MAPK Signaling Influenced by Oxygen Environments

Abstract

Mammalian cells thrive in a variety of different environments. In tissue culture there are many variables cells are exposed to; the impact of oxygen gas is one that is not well understood in relation to signaling pathways. In tissue culture, cells are commonly grown in atmospheric oxygen (~21%), yet the environment of many cells in vivo is considerably less (1% – 14% depending on the cells type). The increased oxygen content can cause higher levels of reactive oxide species (ROS). When ROS overwhelm the cell, normal cellular responses are affected. Extracellular kinase 1 and 2 (ERK 1/2) are proteins know to be directly modified by ROS, sometimes resulting in kinase inhibition. In this study we examined responses of the MAPK pathway to epidermal growth factor (EGF) in normoxic (6% O2) and atmospheric (21% O2) conditions. Baby hamster kidney (BHK‐21) or Human Dermal Epithelial (HMEC‐1) cells were grown and maintained in atmospheric conditions. The cells were then seeded and either kept under atmospheric conditions (normal tissue culture conditions) or moved to 6% oxygen in a Bactrox™(with all other conditions remaining constant). These cells were grown for two days until approximately 80–90% confluent and then they were serum starved and treated with EGF (0–30 minutes) in the two environments. They were harvested on ice in their respective environments. The lysates were then removed and frozen. After lysis the two conditions were treated in atmospheric oxygen. We hypothesized that the ERK 1/2 pathway responses would be different under the conditions of 6% versus 21% oxygen. ERK 1/2 phosphorylation status was assessed in parallel with kinase activity. Also explored was the difference in cell growth between the two environments. Our results show subtle differences in the peaks of phosphorylation and in the threshold of responses as well as differences in activity of the harvested ERK. Our work will lead to an enhanced understanding of how cellular signaling pathways are impacted by oxygen levels.Support or Funding InformationNIH grant # R15 GM110634‐01A1 to C.A.C., and Kennesaw State University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.