First, they examined the expression of Arid1a in the developing and adult mouse bladders with immunofluorescent staining. It is broadly expressed across all three bladder layers at the onset of bladder differentiation and then becomes more specifically expressed in superficial cells at the luminal surface in adulthood. To further understand the expression of Arid1a and other SWI/SNF complex components, researchers then isolated mRNA from urothelial cells across development and adulthood (E15.5, E18.5, P60). RNA-sequencing confirmed that Arid1a and other subunits of the complex (Arid1b, Brg1/Smarca4, and Baf47/Smarcb1) were expressed throughout the urothelium, with decreasing expression of Arid1a and Arid1b starting at E18.5.
The investigators generated transgenic mice with conditional Arid1a knockout in urothelial progenitor cells and subsequently confirmed that Arid1a expression was depleted only in urothelial bladder cells but not other cell types. Interestingly, Arid1b expression increased, pointing towards a potential inhibitory effect of Arid1a on Arid1b or a compensatory mechanism upon Arid1a downregulation. Furthermore, in mutant embryos, the urothelium appeared disorganized across all layers and contained a significantly higher number of proliferating cells (Ki67+ and pHH3+) than controls, particularly in the basal layer. In addition, transcription factors required for urothelial cell differentiation showed reduced expression in mutants, further consolidating the role of Arid1a in the proliferation and differentiation of urothelial cells during development.
Importantly, in the adult bladders, more Ki67+ cells were found on the luminal surface rather than the basal layer. The investigators performed whole transcriptome sequencing of mutant and control adult bladders to identify differentially expressed genes (DEGs), focusing on genes related to the epigenetic regulator polycomb repressive complex 2 (PRC2) since SWI/SNF and PRC2 have antagonistic effects on gene expression. Ezh2, a component of PRC2, was upregulated in mutants compared to control. Similarly, genes that were downregulated in Ezh2 mutants were upregulated in Arid1a mutants. Other affected genes were involved in mitosis, the p53 pathway, proliferation, and oxidative phosphorylation.
Bladders in injured mice had a urothelium of four or more layers rather than three, and cell proliferation increased, particularly over the first three days. The researchers identified enrichment in genes involved in inflammatory pathways (TNFα, NF-κB, and IL6-JAK-STAT3) in mutants relative to controls and genes involved in the MTORC1 pathway, reaction oxygen species, and oxidative phosphorylation.
This study shows that Arid1a plays distinct roles in the development, maintenance, and injury response of bladder urothelium. Interestingly, in the context of cancer, the urothelium in Arid1a mutants did not show abnormal growth or thickening, indicating that it may not be sufficient for carcinogenesis. In addition, the finding that Arid1a depletion increases cell proliferation is consistent with the finding that human ARID1A may function as a tumor suppressor gene.
Written by: Bishoy M. Faltas, MD, Director of Bladder Cancer Research, Englander Institute for Precision Medicine, Weill Cornell Medicine, New York City, New York
References:
- Guo, C., Zhang, Y., Tan, R., Tang, Z., Lam, C. M., Ye, X., Wang, Z., & Li, X. (2022). Arid1a regulates bladder urothelium formation and maintenance. Developmental biology, 485, 61–69. Advance online publication. https://doi.org/10.1016/j.ydbio.2022.02.008
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