Effect of Hypotonic Osmotic Stress on Apoptosis in Cultured Human Skin Keratinocytes

Abstract

While it is known that a cell in a hypotonic environment swells from an influx of water due to an osmotic imbalance, the pathways activated inside the cell are not as clearly observable or thoroughly investigated. To determine whether apoptotic and cell survival signaling pathways are activated, cultured human skin keratinocytes (HaCaT cell line) were exposed to increasing levels of hypotonicity from 0% dilution up to 50%. Western blotting analysis was used after 30 minutes of incubation. The results show that AMPK is activated when the dilution is increased, and the activity is peaked at 40% dilution and then dropped slightly at 50% dilution. Similarly, AKT is activated and the activity is peaked at 40% and 50% dilutions, suggesting that at this level of hypotonicity these pathways are activated for cell survival. The results also demonstrate that p38 and JNK are activated as the dilution is increased, and the activity is peaked at 40% dilution. Interestingly, the data from cell migration assay show that cell migration is increased gradually as dilution is increased, but dropped significantly at 50% dilution. Collectively, our data suggest that hypotonic stress affect both apoptotic and survival pathways and to certain degree enhance cell migration.