 h after plating medium was changed, and fresh media containing no addition (control), 20 μg/ml CHX, or CHX and proteasome inhibitors as indicated in the figure were added. The left panel shows the quantification of relative fluorescence and protein levels as determined by flow cytometry and Western immunoblot. Data are expressed as mean ± S.E. from three different experiments. The middle panel shows a representative Western and immunoblot experiment of proteins (EGFPd2 and tubulin, which was used as control) immunoblotted with anti-EGFP and anti-tubulin antibodies. Φ, untransfected PC12. Right panel, total RNA was isolated and analyzed by qPCR as described under “Experimental Procedures.” Graphs show the relative -fold change and the corresponding ranges (upper and lower bars) as calculated from three different experiments from −ΔΔCT values. Lacta, lactacystin.
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