Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2 Signaling in Mice.

Shiori Hiramoto,Aya Sakaegi,Takuya Okada,Atsufumi Kawabata,Hiroyasu Ishikura,Naoki Toyooka,Junichi Tanaka,Junichi Tanaka,Hidenori Wake,Fumiko Sekiguchi,Yuki Toriyama,Huy Du Nguyen,Masahiro Nishibori,Kyoko Okazaki,Kaoru Yamaguchi,Maho Tsubota

doi:10.3390/cells9081748

Shiori Hiramoto, Aya Sakaegi + Show 14 more

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https://doi.org/10.3390/cells9081748

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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.

Highlights

Interstitial cystitis/bladder pain syndrome (IC/BPS) is common to women, and characterized by pelvic/suprapubic pain, bladder discomfort or pressure, as well as increased frequency of urination, which are largely resistant to drug therapy
We have demonstrated that the activation of receptor for advanced glycation end products (RAGE) by high mobility group box 1 (HMGB1) derived from unknown cells plays a role in the development of the bladder pain accompanying CPA-induced cystitis in mice [2]
To test whether the HMGB1/RAGE pathway [2] would be upstream of the CSE/H2 S/Cav 3.2 pathway [3], we examined the effects of an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of inactivating HMGB1 and FPS-ZM1, a RAGE antagonist, on the CPA-induced CSE

Summary

Introduction

Interstitial cystitis/bladder pain syndrome (IC/BPS) is common to women, and characterized by pelvic/suprapubic pain, bladder discomfort or pressure, as well as increased frequency of urination, which are largely resistant to drug therapy. To identify potential therapeutic targets to treat bladder pain in IC/BPS patients, we have been studying the mechanisms underlying bladder pain in a cyclophosphamide (CPA)-induced cystitis mouse model, and have shown the two different mechanisms involved in the cystitis-related bladder pain, i.e., the activation of the receptor for advanced glycation end products (RAGE) by high mobility group box 1 (HMGB1) [2], and the elevated. Extracellular HMGB1 in the peripheral tissue is considered to participate in inflammatory and neuropathic pain through direct or indirect activation of RAGE, Toll-like receptor 4 (TLR4) or CXCR4 [2,7,8,9,10]. We have demonstrated that the activation of RAGE by HMGB1 derived from unknown cells plays a role in the development of the bladder pain accompanying CPA-induced cystitis in mice [2]

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