Differential Effect of the Protein Synthesis Inhibitors Puromycin and Cycloheximide on Vascular Smooth Muscle Cell Viability

Abstract

Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycintreated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2␣. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.

Quinotrierixin Inhibited ER Stress-Induced XBP1 mRNA Splicing through Inhibition of Protein Synthesis

“…3). Rather, consistently with a previous report, 20) single treatment of cells with puromycin induced XBP1 mRNA splicing (Fig. 3B).…”

Translocon closure to Ca2+ leak in proliferating vascular smooth muscle cells

“…Nevertheless, the translocon can readily be made leaky to Ca 2ϩ by puromycin. Puromycin is used therapeutically as an antiprotozoal agent and has been compared with cycloheximide as a potential pharmacological approach to stabilizing atherosclerotic plaques (6). Intriguingly, the latter study showed that puromycin, but not cycloheximide, evoked VSMC apoptosis.…”

Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells

“…Cycloheximide, a protein translation inhibitor that is well tolerated by SMCs and does not cause cell death (17,18), was administered to block protein elongation, and contractile proteins levels were followed over time. In NTG cells, the contractile proteins remain highly stable for up to 48 h of exposure to cycloheximide (Fig.…”