SMG-1 kinase attenuates mitochondrial ROS production but not cell respiration deficits during hyperoxia

ABSTRACT

Purpose: Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia. Materials and Methods: Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes. Results: Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53. Conclusions: Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.

KEYWORDS:

• Hyperoxia
• mitochondrial dysfunction
• p53
• reactive oxygen species
• SMG-1

Acknowledgments

We would like to thank Dr. Gail Johnson for her discussions regarding this research, Min Yee for assistance with sample collection, and Dr. William Domm for critically reviewing the manuscript.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Funding

This work was supported by the National Heart, Lung and Blood Institute grants R01-HL067392 and R01-HL091968 (M.A.O.) and R01-HL071158 (P.S.B.) and the NIH Training Grant T32-ES07026 (E.A.R.). The University of Rochester's Department of Pediatrics provided financial support through the Perinatal and Pediatric Origins of Disease Program.