P21 Cip1 Expression is Increased in Ambient Oxygen, Compared to Estimated Physiological (5%) Levels in Rat Primary Muscle Precursor Cell Culture

Abstract

Oxidative stress has been implicated as a key regulator of skeletal muscle dysfunction in a variety of conditions including sarcopenia, heart failure, cancer, and muscular dystrophy. While it is common practice to culture cells in the presence of ambient oxygen (~21% O2), the O2 level observed in the physiological environment of skeletal muscle cells is much lower. Previous efforts to culture a variety of different stem cells, including muscle precursor cells (MPCs), under O2 conditions that better mimic in vivo conditions have resulted in enhanced proliferation. In the present study, we determined whether 21% O2 in culture represents a sufficient stimulus to cause increased expression of two key negative regulators of the cell cycle, p21Cip1 and p27Kip1. Markers of cell proliferation were decreased after 48 hr culture in 21% O2, compared to 5% O2. 21% O2 had no effect on either p27Kip1 promoter activity or protein expression. Although p21Cip1 promoter activity remained unchanged between 5% and 21% O2, there were significant increases in both p21Cip1 mRNA and protein expression. In summary, increasing O2 levels in primary MPC culture from 5% to 21% specifically increases p21Cip1 expression, while there was no observed difference in p27Kip1 expression, suggesting that p21Cip1 may be more sensitive to changing O2 levels in culture. Supported by AG18780.