ABSTRACT: Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-alpha). For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background) were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA) for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-alpha, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR) was used to confirm some of the BCG-specific transcripts.Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/beta-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the development of the lymphatic system. Interestingly, TNF-alpha-specific networks overlapped with the following canonical signaling pathways: PPAR, death receptor, and apoptosis. Finally, LPS-specific networks overlapped with the LPS/IL-1 mediated inhibition of RXR. Because NF-kappaB occupied a central position in several networks, we further determined whether this transcription factor was part of the responses to BCG. Electrophoretic mobility shift assays confirmed the participation of NF-kappaB in the mouse bladder responses to BCG. In addition, BCG treatment of a human urothelial cancer cell line (J82) also increased the binding activity of NF-kappaB, as determined by precipitation of the chromatin by a NF-kappaB-p65 antibody and Q-PCR of genes bearing a NF-kappaB consensus sequence. Next, we tested the hypothesis of whether small GTPases such as LRG-47 are involved in the uptake of BCG by the bladder urothelium.As expected, BCG treatment induces the transcription of genes belonging to common pro-inflammatory networks. However, BCG also induces unique genes belonging to molecular networks involved in axonal guidance and lymphatic system development within the bladder target organ. In addition, NF-kappaB seems to play a predominant role in the bladder responses to BCG therapy. Finally, in intact urothelium, BCG-GFP internalizes in LRG-47-positive vesicles. These results provide a molecular framework for the further study of the involvement of immune and nervous systems in the bladder responses to BCG therapy.
