Seventy five genes fulfilled both the criteria of being expressed 3-fold higher after stimulation with SP or LPS and of not changing expression in response to the same stimuli of PAR1^-/- mice, and were considered to be PAR1-dependent (Additional files 1 and 2, Table 1A and 1B). To further annotate this set of genes, we used a web-based entry tool developed by Ingenuity Pathways Analysis [IPA] 27to query their knowledge database 272829. The resulting networks contain: A- Sub-cellular layout indicating the predominant location for the expression of encoded proteins (Figure 1); B-Analysis of canonical pathways significantly associated with this group of genes (Figure 2), and C-D- Biological functions across the entire dataset most significantly associated with this set of genes (Figure 3 and 4).

Genes (green) were associated with function (magenta) and localized to each compartment according to IPA and gene ontology. Dotted lines further group the genes by activity or function such as signal transduction, cytoskeleton re-organization and cell motility, carbohydrate metabolism, proteins and enzymes, transcription, and RNA editing. Of note, a particular set of genes such as dctn1 and arf6 are involved in PAR trafficking, and their function is associated with other PAR1-dependent transcripts with function in the cytoskeleton reorganization.

Overall, PAR1-dependent transcripts belong to several canonical pathways (Figure 2). This type of IPA also revealed key genes that are significantly associated with more than one canonical pathway. This is the case of elk1, nfkbia, hspb1, dusp1, and akt2. In addition, this analysis indicates pathways such as VEGF and integrins which share common genes, as is the case of those encoding cytoskeleton proteins actg1 and actb.

Figures 3 and 4 represent biological functions significantly (p < 0.01) associated with PAR1-specific genes. Those included: apoptosis (n = 26 genes); cell death (n = 29); cell survival (n = 10); cancer (n = 29); cellular growth and proliferation (n = 29); cell-to-cell signaling (n = 15); hematological disease (n = 9); cellular movement (n = 16); gene expression (n = 18); immune and lymphatic system development and function (n = 9); immune response/disease (n = 19); and inflammation and inflammatory disease (n = 22). This type of analysis also revealed PAR-1 dependent genes (upk2, jundD, ptprc, and nfkbp1a) encoding proteins associated with renal and urological diseases. Figure 1D also indicates common pathways of PAR1-dependent transcripts shared by inflammation (red) and cancer (green) and revealed possible targets uniquely associated with some of the biological functions.

Of the 19 genes tested by Q-PCR analysis of CHIP isolated from wild type mice challenged with control peptide, PAR1- and PAR2-AP, 4 genes (adam-3, dctn1, elk1, and mmp2) had their control levels 1.5 times below background (un-transcribed region) and, therefore, their results are not being presented. Results are presented in figure 5 as averaged Transcription Events Detected Per 1000 Cells for each gene tested and their standard deviations. With the exception of pla2G1b, these results indicate that treatment of wild type mice with PAR1- and PAR2-AP induced up-regulation of the following PAR1-dependent genes: actb, akt2, arf6, ccl7, cd63, dusp1, fkbp1a, nfkbia, phlda1, plaur, s100a10, tnfaip3, ube2h, and upk2.

Our cDNA array results indicate that pla2G1b encoding a calcium-dependent PLA₂ was a PAR1-dependent transcript. However, this particular transcript was not validated by Q-PCR of actively-transcribed DNA (Figure 5). Because of the prominent role of the calcium-independent phospholipase A₂ (iPLA₂) in mediating tryptase-activated PAR in bladder microvascular endothelial cells 30, bladder urothelial cells 3132, and PAR2-AP in bladder contractions 33, we decided to determine whether bladder instillation with PAR-APs also induced up-regulation of iPLA₂ in the mouse bladder mucosa. The rationale for using only the mucosa was to decrease the complexity of the bladder system, getting closer to the urothelial cell in culture used by the other authors 3132. For this purpose, cytoplasmic extracts from the bladder mucosa of WT mice instilled with PAR-APs- or control peptide- were subjected to Western Blot analysis to measure relative concentration of iPLA₂. Results are representative of 4 separate experiments (Figure 6). The bar graph shows the average densitometric values (Figure 7). When data was normalized with beta-actin used as loading control, the same relationship was observed (data not shown). These data indicate that in the mouse urinary bladder, all PAR-APs induced up-regulation of iPLA₂.

In terms of mechanism of action, it is well established that after stimulation, PARs couple to various G proteins and activate signal transduction pathways resulting in the rapid transcription of genes that are involved in inflammation 13263437. However, the response of the transcriptome downstream of PAR activation is not known. Here, we used an approach involving the combination of cDNA arrays, CHIP assay, and in silico querying of knowledge databases in order to identify functions dependent of PAR1 activation that are modified during bladder inflammation. Although we clearly defined the criterion for a gene to be named PAR1-dependent (see Methods section), the expression of some/many of these genes may only be indirectly dependent on PAR-1, due to the nature of the downstream cascades and interacting pathways.

