Inducible Activation of NFkB on Cultured Normal Human Urothelial Cells

Shin, Myung Sik; Appell, Rodney

Abstract

PURPOSE: We investigated the inducible response of transcription factor Nuclear Factor Kappa B(NFkB) in response to lipopolysaccharide(LPS), interferons(INFs), and tumor necrosis factor(TNF) on the normal urothelial cells derived from the ureter and the bladder.
MATERIALS AND METHODS
Urothelial cells were harvested from the ureter and the bladder, cultured, passaged and expanded in serum free medium. Immunostaining of the urothelial cells with reacting anti-cytokeratin antibodies was done to identify a stable epithelial phenotype. Cultured human urothelial cells were either untreated and treated with LPS, INFgamma, INFalpha and TNFalpha. Cell extracts were prepared and used for electrophoretic mobility shift assay(EMSA) using PRD II-kB as probes for NFkB respectively.
RESULTS
We cultured urothelial cells successfully confirmed by immunohistochemical staining. In both urothelial cell types, NFkB bindings with their respective probe were induced by treatment with LPS, INFgamma and TNFalpha. NFkB was weakly induced by INFalpha. For the NFkB complex, a distinctly different migrating pattern of the NFkB was noted between the diffrent urothelial cells.
CONCLUSIONS
Many urothelial diseases are unique to specific areas of the urinary collecting system. The characterization of different inducible responses of transcription factors involved in cytoplasmic and nuclear signaling of genes encoding immunologically relevant proteins provide a unique opportunity for understanding disease presentation and designing specific treatment interventions.