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Abstract
Characterization of the phosphoinositide 3-kinase-signaling pathway in a human renal tubular epithelial cell (TEC) line HKC-8 revealed high levels of Akt phosphorylation in serum-starved cultures. In contrast to Erk1/2, little additional phosphorylation of Akt was observed after cytokine or serum stimulation. Replacement of the conditioned medium attenuated Akt phosphorylation such that 90 min after the addition of warmed serum-free media, Akt phosphorylation had fallen sufficiently to allow an epidermal growth factor-stimulated increase to be detected readily. Although the mechanism by which the phosphoinositide 3-kinase/Akt pathway is activated in serum-starved TEC is unknown, the mediator responsible is secreted from these cells. Thus, conditioned media removed from a dish of quiescent TECs stimulated Akt phosphorylation in washed TEC cultures within 10 min. Biochemical characterization of the bioactive agent identified a heat labile factor of small apparent molecular mass. The basal level of Akt phosphorylation observed in serum-starved cultures was inhibited by wortmannin at concentrations that demonstrated its dependence on 3-phosphoinositide synthesis (IC(50) = 8 nm). Regular removal of conditioned media from TEC cultures and its replacement with serum free media resulted in a sustained attenuation of Akt phosphorylation. Interestingly, after 5 days of this treatment, washed TEC cultures contained a greater number of viable cells than cultures maintained in conditioned media throughout. This observation was not explained by a difference in the rate of DNA synthesis. Instead, the number of cells undergoing apoptosis increased markedly in the unwashed cultures. Consequently, we propose that in HKC-8 cells Akt phosphorylation is up-regulated in an effort to minimize cell death. This stress-activated response is initiated by a factor secreted into the conditioned medium that stimulates the phosphoinositide 3-kinase signaling pathway.
Highlights
The mechanism by which the phosphoinositide 3-kinase/Akt pathway is activated in serum-starved tubular epithelial cell (TEC) is unknown, the mediator responsible is secreted from these cells
In this study we investigate the significance of markedly elevated levels of Akt phosphorylation found in serum-starved cultures of the renal TEC line HKC-8
The results of this study reveal an inverse correlation between the basal level of Akt phosphorylation and long term viability of HKC-8 cells under serum-free conditions
Summary
The anti-apoptotic role of the PI3K/Akt pathway is widely acknowledged, few studies demonstrate increased levels of Akt phosphorylation under conditions where cell viability has been compromised. This is somewhat surprising given that activation of this signaling pathway might be expected in response to stress and cell damage. In this study we investigate the significance of markedly elevated levels of Akt phosphorylation found in serum-starved cultures of the renal TEC line HKC-8 We investigate whether this increased Akt phosphorylation correlates with alterations in either cell viability or proliferation
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