Figure 1.

Glucose-6-phosphate dehydrogenase (G6PD) inhibition, G6pd knockdown, and ectopic expression of the loss-of-function Mediterranean G6PD variant increase expression of smooth muscle cell (SMC)-restricted genes and decrease cell growth. A: glucose metabolism through the pentose phosphate pathway (PPP) (schematic, top) and levels of 6-phospho-d-gluconate (6PGL) and sedoheptulose-1-phosphate (Sedo-1-phos), 2 metabolites in the PPP, are decreased in SMCs treated with N-ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea (NEOU, 1 µmol/L; bottom). AU, arbitrary units. B: Western blot data representative of 5 experiments demonstrating that G6PD inhibition with epiandrosterone (EPI; 50 µmol/L) or NEOU (1 µmol/L) increases expression of myosin heavy chain 11 (MYH11) in A7r5 cells. Summary data showing that expression of MYH11, transgelin (TAGLN, also known as SM22α), and α-actin (ACTA2) is increased and Krüppel-like factor 4 (KLF4) decreased in A7r5 cells treated for 48 h with vehicle control (Ctrl; n = 5 samples) or NEOU (1 µmol/L; n = 5 samples). C: Western blot data representative of 5 experiments demonstrating G6pd knockdown with Ad-G6PD-shRNA [10¹² plaque-forming units (PFU]. Scrambled (Scr) RNA was used as the control for G6PD-shRNA, and α-tubulin (TUBA1A) served as a loading control. D and E: expression of smooth muscle myosin heavy chain (Myh11; D) and myocardin (Myocd; E) mRNA in A7r5 cells and primary rat (RSMCs) and mouse (MSMCs) aortic SMCs is higher after treatment for 48 h with NEOU (1 µmol/L; n = 5 samples) vs. vehicle control (n = 5 samples). F: numbers of human coronary artery SMCs (HSMCs) and RSMCs, determined in CyQUANT Cell Proliferation Assays, are decreased after treatment for 48 h with NEOU (1 µmol/L; n = 6 samples) vs. vehicle control (n = 6 samples). G: ectopic expression of enzymatically active human G6PD (wild type) and inactive human G6PD (S210A) mutant in A7r5 cells is shown. A7r5 cells expressing inactive human G6PD mutant grew slower than those expressing active human G6PD 48 h after transfection. H: RT-PCR results showing that relative expression of Myocd and Myh11 was higher in aorta from Mediterranean G6PD variant (S188F) than wild-type rats. I: representative Western blot and summary results demonstrating that relative expression of MYH11 and TAGLN were higher in aortas from Mediterranean G6PD variant (lane 1) than wild-type (lane 2) rats. MW, molecular weight.