EZH2 and matrix co-regulate phenotype and KCNB2 expression in bladder smooth muscle cells

Priyank Yadav; Tabina Ahmed; Suejean Park; Martin Sidler; Annette Schröder; Karen J Aitken; Darius Bägli

EZH2 and matrix co-regulate phenotype and KCNB2 expression in bladder smooth muscle cells

Am J Clin Exp Urol. 2023 Aug 15;11(4):293-303. eCollection 2023.

Authors

Priyank Yadav^{1

2}, Tabina Ahmed¹, Suejean Park¹, Martin Sidler³, Annette Schröder⁴, Karen J Aitken¹, Darius Bägli^{1

5}

Affiliations

¹ The Hospital for Sick Children Toronto, Ontario, Canada.
² Department of Urology and Renal Transplantation, Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow, Uttar Pradesh, India.
³ Paediatric and Neonatal Surgery, Klinikum Stuttgart Stuttgart, Baden-Württemberg, Germany.
⁴ Department of Urology and Pediatric Urology of The University Medical Center Mainz Mainz, Rheinland-Pfalz, Germany.
⁵ Division of Urology, Department of Surgery, University of Toronto Toronto, Ontario, Canada.

PMID: 37645613
PMCID: PMC10461034

Abstract

Background: Partial bladder outlet obstruction (PBO) is a widespread cause of urinary dysfunction and patient discomfort, resulting in immense health care costs. Previously, we found that obstruction is associated with altered regulation of epigenetic machinery and altered function. Here we examined if PBO and chronic bladder obstructive disease (COBD) affect epigenetic marks in a proof of principle gene and explored mechanisms of its epigenetic regulation using in vitro models.

Methods: Archival obstruction tissues from COBD had been created in 200-250 g female Sprague-Dawley rats by surgical ligation of the urethra for 6 weeks, followed by removal of the suture and following animals for 6 more weeks. Obstruction (PBO) is the 6-week ligation only. Sham ligations comprise passing the suture behind the urethra. Histone3 lysine27 trimethylation (H3K27me3) was studied by immunostaining and Chromatin immunoprecipitation (ChIP)/PCR. The interaction of matrix with KCNB2 regulation was studied in human bladder SMC plated on damaged matrix and native collagen and treated with vehicle or UNC1999. Cells were analyzed by immunostaining for cell phenotype, and western blotting for KCNB2, H3K27me3 and EZH2. Effects of conditioned media from these cells were also examined on cell phenotype. siRNA against KCNB2 was examined for effects on cell phenotype and gene expression by RT-qPCR.

Results: H3K27me3 increased by immunofluorescence during PBO, and by ChIP/PCR during COBD in the CpG Island (CGI) as well as 350 bp upstream. Obstruction vs. sham also showed an increase in H3K27me3 deposition. In SMC in vitro, EZH2 inhibition restored KCNB2 expression and partially restored SMC phenotype.

Conclusions: Regulation of KCNB2 at the promoter demonstrated dynamic changes in H3K27me3 during COBD and obstruction. In vitro models suggest that matrix plays a role in regulation of EZH2, H3K27me3 and KCNB2, which may play a role in the regulation of smooth muscle phenotype in vivo.

Keywords: Bladder obstruction; H3K27me3; epigenetics; matrix biology; potassium channels; smooth muscle.