Abstract
To elucidate pathways in bladder inflammation, we employed our physiologically relevant LL-37 induced cystitis model. Based on inflammatory studies involving other organ systems implicating the receptor for advanced glycation end-products (RAGE), we first hypothesized that RAGE is critically involved in LL-37 induced cystitis. We further hypothesized a common RAGE ligand - high mobility group box 1 (HMGB1) is up-regulated in bladders challenged with LL-37. Finally, we hypothesized NF-κB dependent inflammatory genes are activated in LL-37 induced cystitis. Testing our first hypothesis, C57Bl/6 mice were challenged with either saline (control) or 320 μM of LL-37 intravesically for 1 hr. After 12 or 24 hours, tissues were examined with immunohistochemistry (IHC) for RAGE, and both mRNA and protein isolation for respective qRT-PCR and Western Blot analysis. Our second hypothesis was tested by employing HMGB1 IHC. Testing our final hypothesis, qRT-PCR was performed investigating five genes: TNFα, IL-6, IL-1β, GM-CSF, COX-2. In control and LL-37 challenged tissues, IHC for RAGE revealed similar qualitative expression. Evaluation with qRT-PCR and Western Blot for RAGE revealed diminished expression at the mRNA and protein level within LL-37 challenged bladders. IHC for HMGB1 revealed a moderate qualitative increase within LL-37 challenged tissues. Finally, with the exception of TNF α, all NF- κB dependent inflammatory genes yielded substantial up-regulation. We have employed our LL-37 induced cystitis model to gain insight towards a possible mechanistic pathway involved in bladder inflammation. This work provides data for future studies involving the inflammatory ligand HMGB1, RAGE, and receptor pathways that activate NF-κB.
Highlights
In order for the bladder to store urine it must be compliant
We further hypothesized that a common receptor for advanced glycation end-products (RAGE) ligand high mobility group box 1 (HMGB1) is up-regulated in bladders challenged with LL-37
In this study we propose that LL-37 induced bladder inflammation involves RAGE, a common RAGE ligand HMGB1, and converges on NF-κB signaling
Summary
In order for the bladder to store urine it must be compliant (pliable). It is imperative that it can hold urine at low pressures. We hypothesized that LL-37 induced bladder inflammation involves the receptor for advanced glycation end-products (RAGE) and a common RAGE ligand high mobility group box 1 (HMGB1), resulting in the activation of NF-κB dependent inflammatory genes. Most studies suggest that ligand activation of RAGE transmits cell surface signals to various intracellular pathways including NF-κB, a heterodimeric protein complex is responsible for a multitude of transcriptional programs that produce proinflammatory cytokines and enzymes such as TNFα, IL-1β, IL-6, and COX-2 [18,19,20,21,22,23,24]. HMGB1 was first identified as a non-histone chromosomal protein involved in DNA binding [28] and later recognized as a pro-inflammatory cytokine mediating endotoxin lethality in mice [29] It is predominantly a nuclear protein present in most eukaryotic cells where it stabilizes nucleosome formation and facilitates transcription [30,31,32]. We investigated whether activation of these pathways in the bladder could be responsible for the sustained, proinflammatory phenotype
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