All animal experimentation described here was performed in conformity with the "Guiding Principles for Research Involving Animals and Human Beings" (The studies have been reviewed and approved by the institutional review committee protocols 05-088I and 05-081). Genetically mast cell-deficient WBB6F1-Kit^w/Kit^w-v and normal C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

Acute cystitis was induced as we described previously 2123272829. Briefly, 8–10 week old female mice were anesthetized with isoflurane using a precision vaporizer, 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 on two consecutive days with 200 μl of one of the following substances: PAR-activating peptides (PAR1-AP = SFFLRN 30; PAR2-AP = SLIGRL 30; PAR4-AP = AYPGKF 31, and control inactive peptide = LRGILS 30). All peptides were used at 10 μM concentration 22. Substances were infused at a slow rate to avoid trauma and vesicoureteral reflux 18. To ensure consistent contact of substances with the bladder, infusion was repeated twice within a 30-min interval and a 1-ml Tb syringe was maintained on the catheter end retaining the intravesical solution for at least 1 hour. Afterwards, the catheter was removed and the mice were allowed to void normally. Twenty-four hours post the second instillation, animals were euthanized by cervical dislocation and bladders were rapidly removed and frozen for immunohistochemistry or placed in cold PBS with protease (Protease Inhibitor Cocktail, Sigma, St. Louis, MO) and phosphatase inhibitors (20 mM sodium fluoride, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate) for separation of the urothelium/submucosa away from the detrusor smooth muscle.

To decrease the complexity of using whole bladder homogenates, the bladder mucosa was separated from the detrusor smooth muscle, as described 32. Briefly, immediately after removal from the animal, bladders were placed in PBS with protease inhibitors on ice and visualized under a dissecting microscope (Nikon SMZ 1500) and the detrusor smooth muscle was separated by blunt dissection away from the mucosa which contained the epithelium and sub-epithelial layers. Isolated layers were flash frozen and stored at -80°C until processing. Tissues were pulverized in a spring-loaded tissue pulverizer (Bio-Pulverizer, Biospec Products, Bartlesville, OK) and chilled with liquid nitrogen. Nuclear and cytosolic extracts were prepared using the Pierce NE-PER kit that enables stepwise separation and preparation of cytoplasmic and nuclear extracts from bladder tissue, as described 26. Addition of the first two reagents (Pierce's proprietary information) to the pulverized tissue causes disruption of cell membranes and release of cytoplasmic contents. After recovering the intact nuclei from the cytoplasmic extract by centrifugation at 16,000 × g for 5 minutes, the nuclei are lysed with a third reagent (Pierce's proprietary information) to yield the nuclear extract. Extracts obtained with this product generally have less than 10% contamination between nuclear and cytoplasmic fractions – sufficient purity for most experiments involving nuclear extracts. A western blot was prepared using the nuclear and cytosolic extracts and probed for the nuclear proteins histone H3 and lamin A/C. No nuclear contamination was shown in the cytosolic fractions (data not shown). Protein concentrations were determined with a Micro BCA kit (Pierce, Rockford, IL) per manufacturer's instructions.

Minimum Information About Microarray Experiments – MIAME 33.

PAR1-activating peptide (AP), PAR2-AP, PAR4-AP, and control peptide were instilled into the mouse bladder at the concentration of 10 μM. This treatment induces a strong and reproducible bladder inflammation, whereas no inflammation was observed in mice instilled with 10 μM control peptide 22. We used a protein/DNA combo array that separated bound DNA/protein complexes from unbound probe. The biotin-labeled probe/protein was hybridized to a membrane containing 345 transcription factor consensus sequences. As this assay closely resembles the membrane cDNA arrays, we used the same methodology developed by our collaborator (ID) to analyze the results 273543. Figure 2 illustrates the TFs that were at least 3-fold up-regulated and Figure 3 represents those TFs that were 3-fold down-regulated by each PAR-AP. Table 2 summarizes all TFs. The relative magnitude of the different TF signals between PAR-APs is illustrated in Figure 4. These results strongly suggested that TFEB, a member of the MiTF family of bHLH-Zip transcription factors, was the only TF commonly altered by PAR1-AP, PAR2-AP, and PAR4-AP and therefore, occupied the center of Venn diagram (Figure 2).

