Mitochondrial PKC epsilon Regulates Mitochondrial Function in Airway Basal Cells from Older Human Donors

Introduction: The lungs undergo dramatic changes with aging, leading to a decline in innate immunity. Aging is a risk factor for chronic lung diseases. Despite the relevance of the normal aging process in the development of chronic airway diseases, little is known about the normal aging process of airway cells. Basal cells are progenitor cells of the airway that play an important role in repairing airway injury. This led to the hypothesis that the mitochondrial function of basal cells diminishes with aging and that elevated PKC𝜀 signaling contributes to this dysfunction.Methods: Primary airway basal cells from younger (19-40 yrs) and older (≥ 65 yrs) healthy donors were isolated and cultured. MitoStress assays, staining for mitochondrial membrane potential, and PKC𝜀 activity assays were used to characterize the cells. To isolate the role of PKC𝜀 in mitochondrial function, a transformed cell line with genetically modulated PKC𝜀 function was also used.Results: Mitochondrial bioenergetics were measured in primary human basal cells from younger and older donors, and there was a significant decrease in the oxygen consumption rate (OCR) in older cells at baseline and during MitoStress tests. The mitochondrial membrane potential was also evaluated. Older cells exhibited a significant decrease in membrane potential compared to that in younger cells. Mitochondrial membrane potential could be improved in older cells by inhibiting PKC𝜀, while membrane potential was diminished in younger cells by activating PKC𝜀. To further explore the role of PKC𝜀 in mitochondrial function, MitoStress tests were performed on the transformed basal cell line 16HBE14o- with modulation of PKC𝜀 activity using a PKC𝜀 dominant negative construct. In these experiments, cells with lower PKC𝜀 activity had improved OCR at baseline and after MitoStress tests. To confirm that PKC𝜀 was present in mitochondrial fractions, catalytic activity assays confirmed measurable PKC𝜀 activity within isolated mitochondria. Electron flow assays were performed to determine which electron transport chain complex was most affected. In these assays, Complex I and IV had the greatest change with modulation of PKC𝜀.Conclusions: PKC𝜀 activity increases with aging in basal airway epithelial cells and is associated with decreased mitochondrial function. Modulating PKC𝜀 activity can modulate mitochondrial function.