Vasopressin-Dependent β-Catenin phosphorylation at Ser552 Regulates Gene Expression and Branching Structure of Mouse Collecting Duct System

Vasopressin binds to V2 receptors in collecting duct principal cells triggering protein kinase A (PKA) –dependent signaling and increased expression of PKA-target genes such as Aqp2. Our previous observations show that vasopressin increases phosphorylation of Ctnnb1 at Ser552, which is prevented by PKA deletion. However, specific roles in regulation of the mature collecting duct have not been ascertained.

Using CRISPR/Cas9, a Ctnnb1 S552A knock-in mutant (Mut) mouse line was generated. Experiments were done in both wild type (WT) mice and Mut mice undergoing administration of vehicle or the vasopressin analog, desmopressin (dDAVP) by osmotic minipumps (2 ng/hour, 5days) without water restriction (n=4 mice for each group). RNA-seq transcriptomics and ATAC-seq- based DNA accessibility assessment were carried out in microdissected cortical collecting ducts (CDs). Immunocytochemistry of kidney section was performed to determine the nephron: CD ratio in mice. 

RNA-seq in microdissected cortical collecting ducts (CCDs) from vasopressin-excess mice showed significant changes in 153 transcripts including the expected increases in aquaporin-2 (AQP2) and aquaporin-3 (AQP3) along with increases in several other known targets of V2-receptor-mediated signaling in principal cells. In addition, there were decreases in abundances of many transcripts known to be targets of regulation by the Wnt/frizzled/β-catenin pathway. The vasopressin-excess model also manifested marked increases in phosphorylation of β-catenin at a known protein kinase A (PKA) site, viz. Ser552. CRISPR-mediated mutation of this PKA-target site in mice resulted in extensive changes in the transcriptomes of microdissected CCDs after dDAVP-infusion, heavily weighted toward increases in known transcriptional targets of the Wnt/β-catenin pathway. At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. ATAC-seq in microdissected CCDs from dDAVP-infused mice revealed decreases in chromatin accessibility at promoters of many genes regulated in the Wnt/β-catenin pathway. The cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 + 0.66 in control mice to 3.33 + 0.82 in Ser552Ala mutant mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between control and Ser552Ala mutant mice (both ~14,000 per kidney).

Vasopressin-excess is associated with vasopressin/PKA-dependent repression of Wnt/Frizzled/β-catenin target gene expression, mediated in part by PKA-dependent phosphorylation of Ser552 of β-catenin. The repression is associated with decreasing chromatin accessibility in the promoter-TSS regions of target genes. The observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of β-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation.