We used a highly effective method for differential gene analysis, termed suppression subtractive hybridization (SSH), which has been developed for the generation of subtracted cDNA libraries. It is based primarily on suppression PCR and combines normalization and subtraction in a single procedure . The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization . Unlike microarrays, which mainly identify moderate to high abundant genes, SSH identifies clones that are expressed at very low levels. It is possible that some of the extremely low-level gene expression is not biologically significant, as it might arise from 'random transcription' . Therefore, we confirmed the differential expression of twenty six SSH-selected transcripts by QRT-PCR and the results were highly correlated. In addition, we introduced a redundancy factor by comparing the data generated from the analysis of multiple SSH libraries (MC, DC, MIC, MIDI, DID, and DIMI). Nevertheles, the question of validation certainly is an important one, and it is one we have considered. For one, we examined properties of the candidate genes that were independent of expression, such as the presence of promoter sequences, to increase the probability that mechanisms suggested by the expression data were not simply statistical anomalies. Moreover, the finding that mechanisms known to be operant emerged from the analysis also increases confidence that novel ones are valid as well.
This study was carried out at a single time point. The 24 hour time point was chosen because it coincides with the peak of acute inflammation [8, 9]. This point characterizes the acute inflammatory responses to LPS and in addition to edema and vasodilation, the peak response of neutrophil infiltration occurred at 24 h in the mucosa and submucosa as well as in the detrusor smooth muscle [8, 9]. The rationale for a single time point was to keep the number of variables within a reasonable limit. We understand that 24 h time point may not identify preceding or proceeding events as we indicated previously [7–9]. Therefore, the results of regulatory network here presented should be viewed as a snapshot of the inflammatory transcriptome at the point of maximal inflammatory response. Nevertheless, genes identified as important at this time point can now be followed over time by other techniques.
By using cDNA subtractions between mucosa and detrusor smooth muscle layers isolated from control and inflamed bladders, several clones identified transcripts that were further annotated. It has to be taken in consideration that the mucosal layer contains the urothelial layer and the lamina propria which involves other cell types (fibroblasts, myofibroblasts, etc.) in addition to urothelial cells that may underlie the transcriptomes identified. In addition, during the inflammation, inflammatory cells are present in the detrusor as well as in the mucosa and may contribute to the inflammatory bladder transcriptome.
Some of the transcripts are RIKEN sequences and therefore, have no biological process attributed. Interestingly, some of these transcripts with unknown function, such as ambladder, have been reported in the adult male urinary bladder by others using SSH . However, the present work indicates that the same transcript was isolated from female urinary bladder as well.
Central to elucidation of hypothetical cis-regulatory networks is the identification and classification of naturally occurring transcription factor-binding sites in subtracted libraries. The combination of SSH-derived transcripts with PAINT-guided query of the TRANSFAC database permitted the generation of regulatory networks containing upstream TREs connecting corresponding TFs to the respective transcripts. Interestingly, many of the genes identified by SSH are transcription factors which emphasize the use of SSH to identify low expression transcripts.
Mucosa control [MC], (figure 2)
The regulatory network for the control bladder mucosa (MC) was obtained in comparison to detrusor control and contained the following over-represented TREs: GA-binding protein (GABP), YY1 (yin yang 1), SF-1 (steroidogenic factor-1), EF2, CRE-BP1, and Sp-3 (trans-acting transcription factor 3).
GABP, also known as nuclear respiratory factor 2 (NRF-2), is a transcriptional coordinator of mitochondrial and nuclear-encoded subunits of cytochrome oxidase genes . NRF-2 responds to increased neuronal activity by translocating from the cytoplasm to the nucleus, where it engages in transcriptional activation of target genes . GABP is abundant in the kidney , however, the information about GABP is scanty in the rest lower urinary tract.
YY1 is a zinc finger TF which is thought to regulate cell growth and differentiation. YY1 normally antagonizes serum response factor (SRF) . Interestingly, in the inflamed mucosa YY1 was not expressed and SRF was considered over-represented (Figure 2). YY1 along with GAPB drives the expression of myosin light chain phosphatase (mylc2b) which can be developmentally regulated in mammalian urinary bladders  and it is involved in the bladder response to obstruction .
