Regulatory network of inflammation downstream of proteinase-activated receptors

Abstract

BackgroundProtease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown.We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency.ResultsHere, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli.ConclusionThe combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation.It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.

Wnt6 Is Expressed in Granulomatous Lesions of Mycobacterium tuberculosis–Infected Mice and Is Involved in Macrophage Differentiation and Proliferation

“…The induction of Wnts has been only poorly described in inflammatory settings: to date, an upregulation of Wnt6 has been almost exclusively observed in microarray-based studies, for example, in M. tuberculosis-infected murine macrophages (46), in the bladder of mice after stimulation with LPS, in a rat renal allograft model, in coloscopic biopsies of ulcerative colitis patients, and most recently also in the lung of asthma patients (14,(47)(48)(49). Of note, Liu et al (50) describe an induction of Wnt6 mRNA by Salmonella typhimurium infection of murine intestinal epithelia cells ex vivo.…”

Cytotoxic Necrotizing Factor 1 and Hemolysin from Uropathogenic Escherichia coli Elicit Different Host Responses in the Murine Bladder

“…Prior to inoculation of mouse bladders, we quantified the amount of LPS in our CNF1 preparation because LPS itself elicits a robust inflammatory response that could potentially confound interpretation of our results. We found that the CNF1 preparation contained 9.9 ng/ml LPS, a concentration that is 10,000-fold lower than the concentration of LPS (100 g/ml) that is typically introduced into the bladder to stimulate an inflammatory response (59,60). We inoculated 12 mice per group intraurethrally with 1 or 2 g CNF1 and sacrificed them 24 h later.…”

VEGF receptors and neuropilins are expressed in the urothelial and neuronal cells in normal mouse urinary bladder and are upregulated in inflammation

“…To assess alterations in the receptor expression, we used chromatin immunoprecipitation (ChIP) combined with quantitative real-time PCR (Q-PCR), a method that uses the DNA transcriptome and, therefore, reflects transcription of active genes (37,40,41). Although we detected statistically significant inflammation-induced increase in the expression of some VEGF receptors and coreceptors, the changes were too small to be considered consequential (Fig.…”

“…In normal bladder this signaling is a key mechanism downstream of protease-activated receptor (PARs) activation in response to proinflammatory stimuli (36,40), while in bladder with the superficial cancer it is part of the response to intravesical therapy with Bacillus Calmette-Guerin (BCG) (34). This new appreciation of VEGF's signaling role in bladder inflammation is supported by emerging evidence that levels of various VEGF proteins are, in general, increased at the site of inflammation (20,26,27,35,49) and that infiltrating lymphocytes and other inflammatory cells represent an additional source of VEGF (24,27).…”