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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.

SUBMITTER: Legesse-Miller A

PROVIDER: S-EPMC3442405 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity.

Legesse-Miller Aster A Raitman Irene I Haley Erin M EM Liao Albert A Sun Lova L LL Wang David J DJ Krishnan Nithya N Lemons Johanna M S JM Suh Eric J EJ Johnson Elizabeth L EL Lund Benjamin A BA Coller Hilary A HA

Molecular biology of the cell 20120808 18

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 ...[more]

PMID: 22875985

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Project description:Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C ? (PKC?) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKC? or ERK1/2 inhibitors due to intrinsic inhibition of PKC? and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-?B binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-?B for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E(2). As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKC?/ERK1/2 signaling pathway.

Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-4 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.

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Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity.

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Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity.

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