Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity

Abstract

Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition-mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition-induced cytotoxicity.

FIGURE 1:

Proliferating fibroblasts are more sensitive to proteasome inhibitors than are quiescent fibroblasts. (A–C) Proliferating (P), contact-inhibited (4dCI), and serum-starved (4dSS) cells were incubated with MG132 or DMSO control, as indicated, for 24 h. Cell viability and induction of apoptosis were determined via annexin V and PI staining, followed by flow cytometry analysis. (A) Representative scatter plot of flow cytometry data. (B) Cell viability data for proteasome-inhibited and control proliferating and quiescent fibroblasts. The fraction of cells that were viable (PI negative) in the DMSO-treated cells was calibrated to 100%, and all other samples were normalized accordingly. Average viability and SE in triplicate samples from three independent experiments are shown (n = 9). (C) The fractions of annexin V–positive, PI-negative (early apoptotic) cells, annexin V– and PI-positive (late apoptotic) cells, annexin V–negative, PI-positive (very late apoptotic or necrotic) cells. The average and SE for three independent experiments, each performed in triplicate (n = 9), are shown. Asterisks indicate a statistically significant difference (p < 0.05) between MG132-treated and control cells.

FIGURE 2:

Proteasome levels and activity are comparable in proliferating and quiescent fibroblasts. (A) Protein lysates from fibroblasts induced into quiescence by contact inhibition or serum starvation were collected at the indicated time points. Constitutive proteasome subunit levels were monitored by immunoblotting. GAPDH was used as a loading control. The ratio of the proteasome subunit to GAPDH, normalized to the proliferating (P) sample, is shown. (B) Total proteins from proliferating (P), contact-inhibited (4dCI), and serum-starved (4dSS) cells were collected, and 20S proteasome activity was monitored using fluorescent substrates for the indicated enzymes. Enzyme activity of the proliferating sample was calibrated to 100% for each enzyme, and the other samples were normalized accordingly. Average fluorescence and standard deviations in triplicate samples from two independent experiments (n = 6) are shown. (C) Cells were treated with MG132 as indicated for 24 h, and the accumulation of ubiquitinated proteins was monitored by immunoblotting. GAPDH was used as a loading control.

FIGURE 3:

Inhibition of autophagy results in increased apoptotic cell death in proteasome-inhibited, serum-starved fibroblasts. (A) Autophagy is induced in contact-inhibited (CI) and serum-starved (SS) fibroblasts. Protein lysates were collected as in Figure 2A and subjected to immunoblot analysis with an antibody to LC3. The ratio of LC3 II to total LC3 (LC3 I + LC3 II) is shown, as well as the ratio of LC3 II to GAPDH, normalized to the proliferating sample. (B) MG132 treatment leads to induction of autophagy in proliferating and quiescent cells. Cells were treated with increasing amounts of MG132 for 24 h, and LC3 levels were monitored by immunoblot analysis. The ratios of LC3 II to total LC3 and LC3 II to GAPDH, normalized to DMSO (–) control, are shown. (C) MG132 treatment leads to active autophagy flux. Cells were treated for 24 h with DMSO (–), MG132, Baf, or MG132 and Baf, as indicated. LC3 levels were monitored by immunoblot analysis. The ratios of LC3 II to total LC3 and of LC3 II to GAPDH normalized to DMSO-treated control cells are shown. (D) Treatment with Baf sensitizes serum-starved fibroblasts to MG132. Proliferating and 4dSS fibroblasts were treated with MG132 (0–10 μM) in the presence or absence of 100 nM Baf for 24 and 48 h. Caspase 3/7 activity was monitored using a luminescent substrate. The fold change of caspase 3/7 activity in MG132-treated compared with DMSO-treated cells is shown. The experiments were performed three times in triplicate; mean fold change and SE are shown (n = 9). Asterisks indicate a significant difference for cells treated with MG132 together with 100 nM Baf compared with cells treated with MG132 alone at the same concentration (p < 0.05).

FIGURE 4:

MG132 treatment results in a widespread transcriptional response for both proliferating and quiescent cells. Proliferating (P), contact-inhibited (4dCI), or serum-starved (4dSS) fibroblasts were treated as indicated for 24 h. From each sample, RNA was collected, fluorescently labeled, and hybridized to whole–human genome microarrays. For each sample, the log₂ of the ratio of expression relative to the DMSO-treated control is shown. The gene expression profile was used to cluster the genes into 10 groups using K-means clustering. Yellow indicates high expression, and blue indicates low expression.

FIGURE 5:

Proteasome-inhibited quiescent fibroblasts exhibit less accumulation of ubiquitin- and p62-positive juxtanuclear aggregates. (A) Fibroblasts were plated on chamber slides under proliferating (top), contact-inhibited (middle), or serum-starved (bottom) conditions and treated with DMSO as vehicle control or MG132 (1 μM) for 24 h. Samples were fixed and stained with primary antibodies to ubiquitin and p62. Fluorescent secondary antibodies (Alexa 488 for p62 and Alexa 633 for ubiquitin) were used for visualization. Samples were stained with Hoechst to visualize DNA. Images were taken at 40× magnification. Volocity software was used to measure the average aggresome length in images of proliferating and quiescent cells from two different experiments (six images were used for proliferating, four images for contact-inhibited, and five images for serum-starved cells). Asterisk indicates a statistically significant difference between P and 4dCI and between P and 4dSS (p < 0.05). (B) Total proteins were prepared in the presence of 1% SDS, and the samples were filtered through a 0.2-μm-pore-size cellulose acetate membrane. Protein aggregates retained by the cellulose acetate membrane were visualized by Western blot analysis with anti-ubiquitin antibody (top). ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify ubiquitinated protein levels for each sample in two independent experiments, and the fold change of ubiquitinated proteins comparing MG132-treated to the DMSO control samples is plotted (bottom). Error bars, SD. p value for proliferating vs. 4dSS is 0.052.

