It has to be taken into consideration that the approach of this study permitted the generation of new testable hypothesis rather than the more traditional hypothesis-driven research. Indeed, the major question being answered by this work is what TREs can be therapeutically targeted for reducing the influence of tachykinins in bladder disorders? The method introduced here, supplements the standard procedure of multiple paired comparisons used in microarray analysis by associating the expression level of each gene in the experimental group with a family transcription regulatory elements and to compare with the occurrence of each TRE in a reference file (all genes in the array). This bottom-up approach builds mechanistic models for each individual case, e.g., identifying the binding sites for selected genes and their respective TREs , then specifies the role of each TRE in the network generating a testable hypothesis for the network downstream of NK1R activation. Next, we used EMSA to confirm that selected TREs (NF-kappaB and NKx-2.5) are indeed part of the molecular network downstream of NK1R activation.
An extended family of TREs was significantly correlated with NK1R-dependent genes. Those included c-Rel, NF-kappaB_Q6, PAX-6, CREB_01, CRE-BP1/c-Jun, and v-Myb (Figure 4). However, most of the studies on transcription regulatory elements in urology are related to oncology, which makes it difficult to further illustrate the clinical relevance of our findings. Therefore, we are discussing only the most relevant TREs that modulate bladder inflammatory responses to SP.
AP1 was among the NK1R-dependent TREs. We have provided evidence for a predominant role for AP1 controlling highly expressed NK1R-dependent genes . In the present work, we confirmed the regulatory relationships between AP1 (AP1_Q2 and AP1_C) and NK1R-dependent genes. It is known that the activation of MAPK (JNK, p38) and NF-kappaB signaling pathways leads to the activation of AP-1 and, consequently, the inflammation . The present results extend these findings to the urinary bladder where these pathways can be explored as potential therapeutic targets to decrease the symptoms of cystitis.
NF-kappaB is believed to trigger both the onset and the resolution of inflammation. NF-kappaB activity is correlated with bladder cancer [50–52] and bladder urothelial cells respond to insults with a translocation of NF-kappaB  leading ultimately to an increased NK1R expression . Our present work confirms previous indication that Tachykinins, such as SP, activate NF-kappaB translocation [55, 56]. Indeed, in the urinary bladder, activation of NK1R by SP induces NF-kappaB translocation, as seen by EMSA results (Figure 9), and up-regulation of pro-inflammatory genes, such as the encoding prostaglandin I2 receptor (Figure 6).
Another TRE over-expressed in the NK1R-dependent cluster was the upstream stimulatory factor (USF). Although widely expressed, USF can mediate tissue-specific transcripts. USF is stimulated by glucose in murine mesangial cells, binds to TGF-β1 promoter, contributes to TGF-β1 expression, and may play a role in diabetes-related gene regulation in the kidney .
However, the most impressive switch between NK1R-dependent and independent transcripts was the one observed with two different matrixes of Nkx-2.5 (_01 and _02). Both Nkx-2.5_01 and _02 are binding sites derived from mouse sequences [58, 59]. Nkx-2.5 is a murine homeobox named tinman homeodomain factor and is considered to be a new member of the sub-family of homeobox genes related to the Drosophila . Nkx-2.5 is proposed as a valuable marker in the analysis of mesoderm development . It was first described as an essential transcription factor for normal heart morphogenesis, myogenesis, and function . However, more recently it was shown that Nkx-2.5 is required for the expression of atrial natriuretic peptide  and, along with NF-kappaB, is part of the brain natriuretic peptide promoter . Outside of the heart, this element is important in vessel remodeling , skeletal myogenesis , and pyloric sphincter development . Other sites of Nkx-2.5 expression include pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver .
To our knowledge, this is the first report describing a role for Nkx-2.5 in the urinary tract. In the presence of NK1R, Nkx-2.5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, NGF, TSP2) (Table 3, [additional file 3]). In the absence of NK1R, the matrix _02 had a predominant participation driving 8 transcripts, which includes those involved in cancer (EYA1, Trail, HSF, and ELK-1), smooth-to-skeletal muscle trans-differentiation, and Z01, a tight-junction protein, expression (Table 3, [additional file 3]).
An interesting finding was the constitutive translocation of NKx-2.5 and NF-kappaB in the bladder mucosa. One possible explanation was that mechanical stimulation caused by instillation of saline caused the shift. Therefore, an additional control group was added in which the bladder was removed without instillation. This group (0 hours) also presented a certain amount of shifted NKx-2.5 and NF-kappaB probes. An alternative explanation for these results is that mechanical isolation of the bladder mucosa caused the translocation of both transcription factors. We, therefore, generated preliminary results using an urothelial cell line (J82) which indicated a constitutive activation of both NF-kappaB and NKx-2.5 in the absence of overt stimulation (data not shown). Therefore, we suggest that the bladder mucosa/urothelium might present a constitutive activation of both transcription factors. The similarity of basal translocation of NKx-2.5 and NF-kappaB translocation in the urinary bladder may be related to an overlap of binding motifs in some genes. Indeed, others have shown an overlap of conserved DNA binding motifs including AP-1 sites, NF-kappaB, GATA, and Nkx-2.5 in promoter regions of genes, such as MMP13 .