SF-1 is a zinc finger motif of nuclear receptors  essential for steroidogenesis as well as for the development of the reproductive axis . CRE-BP1, which is an ubiquitous basic-leucine zipper, is required for normal skeletal development.
YY1, SF-1, and EF2 were found upstream of ambladder (RIKEN clone 9530014P05 or prothymosin alpha). Prothymosin alpha is an oxidative stress-protecting gene  and transgenic mice over-expressing this transcript develop polycystic kidney disease PKD . Another gene downstream of SF-1 was calponin 1 (cnn1). Cnn1 encodes for a multifunctional protein whose expression is tightly restricted to differentiated smooth muscle cell lineages during embryonic and post-natal life .
SF-1 and CRE-BP1 were found upstream of sac1 (a murine homolog of S. cerevisiae suppressor of actin mutations). Finally, an additional TRE, Sp-3 was found upstream of sc1 which is a calcium binging protein and an extracellular matrix protein precursor.
Epithelial-stromal interactions in bladder development have been extensively studied . However, the present results depicted in the MC regulatory network raises the hypothesis that the bladder mucosa exhibits TREs and genes whose proteins may regulate the smooth muscle phenotype (Figure 2).
Detrusor control [DC] (figure 5)
The regulatory network for the control detrusor smooth muscle suggests that Pax-3 plays a central role. In addition to Pax-3, other TREs such as: PITX2, CDP, and c-Myb were also found over-represented (figure 5).
Pax-3 was found upstream of the following transcripts: prion protein (prnp), cathepsin L (ctsl), stromal cell derived factor receptor 1 (sdfr1) , thrombospondin, type I domain containing 6 (thsd6), and uroplakin 1b (upk1b). Prnp seems to be involved in cell-to-cell interaction and its expression has been observed in germ cell differentiation during spermatogenesis . Ctsl is a lysosomal proteolytic enzyme and an imbalance between ctsl and its inhibitors is believed to correlate with bladder tumor progression .
In addition to ctsl, the detrusor also presents ctsh under the control of c-Myb. Interestingly, thsd is involved in bladder cancer development  and is a target for methylation . In addition, thsd has been described as inhibitor of angiogenesis. Finally, upk1b gene is highly expressed in normal human urothelium and its mRNA was undetectable or markedly reduced in bladder carcinoma .
Interestingly, the control detrusor normally expresses genes in the Pax-3 pathway that maintain neural progenitor cells  and myoblasts  undifferentiated. Therefore, our data suggests that Pax-3-regulated suppression of neural development in control detrusor changed substantially during inflammation and genes involved in neuronal development such as syn were found to be up-regulated. The later implies that detrusor instability may be a consequence of alterations in Pax-3 pathway leading to increased maturation of neural progenitor cells within the bladder smooth muscle.
Mucosa inflamed versus control [MIC]. (figure 3)
Of all regulatory networks (Figures 2, 3, 4, 5, 6, 7), MIC was the only one presenting NF-κB (p < 0.01), (Figure 3). These results confirmed our previous observation that it is the bladder mucosa and in particular, the urothelium, that responds to LPS with NF-κB translocation . This independent identification of NF-κB , which is known to play a key role in bladder inflammation  strengthens confidence in the identification of novel pathways.
Three transcripts were found downstream of NF-κB: catenin (c adherin-associated protein, delta 1), cox7b, and pIs3 (plastin 3 T-isoform). Regarding catenin, others have described its presence in the bladder mucosa and in particular in the urothelium . Indeed, alpha1-catenin was reported to be reduced in bladder urothelial cells treated with anti-proliferative factor  which make this transcript a possible target in cystitis. Moreover, p120-catenin is frequently altered and/or lost in tumors of bladder . Finally, bladder cancers harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity . Cox7b encodes cytochrome C oxidase, subunit VIIb which is the terminal component of the mitochondrial respiratory chain and catalyzes the electron transfer from reduced cytochrome C to oxygen. Finally, pls3 was found by differential display to have increased expression in cisplatin-resistant human cancer cells .