The use of cDNA arrays has contributed immensely to the understanding of inflammation, in general, and the bladder inflammatory transcriptome, in particular 7202138394041. In addition, the use of curated networks such as the Ingenuity Pathways Analysis leads to a comprehensive integration of how PAR1-dependent transcripts correlate with canonical pathways and known biological functions. Because our results were obtained with whole bladders, it is not clear whether any single network may be operative in a particular cell type. However, this approach can potentially identify previously unrecognized connections among pathways and, therefore, suggests new hypotheses for the mechanisms of bladder inflammation. Therefore, the value of our approach is to raise testable hypotheses that can be tested in isolated cells or in individual bladder layers. Aberrant expression of protease-activated receptors (PARs) has been associated not only with inflammation but with increased angiogenesis, tumor growth, and metastasis of various cancers 424344454647. Therefore, our approach outlined a global visualization of PAR-dependent transcripts (Figure 1) as well as their interaction with several pathways (Figures 2, 3, 4).

Our results indicated that several genes known to be part of the inflammatory response were found downstream of PAR1 activation (b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2). The present work adds to this list pro-inflammatory genes such as wnt6, wisp2, and dvl3 that belong to the WNT family known to be altered in human interstitial cystitis 4849. In contrast, some of the proteins encoded by PAR1-dependent transcripts play a role in shutting down the inflammatory cascade. This is the case with type-1 membrane glycoprotein encoded by Cd200, which delivers an inhibitory signal cancelling the inflammation-induced macrophage activation 50. Within this group, we also highlight nfkbia, which encodes IkappaB-alpha, an inhibitor of the NF-kappaB cascade 5152.

In addition to genes involved in inflammation, we found upregulation of members of the tetraspanin family (cd63, and adam3) and upk2 that dimerises with the tetraspanin uroplakin 1a. Cd63 encodes a protein localized in the membrane of mast cells 53, and anti-CD63 antibodies inhibit mast cell adhesion to fibronectin and vitronectin 53. Adam-3 may have a role in inflammation by regulating the expression of allograft inflammatory factor-1 and iNOS 54. Uroplakin 2 is a major component of the surface plaques of the urothelium 55, is found exclusively in differentiated mammalian urothelium 56, and is a product of terminally-differentiated apical cell layer 5758. In addition to differentiation, uroplakins play a fundamental role in the bladder permeability barrier 59, wound healing 60, bacterial adherence and infection 61, and possibility bladder inflammation 62. The present results are the first direct evidence indicating that inflammation per se alters the message for uroplakin 2.

Among the secreted enzymes regulated by PAR1-dependent transcripts is the calcium-dependent phospholipase A₂, group IB that was up-regulated by LPS- and SP-induced bladder inflammation in wild type mice. Our Q-PCR results did not confirm an up-regulation of transcription in response to PAR1-AP and PAR2-AP. There are several explanations for this discrepancy. One possibility is that the differential expression of RNA was not due to active transcription and therefore, reflects RNA processing. As others have shown that the calcium independent phospholipase A₂ (iPLA₂) mediates bladder urothelial 31, microvascular 30, and smooth muscle 33 responses to PAR activation, and has a role in promoting mmp-2-induced cell migration via the phosphatidylinositol 3-kinase – Akt pathway 63, we went further to test whether this calcium-independent PLA is also up-regulated in response to PAR-APs. Our results confirmed upregulation of iPLA₂ protein in the bladder mucosa of mice instilled with PAR-APs, supporting the upregulation of message levels.

Another group of genes encodes proteins with roles in signal transduction pathways. Those include: plaur, actR-IIb, dusp1, and akt2. Plaur encodes the receptor for urokinase plasminogen activator (u-PA receptor) and promotes plasmin formation 64. Plaur is most closely associated with cancer invasion 65. A strong correlation exists between PAR mediating the response of thrombin and u-PAR-mediated cancer cell migration/invasion 42. Dusp1 encodes a dual specificity phosphatase 1 or mitogen-activated protein kinase [MAPK] phosphatase 1, which dephosphorylates and inactivates MAPKs 6667.Dusp1 regulates a subset of LPS-induced genes 68 and modulates the anti-inflammatory effects of dexamethasone in macrophages 66. Akt2 is a putative oncogene encoding a protein belonging to a subfamily of serine/threonine kinases containing SH2-like (Src homology 2-like) domains. Others have shown that thrombin and PAR- APs induce Akt2 phosphorylation 6970. Akt2 is a general protein kinase capable of phosphorylating several known proteins and, therefore, occupies a central position in several signal transduction pathways (Figures 1B and 1C). Moreover, PAR activation in platelets leads to Akt2 phosphorylation by a mechanism involving either G(12/13) 69 or G_i 71 and Src kinase activation 69.