To confirm whether the urinary bladder expresses TFEB, we used a commercially available antibody to localize its expression. Figure 5A–B represents the expected morphology of the urinary bladder in a cross-section. Figure 5A is a composite of 18 H&E stained photomicrographs of the same cross-section at low magnification (×100), and illustrates the localization of the different layers: urothelium and submucosa, as well as the detrusor muscle. Figure 5B is a high magnification of the mouse bladder urothelium showing a multi-layered urothelium with apical umbrella cells (arrow). Figure 6 is a representative image of a bladder isolated from a mouse challenged with PAR2-AP (10 μM), which indicates the expression of TFEB, particularly in the nucleus (DAPI-positive) of the urothelial cells. Figure 7 is a representative photomicrograph of the mouse bladder stained with secondary antibody. Figures 8A–I are representative photomicrographs illustrating TFEB expression in the bladder cross sections. Figures 8A–C show the constitutive expression of TFEB in the urinary bladders isolated from mice stimulated with the control peptide, including the urothelium (Figures 8A–C) and detrusor muscle. A higher magnification insert depicts the expression in the smooth muscle (Figure 8B). Figures 8D and 8E are representative images of tissues isolated from PAR1-AP-treated mice and illustrate an overwhelming TFEB expression throughout the urothelial layer, including umbrella cells (Figure 8E). Figures 8F–G and 8H–I are representative of responses to PAR2-AP and PAR4-AP, respectively. Overall, our results indicate a constitutive, although low, expression of TFEB in the control bladder urothelium. Bladder instillation with PAR-APs induced a substantial induction of TFEB expression, primarily in the urothelium, that was clearly observed in response to PAR2-AP and PAR4-AP, and to a lesser extent, with PAR1-AP. Figure 9 presents the quantification of 3 replications of TFEB immunohistochemistry.

Direct evidence for an essential role for mast cells in cystitis was reported by our laboratory in animal models of bladder inflammation 7844. However, the responses to PAR-APs were not investigated. Therefore, we compared the bladder inflammatory responses to PAR1-AP in wild type and Kit^w/Kit^w-v mice. As shown in Figure 10, PAR1-AP induces inflammation in wild type mice, consistent with our previous published results 2122. This inflammation was characterized by intense inflammatory infiltrate in the submucosa and vasodilation (Figure 10A). In contrast, Kit^w/Kit^w-v did not develop an inflammatory response to PAR1 activation (Figure 10B).

We used the same binding sequence present in the Panomics array 34, as the TFEB EMSA probe. Our results indicate that intravesical instillation of C57BL/6 mice with all PAR-APs increased the binding activity to the TFEB probe primarily in extracts of the bladder mucosa (Figure 11A). In contrast, no significant increase was observed in mast cell deficient Kit^w/Kit^w-v mice challenged with PAR1-AP (Figure 11C). In addition to TFEB, other members of the microphthalmia (MiTF)-TFE subfamily of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors are known to bind to this E-Box sequence 454647. Therefore, we used a TFEB antibody to reveal a specific TFEB activity, as illustrated by the super shift bands in tissues isolated from C57BL/6 (Figure 11B) and Kit^w/Kit^w-v mice (Figure 11D). Next, we determined the variability of the EMSA by performing additional experiments in tissues isolated from C57BL/6 mice that were challenged with the control peptide and PAR1-AP (10 μM), Figure 12. ImageJ software was used to quantify the EMSA signals and the integrated density is presented on Figures 13A–C

In order to investigate whether the increased expression of TFEB would translate into an up-regulation of genes known to contain an E-box on their promoter, we used a combination of chromatin immunopreciptation (CHIP) and real-time polymerase chain (Q-PCR). For this purpose, we used the same TFEB antibody employed for IHC. CHIP was obtained from the whole bladder isolated from PAR2-AP- and control peptide-treated bladders. We queried the expression of selected genes known to have an E-box on their promoter and compared the results with an un-transcribed region of the DNA. Figure 14 indicates an increased number of events (p < 0.05) on the CHIP precipitated with TFEB antibody when compared to the un-transcribed region for the following genes: e-cadherin, eif4bp2, serpine 1, IGF1R, and cyclin D1. In addition, inflammation associated with PAR2 stimulation led to an up-regulation of serpine 1, WT1, cyclin D1, IGF1R, and cathepsin K.

The only other finding implicating this family in the urinary tract is their involvement in renal cell carcinoma. In this regard, TFE3 is involved in chromosomal translocations recurrent in renal cell carcinoma 53. Translocations of the genes encoding the related transcription factors TFE3 and TFEB are almost exclusively associated with a rare juvenile subset of renal cell carcinoma and lead to over-expression of TFE3 or TFEB protein sequences. TFE3 and TFEB have been identified as cell type-specific leukemia inhibitory factor-responsive activators of E-cadherin 54.

MiTF mRNA and protein levels are higher in human mast cells than monocytes and granulocytes 55. MiTF seems to be involved in the migration, phenotypic expression, and survival of mast cells 56. As an essential transcriptional effector of the c-kit pathway, MiTF is critical for mast cell 4956 and melanocyte development 49575859. Mice deficient in MiTF harbor a severe mast cell deficiency, and MiTF-mutant mast cells cultured ex vivo display a number of functional defects 60. In addition, c-kit- and m-Csf-dependent MAPK phosphorylation transcriptionally activates both TFE3 and MiTF, which is necessary for mast cell developmental functions 54. Our findings indicate that the bladder inflammatory responses mediated by PAR1 are dependent on the presence of mast cells by a mechanism that involves TFEB. The latter adds to a list of evidence published by this laboratory indicating that Kit^w/Kit^w-v mice do not develop bladder inflammation in response to antigen challenge 7, and substance P 7 stimulation, whereas reconstitution Kit^w/Kit^w-v with mast cells from wild type mice restores the inflammatory response 78. Therefore, we did not test whether the responses mediated by PAR2 and PAR4 are also dependent on the presence of mast cells.

Thrombin and tryptase are endogenous agonists of PARs in humans. Along with serine proteases, it seems that tissue factor and element of the coagulation cascade are also endogenous activators of PARs 6162. In human vascular endothelial cells both thrombin and PAR1-AP elicited gene regulation via E-box signaling 62. However, the information regarding endogenous activators of PARs in the mouse bladder is, unfortunately, scanty. While human connective tissue mast cells contain the enzymes chymase and tryptase, mice contain numerous related proteases 636465. Mouse mast cell protease-7 is a tryptase predominantly expressed in differentiated connective tissue-type mast cells 66. Mast cell proteases mcpt-5 (chymase), and the tryptases mcpt-6 and mcpt-7 are all expressed during the development of the mouse embryo 63. However, to the best of our knowledge, there is no information regarding which particular tryptase is expressed in the mouse bladder and/or upregulated in the mouse model during inflammation. Although thrombin is a recognized physiological activator of PAR1 and PAR4, the endogenous enzymes responsible for activating PAR2 in urinary bladder are not known. Recently, it was demonstrated that the kallikrein family of proteinases are able to regulate PAR signaling and may represent important endogenous regulators PAR1, PAR2, and PAR4 67. The latter are likely to be confirmed in the urinary tract, since members of the kallikrein family play a fundamental role in bladder physiology 68. It is interesting to note that activation of PARs on the bladder urothelium leads to activation of one of the members of the MiTF family. Others have shown that mast cell tryptase and MiTF are highly sensitive and specific markers for mast cell disease 69. Moreover, MiTF regulates the expression of mouse tryptases, such as mcpt-7 70 and mcpt-6, in response to stem cell factor 64 or transforming growth factor-beta stimulation 71.

Mast cells are strategically located for optimal interaction with the environment and for their putative functions in host defense 72. Among the mast cell mediators, tryptase is expressed by most human mast cells comprising as much as 25% of the cells' protein 73, and it is released in response to inflammatory stimuli 7374. Indirect evidence for a role of mast cells in cystitis was reviewed in Ref. 75 and includes increased concentration of tryptase in the urine of interstitial cystitis (IC) patients 9, presence of mast cells containing tryptase in the bladder of IC patients 7576, and that mast cell counts in IC patients are one of the few features significantly associated with night-time frequency (p < 0.01; ref. 77). We published direct evidence that mast cells are essential for both bladder inflammation and gene-regulation in experimental cystitis 78. Our publications show that bladder inflammation is induced in normal mice and not in mast cell-deficient Kit^w/Kit^w-v mice. The differences observed were abolished when Kit^w/Kit^w-v mice had their mast cell deficiency selectively repaired by transplantation of bone marrow-derived mast cells from the normal congenic +/+ mice 78. As the bladder response in these mice is similar to the normal +/+ mice, we concluded that mast cells are essential for the development of cystitis 78.

PARs provide an answer to the question of how mast cell products, such as tryptase, produce signals and induce bladder inflammation. Recently, we characterized the bladder responses to PAR-APs and presented evidence of a mandatory role for PARs in experimental cystitis 22. We went further to determine the regulatory network downstream of PARs 25. Our present findings further emphasize that products of mast cells such as tryptase inducing activation of PARs can lead to increased activity of transcription factors such as TFEB that play a fundamental role in mast cell survival. This positive feedback loop might be responsible for the mast cell-dependent perpetuation of bladder inflammation. However, the precise mechanisms involved in this feed back remain to be determined. One possibility is that the mast cells themselves express PARs and are susceptible to PAR-AP activation 7879. Indeed, PAR1-AP induces mast cell adhesion to fibronectin 79. In contrast, messenger RNA for PAR-1 was detected in peritoneal mast cells 80, but PAR-APs failed to induce histamine release from these cells 80. Another possibility is that inflammatory mediators can modulate the mast cell sensitivity to PAR-APs. Indeed, inflammatory cytokines such as IL-4 and IL-12 were suggested to regulate PAR receptor expression on the mast cells 81.