PURPOSE: To characterize the basal and activated form of nuclear factor-kappaB (NF-kappaB) complex in normal urothelial cell cultures after stimulation with tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and double-stranded ribonucleic acid (dsRNA). MATERIALS AND METHODS: Human urothelial cells cultured from normal bladder specimens underwent immunohistochemical staining and cellular extracts were prepared for Electrophoretic Mobility Shift Assays (EMSA), Western blot analyses, RNA isolation and Northern Blot analyses before and after stimulation with TNF-alpha, LPS, and dsRNA. RESULTS: In normal human urothelial cells, activation of the NF-kappaB complex in response to stimulation with TNF-alpha, LPS, and dsRNA was detected by immunohistochemical methods and EMSA. Depending on the stimulus, a specific NF-kappaB complex was activated as seen by supershift experiments in EMSA. By Western blot, the inhibitor of NF-kappaB complex, IkappaB-alpha, degraded in response to stimulation. Northern blot analysis from total RNA revealed subsequent inducible interleukin-8 (IL-8) mRNA expression of normal urothelial cells when treated with TNF-alpha, LPS, and dsRNA. CONCLUSIONS: Normal human urothelial cells contain basal NF-kappaB complexes in an inactivated state. When these cells are challenged by different agents such as TNF-alpha, LPS, and dsRNA, the cells respond by activation of the NF-kappaB signal transduction pathway, degradation of its inhibitor, IkappaB-alpha, and translocation of this primary factor into the nucleus to induce specific genetic responses such as IL-8 expression.
PURPOSE: To characterize the basal and activated form of nuclear factor-kappaB (NF-kappaB) complex in normal urothelial cell cultures after stimulation with tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and double-stranded ribonucleic acid (dsRNA). MATERIALS AND METHODS:Human urothelial cells cultured from normal bladder specimens underwent immunohistochemical staining and cellular extracts were prepared for Electrophoretic Mobility Shift Assays (EMSA), Western blot analyses, RNA isolation and Northern Blot analyses before and after stimulation with TNF-alpha, LPS, and dsRNA. RESULTS: In normal human urothelial cells, activation of the NF-kappaB complex in response to stimulation with TNF-alpha, LPS, and dsRNA was detected by immunohistochemical methods and EMSA. Depending on the stimulus, a specific NF-kappaB complex was activated as seen by supershift experiments in EMSA. By Western blot, the inhibitor of NF-kappaB complex, IkappaB-alpha, degraded in response to stimulation. Northern blot analysis from total RNA revealed subsequent inducible interleukin-8 (IL-8) mRNA expression of normal urothelial cells when treated with TNF-alpha, LPS, and dsRNA. CONCLUSIONS: Normal human urothelial cells contain basal NF-kappaB complexes in an inactivated state. When these cells are challenged by different agents such as TNF-alpha, LPS, and dsRNA, the cells respond by activation of the NF-kappaB signal transduction pathway, degradation of its inhibitor, IkappaB-alpha, and translocation of this primary factor into the nucleus to induce specific genetic responses such as IL-8 expression.
Authors: Eugene V Vykhovanets; Gregory T MacLennan; Olena V Vykhovanets; Edward E Cherullo; Lee E Ponsky; Sanjay Gupta Journal: J Urol Date: 2011-11-17 Impact factor: 7.450