DNA Methylation Reduces the Yes-Associated Protein 1/WW Domain Containing Transcription Regulator 1 Pathway and Prevents Pathologic Remodeling during Bladder Obstruction by Limiting Expression of BDNF

Abstract

Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction in vivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P < 0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change in vitro.

Persistent myopathy despite release of partial obstruction: in vivo reversal of dysfunction and transcriptional responses using rapamycin

“…Only samples with RIN >8 were processed further. We designed a custom high‐throughput QPCR (HT‐QPCR) array similar to the array published in Sidler et al Candidate genes were selected through curation of the literature for genes related to bladder SMC differentiation, mechanical strain, and bladder obstruction (Table S1). This custom‐ordered PCR array (RT 2 Profiler PCR Array, Qiagen, Hilden, Germany) including five reference genes and built‐in quality controls was standardized and highly quantitative.…”

“…QPCR primers for cDNA of reference genes B2M, Ubc, Sdha, Rpl13, Rpl32, and Hprt were previously described. 41 Pre-designed primers for target genes Igfbp7, Bmp2, Dec1+2, Clock, Npas2, Per1+2, Cry1+2, Nr1d1, Bmal, and Sod3 were purchased from IDT.…”

Inhibition of DNA methylation during chronic obstructive bladder disease (COBD) improves function, pathology and expression

“…A sequence of inflammation, hypertrophy and hyperplasia leads to an almost intractable state which persists despite adequate treatment of the initial obstruction 3 , 9 , 23 – 25 . During obstruction, this state is exacerbated by the epigenetic drug, decitabine (DAC) 26 .…”

“…While epigenetics works through many mechanisms, DNA methylation is a generally stable form of epigenetic change, which can be targeted pharmacologically and genetically 27 , 28 . DNA methylation plays a role in gene expression of key obstructive factors, with implications for downstream signaling and pathology 26 . For example, we have seen that DNA methylation alters Brain-derived neurotrophic factor (BDNF) and Connective tissue growth factor (CTGF) during obstruction 26 .…”

“…DNA methylation plays a role in gene expression of key obstructive factors, with implications for downstream signaling and pathology 26 . For example, we have seen that DNA methylation alters Brain-derived neurotrophic factor (BDNF) and Connective tissue growth factor (CTGF) during obstruction 26 . We also know that several genes dysregulated during COBD respond to rapamycin 1 , a drug affecting the mammalian target of rapamycin (mTOR) pathway, which can affect DNA accessibility and chromatin 29 , 30 .…”