In addition to NF-κB, MIC also had SRF that along with several zinc finger TFs (SF-1 [Steroidogenic factor 1]; Gfi-1 (growth factor independent 1); ik-3 (Ikaros 3); and vMaf [basic region leucine zipper]), drive the activity of actg1. Actg1 is a highly conserved protein involved in various types of cell motility, and maintenance of the cytoskeleton.
The MIC regulatory network also includes the following unique transcripts: smoc2 (secreted modular calcium-binding protein 2), amcq (adult male corpora quadrigemina cDNA; RIKEN clone:B230340L02), prss11 (Serine protease 11), and ifit3 (Interferon-induced protein with tetratricopeptide repeats 3). Smoc2 is a widespread glycoprotein with a calcium-dependent conformation . Prss11 encodes a secreted trypsin (HtrA1) that regulates the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins and therefore, may function as a regulator of cell growth . HtrA is involved in stress response pathways . Interestingly, down-regulation of prss11 expression may be an indicator of melanoma progression .
Ifit3 was originally cloned from a cDNA library prepared from the murine cell line, RAW 264.7, after bacterial LPS stimulation . Although it is a transcript induced by IFN , its function is still unknown.
Grp58 encodes a ubiquitously expressed chaperone protein that resides in the endoplasmic reticulum and is part of the protein folding machinery . It is likely that grp58 is involved in the oncogenic transformation since expression analysis revealed an up-regulation of grp58 in breast, uterus, lung, and stomach tumors .
Together these results confirmed that the bladder mucosa expresses unique transcripts involved in cell growth, motility, and cytoskeleton, protein folding, and proteases. Some of these transcripts have been described to be altered in cystitis as well as in LPS-induced bladder inflammation. However, the most striking result was that of 6 different networks, the MIC library was the only one to have over-representation of NF-κB.
Mucosa inflamed versus detrusor inflamed [MIDI], (figure 4)
The question being answered by this experiment was whether or not the bladder mucosa sets the stage for LPS-induced inflammatory responses by up-regulating a unique set of genes and TFs distinct from the detrusor muscle.
In terms of TFs, the upstream stimulatory factor (USF) was the most significantly over-represented (p < 0.001) and driving unique transcripts (figure 4). USF dimerizes to regulate transcription through E-box motifs in target genes. Although widely expressed, they 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 . Others have shown that USF binding activity is enhanced in response to LPS .
Another interesting TF was the liver X-activated receptors (LXR) found upstream of several transcripts bearing USF sequence. LXR is a member of the nuclear receptor superfamily  is a negative regulator of macrophage inflammatory gene expression , and a putative therapeutic agent for the treatment of inflammation , diabetes , and neurodegenerative diseases [57, 58, 60].
LIM-only proteins (LMO), which consist of LMO1, LMO2, LMO3, and LMO4, are involved in cell fate determination and differentiation during embryonic development . LMO2 was originally identified through its involvement in T-cell leukemia and subsequently shown to be critical for normal hematopoietic and endothelial development . Accumulating evidence suggests that LMO1 and LMO2 act as oncogenic proteins in T-cell acute lymphoblastic leukemia, whereas LMO4 has recently been implicated in the genesis of breast cancer.
MAZ (Myc-associated zinc finger protein), also known as serum amyloid A-activating transcription factor-1 (SAF-1), plays a major role in regulating transcription of several inflammation-responsive genes, including matrix metalloproteinase-1 (81), the mouse mast cell protease (mMCP)-6 , and function as growth suppressor in fibroblasts . SAF-1 transgenic mice are prone to develop a severe form of inflammation-induced arthritis . MAZ is upstream of Aplp2 which is a key regulator of structure and function of developing neuromuscular synapses .
