Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Abstract

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; i.p.) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; i.p.) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.

Fig 1. The flow chart demonstrates in vivo study in mice with treatments.

Fig 2. PAR4 activation elicits HMGB1 release in vitro and in vivo.

Western blotting determined HMGB1 levels in culture media collected from human immortalized urothelial cultures (UROtsa) exposed to PAR4-AP for 2 hours (A; representative blot), and in intravesical fluid collected from mice receiving intravesical PAR4-AP for 1 hour (B). Densitometry revealed significantly more HMGB1 release from bladders receiving intravesical PAR4-AP than those receiving control peptide (C; ** p = 0.011; Wilcoxon rank test). Caspase-3 immunostaining (arrows) revealed few labeled nuclei in each treatment group (D).

Fig 3. HMGB1 mediates urothelial PAR4-induced mechanical hypersensitivity without inflammation.

Responses to abdominal mechanical stimulation with von Frey filaments before (baseline) and 24 hours after each treatment are shown (A-C). Intravesical PAR4-AP (B) increases abdominal hypersensitivity over corresponding baseline values (* p ≤ 0.025 after Bonferroni correction for multiple one-tailed paired t-tests). HMGB1 inhibitor, glycyrrhizin (50 mg/kg, ip), abolished PAR4-mediated responses (C). H&E stained paraffin bladder sections showed normal urothelial morphology in all treatment groups (D-F). No inflammatory cells were observed in any of the treatment groups. Submucosal fibrosis with lamina propria expansion (arrow in panel E) was observed in animals treated with PAR4-AP and vehicle, but not in the other groups.

Fig 4. Urothelial PAR4-induced HMGB1 intensity decrease is mediated through MIF.

Panels (A-C) show urothelial HMGB1 immunofluorescence 1 hour after intravesical exposure to control peptide, PAR4-AP, or PAR4-AP after pretreatment with MIF antagonist, ISO-1 (ip). White arrows identify the intravesical surface of the urothelium. Less urothelial HMGB1 immunofluorescent labeling is apparent in PAR4-AP exposed bladders (B) than in control peptide-treated bladders (A). Pretreatment with MIF-1 antagonist ISO-1 prevented this decrease (C). Quantitative image analysis (D) revealed that average urothelial HMGB1 immunofluorescence significantly decreased after intravesical PAR4-AP administration in comparison to control peptide-treated animals (** p ≤ 0.01), indicating urothelial release of HMGB1. In contrast, ISO-1 pretreatment elevated urothelial HMGB1 immunofluorescence from PAR4-AP administration (*** p ≤ 0.001), suggesting MIF antagonism blocks HMGB1 release.