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Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2
Shiori Hiramoto1, Maho Tsubota1, Kaoru Yamaguchi1
1Laboratory of Pharmacology and Pathophysiology, Faculty of Pharmacy, Kindai University (Formerly Known as Kinki University), Higashi-Osaka 577-8502, Japan.
Abstract:
Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.
Insights
Cyclophosphamide-induced bladder pain in mice is driven by HMGB1/RAGE signaling, leading to increased H2S production and CaV3.2 channel activity. Targeting these pathways may alleviate bladder pain.
Area of Science:
- Urology
- Pain Research
- Cell Biology
Background:
- Cyclophosphamide (CPA) induces cystitis-related bladder pain.
- This pain involves receptor for advanced glycation end products (RAGE) activation by high-mobility group box 1 (HMGB1).
- Increased Cav3.2 T-type Ca2+ channel activity, mediated by hydrogen sulfide (H2S) from cystathionine-γ-lyase (CSE), is also implicated.
Purpose of the Study:
- To investigate the crosstalk between HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in CPA-induced bladder pain.
- To elucidate the molecular mechanisms underlying bladder pain development.
Main Methods:
- Assessed bladder pain and CSE expression in CPA-treated female mice.
- Analyzed cell signaling in urothelial T24 and macrophage-like RAW264.7 cells.
- Utilized pharmacological inhibitors and genetic deletion of Cav3.2.
Main Results:
- CPA-induced bladder pain was abolished by inhibiting T-type Ca2+ channels, CSE, or deleting Cav3.2.
- HMGB1 inactivation, RAGE antagonism, and antioxidant N-acetylcysteine prevented CPA-induced CSE upregulation and bladder pain.
- Acrolein (CPA metabolite) triggered ATP release, leading to HMGB1 release via P2X7/p38MAPK/NF-κB/ROS signaling.
Conclusions:
- CPA metabolism to acrolein initiates a cascade involving urothelial ATP release and macrophage HMGB1 release.
- This HMGB1 activates RAGE, upregulating CSE and leading to H2S-driven Cav3.2 channel activation.
- This pathway culminates in nociceptor excitation and bladder pain, highlighting potential therapeutic targets.
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