Hyperglycemia-induced alterations in mesangial (MES)cell function and extracellular 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 pathway (HBP), in which fructose-6-phosphate is converted to glucosamine 6-phosphate by the rate-liming enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). In this study, we investigated the role of HBP on HG-stimulated fibronectin protein synthesis, a matrix component, in SV-40 -transformed rat kidney MES cells. Treatment of MES cells with 25 mmol/l glucose (HG) for 48 h increases cellular fibronectin levels by two-to threefold on Western blots when compared with low glucose (5 mmol/l). Glucosamine (GlcN; 1.5 mmol/l), which enters the hexosamine pathway distal to GFA action, also increases fibronectin synthesis. Azaserine (AZA; 0.5 mol/l), an inhibitor of GFA, blocks the HGbut not the GlcN-induced fibronectin synthesis. Fibronectin contains cAMP responsive element (CRE) consensus sequences in its promoter and the phosphorylation of CRE-binding protein (CREB) may regulate its expression. On Western blots, HG and GlcN stimulate two-to threefold the phosphorylation of CREB at Ser 133, whereas CREB protein content was unaltered by either HG or GlcN. In addition, nuclear CREB activity was increased by HG and GlcN on gel-shift assays using 32 P-CRE oligonucleotides. AZA impeded the HGenhanced CREB phosphorylation and CRE binding but had no effect on GlcN-mediated CREB phosphorylation and CRE binding. Pharmacologic inhibition of protein kinase C (PKC) and protein kinase A (PKA), which are involved in hexosamine-mediated matrix production, blocked the CREB phosphorylation and fibronectin synthesis seen in HG and GlcN conditions. We conclude that the effects of HG on fibronectin synthesis in the mesangium are mediated by the HBP possibly via hexosamine regulation of CREB and PKC/PKA signaling pathways. These results support the hypothesis that the HBP is a sensor and regulator of the actions of glucose in the kidney. Diabetes 50:2355-2362, 2001D iabetic nephropathy is characterized by the accumulation of extracellular matrix (ECM) proteins in the glomerulus and is represented morphologically by thickening and expansion of the glomerular basement membrane and the mesangium (1,2). Hyperglycemia is an important contributor to the development of diabetic nephropathy in both type 1 and type 2 diabetes (3). However, the mechanisms underlying the effects of chronic hyperglycemia on the kidney are not fully understood. Transforming growth factor  (TGF-), protein kinase C (PKC), and protein kinase C (PKC) have been implicated in ECM production and the development of diabetic nephropathy (4 -7). The addition of neutralizing antibodies against TGF- in mesangial (MES) cells cultured in high-glucose (HG) medium blocks the synthesis of matrix proteins (5). Recent studies indicate that PKC is activated in diabetic glomeruli (5,6) an...
Although gammadelta T cells have been implicated in various aspects of the dermal wound healing process, their role in postburn wound healing processes has not been investigated. To study this, we subjected mice deficient in gammadelta T cells (ie, T-cell receptor delta gene [delta TCR]) and wild-type (WT; C57BL6J) mice to burn injury (25% TBSA) or sham treatment; skin samples were isolated 3 days later. Marked inflammation of the injury site was observed in WT mice but was markedly reduced in delta TCR mice. Postinjury fibroblast growth factor, platelet-derived growth factor granulocyte-colony stimulating factor levels, and nitrite/nitrate were elevated in skin samples from injured WT mice, whereas skin tissue levels of these growth factors and inflammatory mediators was significantly atteunuated in delta TCRmice. In conclusion, these findings support the concept that gammadelta T cells are important to postburn wound healing via the production of growth factors and, potentially, regulation of inducible nitric oxide synthase activation.
Hyperglycemia-induced alterations in mesangial (MES) cell function and extracellular matrix (ECM) protein accumulation are seen in diabetic glomerulopathy. Transforming growth factor-beta1 (TGF-beta1) mediates high-glucose-induced matrix production in the kidney. Recent studies demonstrated that some of the effects of high glucose on cellular metabolism are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine (GlcN) 6-phosphate. We previously showed that the high-glucose-mediated fibronectin and laminin synthesis in MES cells is mediated by the HBP and that GlcN is more potent than glucose in inducing TGF-beta1 promoter luciferase activity. In this study, we investigated the hypothesis that the effects of glucose on MES matrix production occur via hexosamine regulation of TGF-beta1. Culturing simian virus (SV)-40-transformed rat kidney MES cells in 25 mM glucose (HG) for 48 h increases cellular fibronectin and laminin levels about twofold on Western blots compared with low glucose (5 mM). GlcN (1.5 mM) or TGF-beta1 (2.5-5 ng/ml) for 24-48 h also increases ECM synthesis. However, the effects of HG or GlcN with TGF-beta1 are not additive. The presence of anti-TGF-beta1 antibodies (20 microg/ml) blocks both TGF-beta1- and GlcN-induced fibronectin synthesis. TGF-beta1 increased ECM levels via PKA (laminin and fibronectin) and PKC (fibronectin) pathways. Similarly, TGF-beta1 and hexosamines led to nonadditive increases in phosphorylation of the cAMP responsive element binding transcription factor. These results suggest that the effects of excess glucose on MES ECM synthesis occur via HBP-mediated regulation of TGF-beta1.
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