Disulfide high mobility group box-1 causes bladder pain through bladder Toll-like receptor 4

Abstract

BackgroundBladder pain is a prominent symptom in several urological conditions (e.g. infection, painful bladder syndrome/interstitial cystitis, cancer). Understanding the mechanism of bladder pain is important, particularly when the pain is not accompanied by bladder pathology. Stimulation of protease activated receptor 4 (PAR4) in the urothelium results in bladder pain through release of urothelial high mobility group box-1 (HMGB1). HGMB1 has two functionally active redox states (disulfide and all-thiol) and it is not known which form elicits bladder pain. Therefore, we investigated whether intravesical administration of specific HMGB1 redox forms caused abdominal mechanical hypersensitivity, micturition changes, and bladder inflammation in female C57BL/6 mice 24 hours post-administration. Moreover, we determined which of the specific HMGB1 receptors, Toll-like receptor 4 (TLR4) or receptor for advanced glycation end products (RAGE), mediate HMGB1-induced changes.ResultsDisulfide HMGB1 elicited abdominal mechanical hypersensitivity 24 hours after intravesical (5, 10, 20 μg/150 μl) instillation. In contrast, all-thiol HMGB1 did not produce abdominal mechanical hypersensitivity in any of the doses tested (1, 2, 5, 10, 20 μg/150 μl). Both HMGB1 redox forms caused micturition changes only at the highest dose tested (20 μg/150 μl) while eliciting mild bladder edema and reactive changes at all doses. We subsequently tested whether the effects of intravesical disulfide HMGB1 (10 μg/150 μl; a dose that did not produce inflammation) were prevented by systemic (i.p.) or local (intravesical) administration of either a TLR4 antagonist (TAK-242) or a RAGE antagonist (FPS-ZM1). Systemic administration of either TAK-242 (3 mg/kg) or FPS-ZM1 (10 mg/kg) prevented HMGB1 induced abdominal mechanical hypersensitivity while only intravesical TLR4 antagonist pretreatment (1.5 mg/ml; not RAGE) had this effect.ConclusionsThe disulfide form of HMGB1 mediates bladder pain directly (not secondary to inflammation or injury) through activation of TLR4 receptors in the bladder. Thus, TLR4 receptors are a specific local target for bladder pain.

Protease activated‐receptor 4 activation as a model of persistent bladder pain: Essential role of macrophage migration inhibitory factor and high mobility group box 1

“…A total of 24 h after the first PAR4 instillation, the 50% mechanical threshold was markedly decreased compared with the PAR4 scrambled peptide group (Fig. a; day 1), in agreement with our earlier reports . The mechanical threshold was increased at day 2 before the second intravesical treatment (Fig.…”

“…We recently reported that intravesical PAR4 instillation generated an acute (24 h) bladder pain model where mice showed bladder hypersensitivity without overt bladder inflammation, an important feature, as most IC/BPS patients do not show bladder inflammation . Furthermore, we examined the mechanisms responsible for the development of bladder hypersensitivity, and observed that PAR4 activation led to the release of urothelial MIF and HMGB1 . Both MIF and HMGB1 are necessary for the development of bladder pain in this model and these observations agree with reports showing a role for MIF and HMGB1 in nociception …”

“…We now show that established bladder pain treatment with a MIF receptor inhibitor partially reversed bladder pain. In our acute PAR4 bladder pain model, MIF was released after urothelial PAR4 activation followed by urothelial HMGB1 release, and blocking either of these substances prevented abdominal mechanical hypersensitivity . MIF interacts with several receptors to mediate its effect including CD74 (cognate receptor), CXCR4 and CXCR2 .…”

“…Isoflurane‐anesthetized mice were transurethrally catheterized (PE10, 11‐mm length) and the bladder drained of urine. PAR4‐AP or PAR4 scrambled peptide control (100 μmol/L in PBS; 150 μL; Peptides International, Louisville, KY, USA) were instilled into the bladder lumen and held for 1 h, and then the mouse was allowed to recover from anesthesia and returned to its cage . Intravesical instillation was repeated twice every 48 h on day 0, and days 2 and 4 (Fig.…”

Sterile Inflammatory Role of High Mobility Group Box 1 Protein: Biological Functions and Involvement in Disease

“…Disulfide (or isomerized) HMGB1, characterized by 2 cysteine residues that are oxidized to form an S-S bond between C23 and C45, was found to induce NF-κB translocation and the secretion of inflammatory factors via TLR-4 in activated immune cells, while mutations of C23 or C45 abrogated the cytokine activity of HMGB1 [14]. It also induced bladder pain via TLR-4 [15]. Frank et al [16] found that it potentiated the neuroinflammatory response in vivo, but that thiol HMGB1 failed to promote the microglial proinflammatory response to lipopolysaccharide (LPS).…”