αB-Crystallin Accumulation in Human Astroglioma Cell Line U373MG Is Stress-Dependent and Phosphorylation-Independent

Abstract

αB-crystallin, a major lens protein, is present in non-lenticular tissues. Although αB-crystallin possesses chaperone-like activity, its physiological significance outside the lens is unclear. Recent studies suggest that stress-induced mechanisms of αB-crystallin accumulation are dependent upon the type of insult. To further investigate this possibility, the response of αB-crystallin to different stress conditions was examined in the human astroglioma U373MG cell line which constitutively expresses αB-crystallin. Exposure of this cell line to 25 μM cobalt chloride and heat stress resulted in increased accumulation of αB-crystallin. Exposure to cobalt chloride resulted in a 6-fold (water-soluble) and 5-fold (water-insoluble) increase in αB-crystallin, 4 h after the initial exposure to cobalt. The water-soluble fraction of U373 cells subjected to heat shock at 42°C for 30min and harvested after 1 h exhibited a 4.5-fold increase in the level of αB-crystallin over the control levels.
Approximately 2 h after the removal of the cells from the heat stress, the level of αB-crystallin in the urea-soluble fraction increased 3-fold. Although αB-crystallin accumulation initially increased in response to both the cobalt and heat shock challenges, analysis of the pattern of protein accumulation indicates that there are differences in the mechanism of αB-crystallin induction which are dependent on the type of stress. The concentration of αB-crystallin in both the water-soluble and water-insoluble fractions of heat-stressed cells showed that αB-crystallin concentrations returned to basal levels within 4 h after the initial shock, while levels of αB-crystallin remained high in the water-soluble fraction of cobalt-stressed cells 72 h after the initial stress. Since the role of phosphorylation in chaperone function is controversial, U373 cells were examined for induced forms of phosphorylated αB-crystallin. Isoelectric focusing gel electrophoresis followed by Western analysis indicated that the predominant form of αB-crystallin was unphosphorylated although small amounts of the phosphorylated form were found in all conditions. The phosphorylation state of αB-crystallin did not change after exposure to stress conditions indicating that the protective role of αB-crystallin is phosphorylation-independent under these conditions.