GLUT1 induced cFLIP expression promotes proliferation and prevents apoptosis in VSMCs

Enhanced GLUT1 has been shown to inhibit apoptosis in several cell systems including vascular smooth muscle cells (VSMCs). A decrease in apoptosis could lead to increased VSMC numbers in neointimal and medial arterial layers under several pathologic conditions. The hypothesis underlying these studies is that GLUT1 induces expression of anti-apoptotic and pro-survival genes that increase VSMC survival. Transcriptomic analysis of A7r5 VSMCs, in which GLUT1 was acutely overexpressed, showed a 2.14-fold increase in cFLICE Inhibitory Protein (cFLIP), which promotes cellular growth and prevents apoptosis through caspase 8 binding. We confirmed that overexpression of GLUT1 results in enhanced mRNA and protein expression of both cFLIPL and cFLIPS isoforms, in primary and stable immortalized VSMC lines, as well as in aortae from GLUT1 transgenic mice. Increased GLUT1 reduced VSMC death by more than 2-fold after serum withdrawal, as evidenced by decreased caspase 3 activity and trypan blue exclusion studies. GLUT1 overexpression also resulted in a greater than 2-fold increase in PCNA expression and live cell numbers, consistent with augmented VSMC proliferation. Lentiviral knock-down of cFLIPL showed that cFLIPL was necessary for the pro-proliferative and anti-apoptotic effects of GLUT1 overexpression. In addition, exposure to TNF induced activated NFkappaB, as demonstrated by IkB alpha phosphorylation, and induced mRNA expression of Inhibitor of Apoptosis 1 (IAP-1). IAP is a molecule for which NFkappaB acts as a transcription factor, only in GLUT1-overexpressing cells. Further, overexpression of human cFLIPL was sufficient to increase IAP-1 expression in the presence of TNF, which was compounded in GLUT1-overexpressing cells. This suggests that cFLIPL is sufficient to restore TNF signaling and to increase TNF-induced IAP-1 expression in response to GLUT1. Increased IAP-1 expression was also observed in aortae excised from GLUT1 transgenic mice. Taken together, these data suggest that GLUT1 induction of cFLIPL expression augments proliferation and prevents apoptosis in VSMCs. We found that GLUT1-enhances signaling via the PI3K/AKT/GSK3 pathway and by inhibiting metabolism with 2-deoxy-glucose we were able to confirm that both AKT (S473) phosphorylation and cFLIPL mRNA expression were dependent on glycolytic metabolism. The PI3K/AKT/GSK3 and NFkB pathways can account, in part, for some of the anti-apoptotic and pro-proliferative effects of GLUT1 and cFLIP.