Carbon monoxide releasing molecule-2 CORM-2 represses global protein synthesis by inhibition of eukaryotic elongation factor eEF2

Abstract

Carbon monoxide (CO) is an endogenous gaseous transmitter that exerts antiproliferative effects in many cell types, but effects of CO on the translational machinery are not described. We examined the effects of the carbon monoxide releasing molecule-2 (CORM-2) on critical steps in translational signaling and global protein synthesis in pancreatic stellate cells (PSCs), the most prominent collagen-producing cells in the pancreas, whose activation is associated with pancreatic fibrosis. PSCs were isolated from rat pancreatic tissue and incubated with CORM-2. CORM-2 prevented the decrease in the phosphorylation of eukaryotic elongation factor 2 (eEF2) caused by serum. By contrast, the activation dependent phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1) was inhibited by CORM-2 treatment. The phosphorylation of eukaryotic initiation factor 2α (eIF2α) and eukaryotic initiation factor 4E (eIF4E) were not affected by CORM-2 treatment. In consequence, CORM-2 mediated eEF2 phosphorylation and inactivation of 4E-BP1 suppressed global protein synthesis. These observations were associated with inhibition of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling and increased intracellular calcium and cAMP levels. The CORM-2 mediated inhibition of protein synthesis resulted in downregulation of cyclin D1 and cyclin E expression, a subsequent decline in the phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and cell growth arrest at the G0/G1 phase checkpoint of the cell cycle. Our results suggest the therapeutic application of CO releasing molecules such as CORM-2 for the treatment of fibrosis, inflammation, cancer, or other pathologic states associated with excessive protein synthesis or hyperproliferation. However, prolonged exogenous application of CO might also have negative effects on cellular protein homeostasis.