NRF2 Deficiency in Bladder Epithelial Cells Owing to Ubiquitination by N6-Methyladenosine-Modified TRIM21 Induces Oxidative Stress and Inflammation to Aggravate IC/BPS

Abstract

Background: Interstitial cystitis/bladder pain syndrome (IC/BPS) has become a pressing clinical issue due to its unclear etiology and severe, persistent pelvic pain. Despite extensive research, the pathogenesis of IC/BPS remains unresolved, and current treatments primarily target symptom relief rather than addressing underlying disease mechanisms. This study aimed to investigate the effects of nuclear factor erythroid 2-related factor 2 (NRF2) on IC/BPS and the potential molecular mechanisms.

Methods: Bladder mucosal biopsies from IC/BPS patients were subjected to RT-qPCR and immunoblotting to quantify NRF2 mRNA/protein expression. In vivo modeling, WT and NRF2 gene knockout mice received intraperitoneal cyclophosphamide to induce cystitis. Bladder function was assessed via Void Spot Assays, and Urodynamic. In vitro validation, LPS-stimulated SV-HUC-1 cells were transduced with NRF2 knockdown or overexpression, and oxidative stress and inflammation levels were evaluated. Then, the molecular mechanism of NRF2 in IC/BPS was determined by conducting Western blot, mass spectrometry, co-immunoprecipitation, and RT-qPCR analyses.

Results: This study identified markedly reduced expression of NRF2 in the lesional bladder mucosa of patients with IC/BPS. By employing NRF2 knockout mice and cellular models of bladder inflammation, the essential role of NRF2 in modulating oxidative stress and inflammation was underscored. Furthermore, tripartite motif-containing 21 (TRIM21) interacted with NRF2, promoting its degradation via ubiquitination in bladder epithelial cell lines, thus elucidating TRIM21's regulatory role in bladder inflammation. Additionally, N6-methyladenosine (M6A) modifications recognized by IGF2BP2 enhanced TRIM21 expression by stabilizing TRIM21 mRNA.

Conclusion: This study positions the TRIM21-NRF2 axis as a key regulator of oxidative stress and inflammation in IC/BPS and suggests it as a promising therapeutic target for future IC/BPS interventions.

Keywords: IC/BPS; N6-methyladenosine; NRF2; TRIM21; ubiquitination.

Figure 1

Effect of NRF2 expression on bladder function in the bladder inflammation model. (A and B) NRF2 protein levels in the lesional and non-lesional bladder mucosa of patients with IC/BPS (Each L and N constituted a paired sample.). (C) Urinary blots from WT and NRF2 KO mice, with each bright dot representing a urination event. (D) Diagram of the urodynamic examination, where each peak signifies a urination. (E) 50% withdrawal threshold of WT and NRF2 KO mice. (F) Representative bladder images. (G) HE staining of bladder tissues. ** P < 0.01.

Figure 2

Effects of NRF2 expression on oxidative stress and inflammation in the cellular inflammation model. (A) ROS levels in cells transfected with vector, NRF2, shCtrl, or shNRF2. (B) Relative GSH/GSSG ratio of cells transfected as indicated. (C) Apoptosis rate of cells under the specified transfections. (D and E) Western blotting analysis of oxidative stress inflammatory and apoptotic indicators in SV-HUC-1 cells following indicated transfections and treatments. ** P < 0.01.

Figure 3

TRIM21 interacts with NRF2. (A) Silver staining of SDS-PAGE was performed through IP assays using either IgG or NRF2 antibodies. Red colored items: silver-stained protein bands (B) MS analysis confirmed the interaction between TRIM21 and NRF2. y-type ions: C-terminal fragments (blue peaks); b-type ions: N-terminal fragments (red peaks); Modified fragments: y⁺-H₂O: Water loss peaks; y⁺-NH₃: Ammonia loss peaks. (C and D) Cell lysates from SV-HUC-1 cells were subjected to IP using antibodies against TRIM21 and NRF2. (E) IF images demonstrate the colocalization of TRIM21 and NRF2 in SV-HUC-1 cells. (F) Structural domains illustrating the binding of TRIM21 and NRF2 are shown through molecular docking analysis. (G) Schematic representation of full-length (FL) Myc-labeled TRIM21 along with various deletion mutants. (H) HEK293T cells were co-transfected with the indicated plasmids, and cell lysates were analyzed by IP using Myc beads, followed by Western blotting analysis with specific antibodies.

Figure 4

TRIM21 ubiquitinates NRF2. (A) HEK293T cells expressing vector or Myc-TRIM21 were treated with CHX (40 μg/mL) and collected at the indicated time, and analyzed by Western blotting. (B) SV-HUC-1 cells transfected with control shRNA (shCtrl) or TRIM21 shRNA were treated with CHX, collected at specified times, and subjected to Western blotting analysis. (C) HEK293T cells were cotransfected and treated as indicated; cell lysates underwent IP followed by Western blotting analysis. (D) SV-HUC-1 cells were cotransfected with the indicated shRNA and HA-ubiquitin (HA-Ub); cell lysates were subjected to IP and subsequent Western blotting analyses. (E and F) SV-HUC-1 cells were cotransfected as indicated, and NRF2 ubiquitination linkage was analyzed. (G) SV-HUC-1 cells transfected with wild-type Ub or Ub-Lys48R were cultured with either control vector or TRIM21; cell lysates were analyzed by Western blotting using specific antibodies. “+”: Treated; “-”: Untreated.

Figure 5

Effect of TRIM21 expression on oxidative stress, and inflammation in the cellular inflammation model. (A) ROS levels in cells transfected with vector, TRIM21, shCtrl, or shTRIM21. (B) Relative GSH/GSSG ratio of SV-HUC-1 cells transfection as indicated. (C) Apoptosis rates of cells transfected as indicated. (D and E) Western blot detected expression levels of oxidative stress inflammatory and apoptotic indicators in SV-HUC-1 after transfection and treated as indicated. * P < 0.05.

Figure 6

IGF2BP2 maintains TRIM21 mRNA stability through m6A modification. (A) Dot blot assay results from lesional and non-lesional bladder mucosa of patients with IC/BPS. (B and C) RT-qPCR and Western blotting analysis of TRIM21 expression following IGF2BP2 silencing in SV-HUC-1 cells. (D and E) RT-qPCR and Western blotting analysis of TRIM21 expression after IGF2BP2 overexpression in SV-HUC-1 cells. (F) IGF2BP2 enhances TRIM21 stability; SV-HUC-1 cells were treated with Actinomycin D at specified times, and the mRNA half-life of TRIM21 was assessed by RT-qPCR normalized to β-Actin. (G) Western blotting analysis of NRF2 following IGF2BP2 overexpression, with or without shTRIM21. (H) Luciferase activities of WT and mutated TRIM21 plasmids. (I) Positive correlation between IGF2BP2 and TRIM21 expression in the lesional bladder mucosa of patients with IC/BPS. “+”: Treated; “-”: Untreated. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 7

Correlation between IGF2BP2, TRIM21, and NRF2. (A) Representative IHC images of IGF2BP2, TRIM21, and NRF2 of clinical patients with IC/BPS. (B) The mechanistic scheme of our study.