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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Activation of the transcription factors nuclear factor-kappaB and activator protein-1 in bladder smooth muscle exposed to outlet obstruction and mechanical stretching.

PURPOSE: Transcriptional control of bladder genes in response to outlet obstruction, growth factors and mechanical force is poorly understood. We analyzed the effects of bladder obstruction, mechanical stretching and platelet derived growth factor on the activation of the major growth controlling transcription factors nuclear factor-kappaB and activator protein-1. MATERIALS AND METHODS: Complete outlet obstruction was created in female rats by proximal urethral ligation and bladders were harvested 3, 6 and 24 hours later, respectively. Bladder cells were grown in culture and stimulated with 10 ng./ml. platelet derived growth factor or 10 cycles per minute of mechanical stretching for 0.5 to 4 hours. Nuclear proteins were high salt extracted and incubated with 32phosphorus double strand oligonucleotides containing a consensus binding sequence for activator protein-1 or nuclear factor-kappaB. The resulting DNA protein complexes were analyzed by electrophoretic mobility shift assay. RESULTS: Nuclear extract isolated from obstructed bladders showed intense activator protein-1 binding activity 3, 6 and 24 hours after obstruction as well as increased nuclear factor-kappaB binding activity after 6 and 24 hours. Binding activity was absent or minimal in sham operated rats. Cultured cells exposed to mechanical stretching for 2 and 4 hours showed increased activator protein-1 and nuclear factor-kappaB DNA binding compared with unstretched cells. Likewise stimulation with platelet derived growth factor caused a consistent increase in activator protein-1 and nuclear factor-kappaB binding activity. The binding of nuclear proteins was abolished by a 40-fold excess of an unlabeled specific oligonucleotide but not by excess irrelevant oligonucleotide. Thus, the assays were specific for the factors involved. CONCLUSIONS: Bladder obstruction and mechanical stretching cause the formation of activator protein-1 and nuclear factor-kappaB DNA complexes, consistent with a role of these transcription factors in the control of hypertrophy associated gene activation.[1]

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