Hexosamine-induced fibronectin protein synthesis in mesangial cells is associated with increases in cAMP responsive element binding (CREB) phosphorylation and nuclear CREB - The involvement of protein kinases A and C

Hyperglycemia-induced alterations in mesangial (MES) cell function and extr acellular matrix protein accumulation are seen in diabetic glomerulopathy. Recent studies have demonstrated that some of the effects of high glucose ( HG) on cellular metabolism are mediated by the hexosamine biosynthesis path way (HBP), in which fructose-6-phosphate is converted to glucosamine 6-phos phate by the rate-liming enzyme glutamine:fructose-6-phosphate amidotransfe rase (GFA). In this study, we investigated the role of HBP on HG-stimulated fibronectin protein synthesis, a matrix component, in SV-40-transformed ra t kidney MES cells. Treatment of MES cells with 25 mmol/l glucose (HG) for 48 It increases cellular fibronectin levels by two- to threefold on Western blots when compared with low glucose (5 mmol/l). Glucosamine (GlcN; 1.5 mm ol/l), which enters the hexosamine pathway distal to GFA action, also incre ases fibronectin synthesis. Azaserine (AZA; 0.5 mu mol/l), an inhibitor of GFA, blocks the HG-but not the GlcN-induced fibronectin synthesis. Fibronec tin contains cAMP responsive element (CRE) consensus sequences in its promo ter and the phosphorylation of CRE-binding protein (CREB) may regulate its expression. On Western blots, HG and GlcN stimulate two- to threefold the p hosphorylation of CREB at Ser 133, whereas CREB protein content was unalter ed by either HG or GlcN. In addition, nuclear CREB activity was increased b y HG and GlcN on gel-shift assays using P-32-CRE oligonucleotides. AZA impe ded the HG-enhanced CREB phosphorylation and CRE binding but had no effect on GlcN-mediated CREB phosphorylation and CRE binding. Pharmacologic inhibi tion of protein kinase C (PKC) and protein kinase A (PKA), which are involv ed in hexosamine-mediated matrix production, blocked the CREB phosphorylati on and fibronectin synthesis seen in HG and GlcN conditions. We conclude th at the effects of HG on fibronectin synthesis in the mesangium are mediated by the HBP possibly via hexosamine regulation of CREB and PKC/PKA signalin g pathways. These results support the hypothesis that the HBP is a sensor a nd regulator of the actions of glucose in the kidney.