The process of desensitization and re-sensitization of PA receptors is being elucidated 72. Agonists of PARs induce an irreversible activation by proteolytic cleavage of the tethered ligand peptides and rapid receptor endocytosis that are targeted to lysosomes 7374. Interestingly, among the PAR1-dependent transcripts, the following encode proteins of the ubiquitin pathway: mid2, ubr1, pxmp3, ube2h, cul3, and skp1a. Little is known regarding the alteration of expression of this set of genes during inflammation. However, it has been reported that TNFα stimulates ube2h expression by a mechanism involving NF-kappaB 75. Together these results raised the intriguing hypothesis that inflammation can induce an increase in the ubiquitin pathway that also modulates the fate of PARs 76.

The Rab family of proteins play a fundamental role in PAR re-sensitization 77. Here, we show that dcnt1 and arf6 also involved in PAR trafficking and are increased during inflammation. Dcnt1 is a subunit of the dynein/dynactin complex and an effector for rab6 78. This protein is required for the retrograde movement of vesicles and organelles along microtubules 79. Arf6 is a member of the human ADP-ribosylation factor (ARF) family of proteins belonging to the Ras superfamily of small GTPase implicated in vesicle trafficking 808182. Arf6 participates in the endosomal pathway that regulates endocytosis of several receptors 83. Post-internalization, arf6 and other membrane components are recycled back to the cell surface 80. In addition to membrane trafficking, arf6 cellular functions include: actin remodeling, cell adhesion, redistribution of β1 integrins, phagocytosis, cell division, and tumor-cell invasion (for a review, see 80).

As alterations in PAR density itself 25 or in the mechanisms involved in re-sensitization of these receptors can have strong consequences in the shift between homeostasis and inflammation, our results raise the intriguing hypothesis that, in contrast to PAR endocytosis and cessation of the stimulus that occurs during normal physiological responses, inflammation may lead to increased re-cycling of PARs back to the plasma membrane and, therefore, perpetuation of the signal transduction downstream of PAR activation.

Although the classification by biological function permits a visual association of genes and cellular processes (Figure 1), it has the disadvantage of over simplification because several genes may belong to more than one group. This is the case of mmp2 (gelatinase A) that is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes such as inflammation and cancer. Therefore, we constructed figures 2, 3, and 4 in order to illustrate common pathways involving PAR1-dependent transcripts. This type of association analyses permits the identification of new pathways such as the interaction of PAR1-dependent transcripts with the VEGF-family of growth factors, and suggests explanations of an active role of PAR in angiogenesis 45848586. Together, the pathway analysis revealed relevant target for therapeutic interventions to control or to prevent disease progression and bladder inflammation.

We are aware of a single publication investigating genes downstream of PAR1 activation. This work was performed in a human endothelial cell line challenged with thrombin with the aim of identifying early genes and comparing to those up-regulated in response to leukotriene D4 87. Unfortunately, the referred work did not provide GenBank accession numbers for the PAR1 activated transcripts, which makes comparisons of the results to the current study difficult. Nevertheless, some similarities could be found between their work and the present one. This is the case of dusp1 that was found upregulated in both manuscripts. In addition we found adam3, whereas Uzonyi, et al. found another disintegrin-like metalloprotease (adamts1) 87.

All animal experimentation described here was performed in conformity with the "Guiding Principles for Research Involving Animals and Human Beings" (OUHSC Animal Care & Use Committee protocol #05-088I). PAR1^-/-89 and C57BL/6J mice were used in this research. C57BL/6J mice were used as wild type since PAR1^-/- was enriched in this background.

Acute cystitis was induced in groups of mice, as we described previously 2021222440. Briefly, female wild type (C57BL/6J), and PAR-1^-/- mice were anesthetized (ketamine 200 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 one of the following substances: pyrogen-free saline, SP (10 μM), or Escherichia coli LPS strain 055:B5 (Sigma, St. Louis, MO; 100 μg/ml). 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 tuberculin syringe was maintained on the catheter to retain the intravesical solution for 1 hour. After that the catheter was removed and mice were allowed to void normally. Twenty-four hours after instillation, mice were euthanized with pentobarbital (200 mg/kg, i.p.) and bladders removed rapidly.

The microarray data have been deposited in the Gene Expression Omnibus (GEO) database 92. The samples can be retrieved with GEO accession numbers: GSM144550, GSM144551, GSM144552, GSM144553, GSM144554, GSM144555, GSM144556, GSM144557, GSM144558, GSM144559 that are included in a series (accession number GSE6286).