In terms of unique transcripts isolated from the MIDI library, our results indicate that transcripts such as zfp364 (zinc finger protein 364, also known as rab7) and sir2 are downstream of series of TFs including: USF, LXR, DEAF1, MAZ, and Lmo2 complex (figure 4). Sir2 gene encodes a member of the sirtuin family of proteins which are NAD-dependent histone/protein deacetylases . Zfp364 is a member of the Rab family of small G proteins, and regulates intracellular vesicle traffic to late endosomes . Another unique transcript was the androgen-regulated tmprss2 protease  known to be expressed in urogenital tissues . Tmprss2 has gained interest owing to its highly localized expression in the prostate and its over-expression in neoplastic prostate epithelium. Once activated, the serine protease domain of tmprss2 is released from the cell surface into the extracellular space and activates PAR (protease-activated receptor)-2 that has a role in prostate cancer and tumor metastasis . Among the proteins correlating with cytoskeleton dynamics, our SSH identified a transcript (wdr1) encoding a 67-kDa WD40 repeat protein 1 which is the vertebrate homologue of actin-interacting protein 1 . Wdr1 is involved in actin dynamics and seems to be required to induce cell morphologic changes, especially mitotic cell rounding . Others have shown that wdr1 was found upregulated in the lung  and cell lines  following exposure to nickel oxide-induced carinogenesis. Finally, MIDI library also contained a RIKEN cDNA 2310015N07 gene that was described to be isolated from developing mouse libraries but no function yet has been attributed .
Interestingly, a comparison between MIDI and DIMI libraries indicates that both share LXR as a TRE. The major difference between these two libraries was found downstream LXR activation. In the inflamed mucosa, LXR preferentially activates zfp364 and tmprrs2 whereas in the inflamed detrusor LXR was found as a co-modulator of actg2.
In conclusion, the mucosa regulatory network presents USF in a central position raising the hypothesis that USF-target promoters such as the TGF-β1 promoter are involved in the mucosal response to inflammation and whether mucosa inflammation follows similar diabetes- related mucosal gene expression.
Detrusor inflamed versus detrusor control [DIC], (figure 6)
The regulatory network of the detrusor muscle, inflamed versus control, selected the following transcripts: syn1 (Synapsin I or ribosomal protein S15a), siva (CD27-binding protein), and eif4ebp2 (negative regulation of translational initiation), (figure 6).
Syn1 is a member of the synapsin gene family which is a neuron-specific phosphoprotein of small synaptic vesicles.Syn1 has been mapped to an evolutionarily conserved linkage group composed of: araf1, syn1, timp, and properdin located at human chromosome Xp11.2  and mouse chromosome X . Of interest, araf1 is a proto-oncogene which is predominantly expressed in mouse urogenital tissues . In contrast, siva has an important role in the apoptotic pathway induced by the CD27 antigen. Others have described that siva is a direct transcriptional target for both tumor suppressors, p53 and E2F1 . Finally, the eukaryotic initiation factor eIF4E and eIF4E-binding proteins (4E-BPs) control the initiation of protein synthesis and are part of a translational signaling pathway sensitive to insulin  and rapamycin . Changes in the state of phosphorylation of eIF4E and 4E-BPs occur at an early stage of apoptosis . Interestingly, eIF4E selectively enhances the translation of powerful angiogenic factors such as FGF-2 and VEGF  and therefore may have a role in oncogenesis  as well as inflammation.
Over represented TFs in DIC regulatory network were NRSF (Kruppel-type zinc-finger transcriptional repressor RE1-silencing transcription factor [REST]; also known as the neuron-restrictive silencing factor), CREB/CRE-BP1, E2F-1, and Evi-1.
CREB/CRE-BP1, also called transcription factor ATF-2, binds to the cAMP response element and its activity is enhanced after phosphorylation by stress-activated protein kinases such as c-Jun N-terminal kinase and p38. ATF-2 plays a central role in TGFβ signaling by acting as a common nuclear target of both Smad and TAK1 pathways .
Nrf-1 (nuclear respiratory factor 1) regulates expression of nuclear-encoded mitochondrial genes and it was shown to be part of the response to LPS in rats .
FOXp3 belongs to the forkhead gene family which comprises a diverse group of "winged-helix" TFs with important roles in development, metabolism, cancer and aging . Recently, several forkhead genes have been demonstrated to play critical roles in lymphocyte development and effector function . FoxP3 is a potential target for treatment of experimental chronic inflammatory renal disease  and type I diabetes . In addition, both FOXp3 and NRSF seems to be downstream of Wnt-Frizzled signaling  which was recently proposed to participate in the pathogenesis of interstitial cystitis .
E2F, E2F-1, and Rb-E2F-1 belong to a family of TFs implicated in the regulation of cell proliferation and their binding sites are present in the promoters of several growth-regulating genes. E2F family members are functionally regulated, in part, by complex formation with one or more members of the nuclear pocket protein family such as the retinoblastoma protein (Rb) and play a role in neuronal development  by acting as negative regulator of cell proliferation. The interplay between Rb and E2F is critical for proper cell cycle progression . Of interest, E2F-1 has a growth-promoting effect in bladder superficial TCC .
ATF-3 (activating transcription factor 3) is transcriptional repressor involved in survival and regeneration of sensory neurons [93, 94] that responds to insulin . ATF3 is also a novel stress-activated regulator of p53 protein stability/function providing the cell with a means of responding to a wide range of environmental insults . In addition, ATF3 represents a novel mechanism in which anti-inflammatory drugs exert their anti-invasive activity .
The proposed role of nrsf/rest is that of a transcriptional silencer that restricts neuronal gene expression to the nervous system by silencing their expression in non-neural tissues . Interestingly, loss of nrst function in human prostate carcinoma cells is associated with neuroendocrine phenotype, tumor progression, and androgen independence . Others investigators indicated that nrst also modulates the cholinergic gene locus [100, 101] which may have some implication in detrusor instability. Recently, it was proposed that activation of the rest/nrsf target genes overrides muscle differentiation pathways and converted myoblasts to a physiologically active neuronal phenotype . It remains to be determined whether nrst promotes the same transformation in the inflamed detrusor muscle. The latter would explain the hyperactivity of detrusor muscle observed in over-active bladder disorders such as obstruction, incontinence, and inflammation.
In conclusion two major networks are proposed to be active in the detrusor inflamed when compared to control. One containing a neuron-specific phosphoprotein of small synaptic vesicles (syn) and the other an important protein of apoptotic pathway (siva). In both cases, analysis of the intense upstream promoter network leads us to the hypothesis that both genes represent a common downstream target of several pro-inflammatory stimuli.
Detrusor inflamed versus mucosa inflamed [DIMI], (figure 7)
The question being answered by this experiment was whether or not the inflamed detrusor muscle expresses unique transcripts and TFs distinct from the bladder mucosa.
Two major pathways could be constructed with the combination of SSH and PAINT results. The first involves key smooth muscle proteins, a myosin light chain encoded by my19 and gamma actin encoded by actg2. Gamma actins are highly conserved proteins that are involved in various types of cell motility, and maintenance of the cytoskeleton. In addition, a role for smooth muscle alpha actin in force generation by the urinary bladder has been suggested . Several TREs upstream of actg 2 and my19 were over-represented in MIDI, including LXR which was described above, SRF, COUP, Pax-9, CP2, and RFX1.
A second pathway involved the transcripts elp3, gus, and ddx3 and two TFs COMP1 and IRF. Ddx3 is a putative RNA helicase and a member of a highly conserved DEAD box subclass. RNA helicases are highly conserved enzymes involved in transcription, splicing, and translation . There are several examples of the involvement of RNA helicases in differentiation of germ cells, particularly in spermatogenesis. Upstream of ddx3, PAINT selected a TF that cooperates with myogenic proteins (COMP1) and Interferon Regulatory Factor (IRF). Transcription of IRF is synergistically activated by products of inflammation such as IFNγ and TNFα .
Two other transcripts were found downstream of COMP1: Gus (beta-glucoronidase) and elp3. Gus is a sensitive indicator LPS activation of macrophages. Elp3 is one of the sub units of the elongator complex, an acetyltransferase important for normal histone acetylation involved in elongation of RNA polymerase II transcription.
By comparing the inflamed detrusor and mucosa (DIMI), the fundamental difference observed was the up-regulation in the detrusor of genes and TFs related to smooth muscle function. It is fair to propose that this network could underlie detrusor instability during inflammation.