Raymond Rackley, Susan Keay *, Mei Kuang, Joseph Abdelmalak, Ashwin Vaze, Sandip Vasavada, Joseph DiDonato ⁺

Glickman Urological Institute, Cleveland Clinic, Cleveland, Ohio

^* Division of Urology, University of Maryland School of Medicine, Baltimore, MD

⁺ Learner Research Institute, Cancer biology, Cleveland Clinic, Cleveland, Ohio

Introduction and Objectives: IC is a chronic debilitating urothelial inflammatory and cytodegenerative condition of the bladder. Our hypothesis is that the NF-kB signaling pathway is an essential signaling mechanism for the pathogenesis of the inflammatory and cellular change/loss response of the bladder urothelium in IC. To test this hypothesis, we have compared NF-kB signaling in primary cultures of normal urothelium (NU) to IC urothelium exposed to TNF-α, as well as, primary cultures of NU exposed to antiproliferative factor, APF, a biomarker produced by IC cells.

Methods: NU and IC cells were established in Keratinocyte-SFM. Expanded cells were switched to serum-free MEM for 24hrs before treated with TNF-α over a 24hr time course and whole cell extracts were prepared for EMSA and Western blot analyses. In situ cell death assays were performed in all cells treated with TNF-α for up to 96 hrs including NU cells first exposed to antiproliferative factor (APF) for 48hrs prior to TNF-α challenge. Apoptosis was detected by TUNEL assay.

Results Obtained: EMSA and Western blot analyses showed that IC cells have dysfunctional NF-kB signaling compared to NU when challenged with a one time dose of TNF-α. The NU cells always have an ~2 fold increase in NF-kB activation over baseline that declines after 30mins, but then is followed by weaker rebound activation around 6hrs. In comparative difference, the IC cells typically have a stronger activation at 30mins, but the second rebound wave never appears. The cytoprotective outcome of comparative NF-kB signaling was tested via in-situ cell death assays. Compared to NU that had no cell death increasing over time, apoptosis was induced in IC cells by TNF-α treatment. For NU cells exposed to APF prior to TNF-α challenge, apoptosis was inducible in the NU when challenged with TNF-α; thus confirming the ability of APF to induce an IC phenotype in NU cells.

Conclusions: Comparison of the TNF-α activated NF-kB signaling pathway in IC represents a dysfunctional urothelial response (aberrant internal cellular control of the signaling pathway) to extracellular stimulation. This dysfunctional signaling results in cytoprotective losses that lead to urothelial apoptosis.

Financial Funding: NIH-NIDDK-R21-DK 066135, NIH-NIDDK-R01-DK 52596 and NIH NCI RO1 CA84406

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