Ingenuity Pathways Analysis [IPA], (Ingenuity Systems, Mountain View, CA) is a robust and expertly curated database containing up-to-date information on over 20,000 mammalian genes and proteins, 1.4 million biological interactions, and one hundred canonical pathways incorporating over 6,000 discreet gene concepts. This information is integrated with other relevant databases such as EntrezGene and Gene Ontology 29. IPA computes a score for each network according to the fit of the set of supplied focus genes (here, PAR1-dependent genes). These scores, derived from p values, indicate the likelihood of focus genes to belong to a network versus those obtained by chance. A score > 2 indicates a ≤ 99% confidence that a focus gene network was not generated by chance alone 93. The experimental datasets of PAR1-dependent genes was used to query the IPA and to compose a set of interactive networks taking into consideration canonical pathways, the relevant biological interactions, and the cellular and disease processes.

Target validation was sought for 19 of the genes identified from the microarray data as being PAR1-dependent. Female C57BL/6J mice (n = 20 per group) were anesthetized (ketamine 200 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 one of the following substances: control inactive peptide (LRGILS 94) or PAR-activating peptides (PAR1-AP = SFFLRN 94; PAR2-AP = SLIGRL 94). 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 tuberculin syringe was maintained on the catheter end to retain the intravesical solution for 1 hour. After that the catheter was removed and mice were allowed to void normally. Twenty-four hours after instillation, mice were euthanized with pentobarbital (200 mg/kg, i.p.) and bladders removed rapidly and frozen.

Frozen bladders were shipped to Genpathway 95 for querying the chromatin for transcription of specific genes (Genpathway's TranscriptionPath Query assay) 96. Bladders were exposed briefly to formaldehyde for cross-linking of the proteins and DNA together, followed by sonication to fragment the DNA into pieces of approximately 300–500 bp. An antibody against RNA polymerase II (Abcam) was then used to precipitate the DNA transcriptome. The Ab-protein-DNA complexes were purified using beads coupled to protein A. The DNA was isolated from the complexes using a combination of heat to reverse cross-linking, RNase and proteases, and then purified using phenol extraction and EtOH precipitation. The final CHIP DNAs were then used as templates for Q-PCR reactions using primer pairs specific for each gene of interest. Q-PCR was carried out using Taq polymerase (iQ SYBR Green Supermix, Bio-Rad). Primer pairs were designed using Primer 3 97. Details of the primer sequences and the Genebank accession numbers are given in additional material 2 (Table 2). The designed primers shared 100% homology with the target sequence but no significant homology with other sequences.

Q-PCRs were run in triplicate and the averaged Ct values were transferred into copy numbers of DNA using a standard curve of genomic DNA with known copy numbers. The resulting transcription values for each gene were also normalized for primer pair amplification efficiency using the Q-PCR values obtained with Input DNA (unprecipitated genomic DNA). Results are presented as Transcription Events Detected Per 1000 Cells for each gene tested.

Twenty-four hours post-instillation, bladders were rapidly removed and placed in ice cold PBS with protease inhibitors (Complete Protease Inhibitor, Roche, Indianapolis, IN) on ice where the mucosa was dissected away from the detrusor smooth muscle, as described 32. Tissues 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. Cytosolic extracts were prepared using the Pierce NE-PER Kit that enables stepwise separation and preparation of cytoplasmic and nuclear extracts from bladder tissue. 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.

Immunoblot analyses were performed using 15 μg cytosolic extract loaded onto a 10% tris-glycine SDS gel and run at 125 V for 1.5 hours in tris-glycine SDS running buffer (BioRad, Hercules, CA). The proteins were then transferred to a 0.45 μm nitrocellulose membrane in 1/2 × tris-glycine SDS running buffer with 20% methanol using a BioRad Mini-TransBlot Cell. After blocking in 2% BSA, blots were incubated with rabbit anti-iPLA₂ (Cayman Chemical, Ann Arbor, MI) at 1:500 overnight at 4°C. An HRP-conjugated anti-rabbit secondary antibody was used for detection at 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA) and an enhanced chemiluminescent detection kit (Chemiglow West, Alpha Innotech, San Leandro, CA) was used to visualize. Images were taken using the FluorChem HD digital darkroom (Alpha Innotech, San Leandro, CA) and quantified using ImageJ 98.

PAR-AP – PAR1-, PAR2-, and PAR4-AP were synthesized at Molecular Biology Resource Facility, William K. Warren Medical Research Institute, OUHSC, as carboxyl-terminal amides, purified by high-pressure liquid chromatography, and characterized by mass spectroscopy. Peptide solutions were made fresh from powder for most experiments. PAR3-AP was not used in this research because of lack of specificity.