Abstract
BackgroundAll four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders.MethodsFor this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kitw/Kitw-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB.ResultsTFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kitw/Kitw-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kitw/Kitw-v mice.ConclusionThis is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders.
Highlights
All four protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered during inflammation
Protein/DNA combo array PAR1-activating peptide (AP), PAR2-AP, PAR4-AP, and control peptide were instilled into the mouse bladder at the concentration of 10 μM
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 occupied the center of Venn diagram (Figure 2)
Summary
All four PARs are present in the urinary bladder, and their expression is altered during inflammation. Other serine proteases, such as thrombin and trypsin, are produced during tissue damage and make important contributions to tissue responses to injury, repair, cell survival, inflammation [10,11,12,13], and pain [14,15,16,17,18]. Tissue responses to these enzymes are modulated by protease-activated receptors (PARs), a unique class of G protein-coupled receptors that use a fascinating mechanism to convert an extracellular proteolytic cleavage event into a trans-membrane signal. These receptors carry their own ligands, which remain cryptic until unmasked by receptor cleavage (for a review, please see references [14,17,19,20])
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