transforming-growth-factor-beta and Galactosemias

Dysmetabolic state in diabetes may lead to augmented synthesis of extracellular matrix (ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin (FN) production and that of its splice variant, EDB(+)FN, is regulated by protein kinase B (PKB, also known as Akt). In this study, we investigated the role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKBalpha knockout mice and wild-type control littermates. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 wk starting at 6 wk of age. We investigated FN mRNA, EDB(+)FN mRNA, and transforming growth factor (TGF)-beta mRNA expression, Akt phosphorylation, Akt kinase activity, and NF-kappaB and AP-1 activation in the retina, heart, and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB(+)FN, and TGF-beta in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which was prevented in the Akt knockout mice. NF-kappaB and AP-1 activation was pronounced in galactose-fed wild-type mice and prevented in the galactose-fed Akt1/PKBalpha-deficient group. In the retina and kidney, Ser473 was the predominant site for Akt phosphorylation, whereas in the heart it was Thr308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation may be an important mechanism leading to NF-kappaB and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.

Topics: Animals; Diabetes Complications; Extracellular Matrix; Fibronectins; Galactosemias; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Mice, Knockout; Proto-Oncogene Proteins c-akt; Transcription Factors; Transforming Growth Factor beta

Osmotic and oxidative stress is associated with the progression and advancement of diabetic cataract. In the present study, we used a cDNA microarray method to analyse gene expression patterns in streptozotocin-induced diabetic rats and galactose-fed cataractous lenses. In addition, we investigated the regulation and interaction(s) of anti-oxidant protein 2 and lens epithelium-derived growth factor in these models.. To identify differential gene expression patterns, one group of Sprague-Dawley rats was made diabetic with streptozotocin and a second group was made galactosaemic. Total RNA was extracted from the lenses of both groups and their controls. Labelled cDNA was hybridised to Atlas Rat Arrays. Changes in gene expression level were analysed. Real-time PCR and western analysis were used to validate the microarray results.. The expression of 31 genes was significantly modulated in hyperglycaemic lenses compared with galactosaemic lenses. Notably, transcript and protein levels of B-cell leukaemia/lymphoma protein 2 and nuclear factor-kappaB were significantly elevated in rat lenses at 4 weeks after injection of streptozotocin. At a later stage, mRNA and protein levels of TGF-beta were elevated. However, levels of mRNA for IGF-1, lens epithelium-derived growth factor and anti-oxidant protein 2 were diminished in streptozotocin-induced diabetic cataract.. These results provide evidence that progression of sugar cataract involves oxidative- and TGF-beta-mediated signalling. These pathways may promote abnormal gene expression in the hyperglycaemic and galactosaemic states and thus may contribute to the symptoms associated with these conditions. Since oxidative stress seems to be a major event in cataract formation, supply of anti-oxidant may postpone the progression of such disorders.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cataract; Diabetes Mellitus, Experimental; DNA, Complementary; Down-Regulation; Galactose; Galactosemias; Gene Expression Profiling; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Lens, Crystalline; NF-kappa B; Oligonucleotide Array Sequence Analysis; Peroxidases; Peroxiredoxins; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation