Substance P receptor-dependent genes and TFs

Tachykinins (TKs) are a family of peptides involved in the peripheral and central regulation of urinary functions through the stimulation of neurokinin (NK) NK1, NK2 and NK3 receptors. Recent evidence indicates that NK1 receptors are required in antigen-induced cystitis. Therefore, determining the regulatory network downstream NK1 receptor activation is a key step toward understanding the role of TKs in inflammation. For this purpose, we used a Transcriptional Regulatory Network Analysis (TRNA) to identify biologically relevant transcriptional regulatory elements (TRE) that underline the NK1-dependent gene expression in bladder responses to inflammation. Gene expression analysis was obtained using the urinary bladder isolated from WT and NK1-R-/- mice that were stimulated with intravesical instillation of saline or antigen challenge (in sensitized mice) in order to promote cystitis. Based on cDNA array results, we selected a cluster of genes that was dependent on NK1 receptors for their up-regulation in response to inflammation. Next, we used PAINT database to retrieve upstream promoter sequences for those NK1-R-dependent genes and to identify TREs on those promoters. Finally, TREs were enriched statistically by selecting only those that were significantly expressed and a regulatory network downstream of NK1 receptor activation was determined. This work indicates an overriding participation of NK1 receptors in bladder inflammation, provides a working model for the involvement of transcription regulators such as AP1, NF-kB, and Nkx-2.5, and evokes testable hypotheses regarding the regulatory network downstream of NK1 receptor activation. Keywords: URINARY BLADDER INFLAMMATION All mice in this study were sensitized with 1 µg DNP4-human serum albumin (HSA) in 1 mg alum on days 0, 7, 14, and 21, intraperitoneally (i.p.). In normal mice, this protocol induces sustained levels of IgE antibodies up to 56 days post-sensitization (20). One week after the last sensitization, cystitis was induced. Briefly, sensitized WT and NK1R-/- mice were anesthetized (ketamine 40 mg/kg and xylazine 2.5 mg/kg, i.p.), then transurethrally catheterized (24 Ga.; 3/4 in; Angiocath, Becton Dickson, Sandy, Utah), and the urine was drained by applying slight digital pressure to the lower abdomen. The urinary bladders were instilled with 200 µl of pyrogen-free saline or DNP4-OVA (1 µg/ml). One, four, and twenty-four hours after instillation, mice were sacrificed with pentobarbital (100 mg/kg, i.p.) and bladders were removed rapidly. Gene expression was determined using Clontech 1.2 K arrays and genes specifically regulated in the WT were compared with those upregulated in NK1 KO. We have employed a bioinformatics approach to hypothesize functionally relevant transcriptional regulatory elements (TREs) of NK1R-dependent and -independent genes. We used the Promoter Analysis and Interaction Network Toolset (PAINT) available online at http://www.dbi.tju.edu/dbi/tools/paint (58). PAINT is a suite of bioinformatics and computational tools that integrate functional genomics information, as is the case of our microarray-based gene expression data, with genomic sequence and TRE data to derive hypotheses on the TREs relevant to the biological function under study. The TRE hypotheses are generated from statistical enrichment analysis and are defined as those TREs that are significantly enriched such as NK1R-dependent and –independent genes over random occurrence in the gene groups (58). The random occurrence is relative to a ‘reference’: all the genes on the microarray. Employing the microarray accounts for any ‘bias’ present in the genes on the microarray relative to entire genome and guards from incorrectly concluding that certain TREs are relevant to the current experiment.