High glucose burden inhibits EGFR/PI3K/AKT1/mTOR signaling pathway in cutaneous fibroblasts
Abstract
Diabetic healing failure is the clinical expression of countless molecular and cellular disorders having hyperglycemia as the proximal trigger. The fibroblast is a critical building-block cell type for the healing process. Under high glucose concentrations fibroblasts physiology is perturbed. The epidermal growth factor receptor (EGFR) signaling system is crucial for different healing events. We examined the effect of high glucose burden on healthy-donor' cutaneous fibroblasts proliferation, so as the EGFR autophosphorylation on a critical tyrosine residue, along with the activation of downstream signaling pathways proximal to cyclin D1 expression. Fibroblasts were cultured in 15 % FBS at either 5 (normal glucose) or 35 (high glucose) mM under standard culture conditions. Concurrent osmotic control was included. After 6 days of incubation under high glucose, doubling time was calculated. Cells suspensions were plated, fixed and immunolabelled with antibodies directed to phosphorylated forms of EGFR (Y1197), AKT1 (S473) and mTOR (S2448), and native forms of PI3K p85 alpha subunit and cyclin D1. The ratio of cells positive to the diaminobenzidine/peroxidase reaction was calculated and its intensity estimated according to published methodologies. High glucose concentration significantly increased doubling time 5-fold, as compared to cells grown in physiological conditions. Hyperglycemia reduced the constitutive EGFR autophosphorylation. Accordingly, PI3K was also significantly attenuated. Downstream switches AKT1 and mTOR were also affected and very significantly on the signal intensity. Cyclin D1 expression was completely abrogated due to high glucose burden. Collectively, these data suggest that high glucose exposure hinders fibroblasts proliferation by disrupting the EGFR/PI3K/AKT1/mTOR/Cyclin D1 axis.