FIGURE 6:

p62 knockdown results in reduced ubiquitinated protein aggregates and decreased apoptosis. (A) p62 knockdown results in fewer and smaller aggregates in response to proteasome inhibition. shp62- or nonsilencing shRNA control–expressing fibroblasts were plated on chamber slides under proliferating, contact-inhibited, and serum-starved conditions and treated with MG132 (1 μM) or DMSO for 24 h. Samples were fixed and stained with antibodies to p62 and ubiquitin. DNA was visualized by Hoechst staining. Images were taken at 40× magnification. Volocity software was used to quantify the number of aggresomes in images of MG132-treated proliferating and quiescent shControl- and shp62-expressing fibroblasts (five images each were used for shControl and shp62 proliferating and serum-starved cells and four images each were used for shControl and shp62 contact-inhibited fibroblasts). Area size cutoffs were 16–100 μm² for proliferating cells, 0.2–2 μm² for contact-inhibited cells, and 1–10 μm² for serum-starved cells. Asterisks indicate a statistically significant decrease in the number of aggresomes, p < 0.05. (B) An shRNA targeting p62 resulted in reduced p62 protein levels. Proliferating and quiescent fibroblasts expressing shp62 or shControl were treated with MG132 or DMSO for 24 h. p62 levels were monitored by immunoblot analysis. The ratio of p62 to GAPDH, normalized to DMSO (–) shControl, is reported. (C) p62 knockdown results in reduced apoptosis. Proliferating and serum-starved fibroblasts expressing shRNA to p62 or shControl were treated with increasing amounts of MG132 (0–10 μM) for 24 h. The experiments were performed three times in triplicate (n = 9). Apoptosis induction was monitored by caspase 3/7 activity assay, and the fold change compared with DMSO-treated control cells is shown. Asterisks indicate a statistically significant difference between p62 knockdown fibroblasts and shControl cells (p < 0.05).

FIGURE 7:

NAC protects fibroblasts from MG132-induced protein aggregation and apoptosis. (A) NAC treatment reduces intracellular, ubiquitin-positive aggregates for both proliferating and serum-starved, MG132-treated cells. Proliferating (P), contact-inhibited (4dCI), and serum-starved (4dSS) fibroblasts were treated with MG132 (1 μM) for 24 h in the presence or absence of NAC (2 mM). Samples were fixed and stained with antibodies to ubiquitin and p62. DNA was visualized by Hoechst staining. Images were taken at 40× magnification. Volocity software was used to quantify the number of aggresomes in ∼10 images each of MG132 and MG132 plus NAC-treated proliferating, contact-inhibited, and serum-starved fibroblasts. Area size cutoffs were 16–100 μm² for proliferating cells, 0.2–2 μm² for contact-inhibited cells, and 1–10 μm² for serum-starved cells. (B) Proliferating and 4dSS fibroblasts were treated as in A for 48 h. For each sample, fold induction of caspase 3/7 activity relative to DMSO-treated cells is plotted. Data shown are averaged from three independent experiments, each performed in triplicate (n = 9). Error bars, SE. Asterisks indicate statistically significant differences between samples treated with MG132 and NAC compared with samples treated with MG132 alone (p < 0.05).

FIGURE 8:

MG132 treatment increases cellular superoxide levels in proliferating but not quiescent fibroblasts, and 2-ME sensitizes serum-starved quiescent fibroblasts to proteasome inhibition. (A, B) MG132 treatment resulted in up-regulation of MnSOD at transcript and protein levels. (A) MnSOD expression was determined by microarray as in Figure 4. Heat map displaying the results and fold change of each treated sample, normalized to DMSO-treated control, is shown. Yellow indicates increased expression compared with the DMSO control for cells in the indicated proliferative state. (B) MnSOD and catalase are expressed at higher levels in quiescent than in proliferating fibroblasts. Cells were treated as indicated for 24 h, and protein expression was monitored by immunoblot analysis. GADPH was used as a loading control. For each protein, the ratio of protein intensity to GAPDH intensity, normalized to proliferating DMSO (–) control, is shown. (C) Total cellular superoxide levels are increased in MG132-treated proliferating but not contact-inhibited or serum-starved fibroblasts. Cells in different proliferative conditions were treated with DMSO or MG132 and monitored for superoxide levels by incubating with the dye DHE, followed by flow cytometry. Average fold change normalized to proliferating DMSO control is shown from two experiments, each performed in duplicate (n = 4). Error bars, SE; the asterisk indicates a statistically significant difference between proliferating MG132 and DMSO-treated cells (p < 0.05). (D) Proliferating and 4dSS fibroblasts were treated with increasing doses of MG132 (0–10 μM) for the indicated times in the presence or absence of 100 μM 2-ME, which was introduced for the last 6 h of MG132 treatment. Fold induction of caspase 3/7 activity is plotted. The average of three independent experiments, each performed in triplicate (n = 9), is shown. Error bars indicate standard errors. Asterisks indicate statistically significant differences between samples treated with MG132 plus 2-ME vs. MG132 alone at each dosage (p < 0.05).