Abstract
Water permeability of the kidney collecting ducts is regulated by the peptide hormone vasopressin. Between minutes and hours (short-term), vasopressin induces trafficking of the water channel protein aquaporin-2 to the apical plasma membrane of the collecting duct principal cells to increase water permeability. Between hours and days (long-term), vasopressin induces aquaporin-2 gene expression. Here, we investigated the mechanisms that bridge the short-term and long-term vasopressin-mediated aquaporin-2 regulation by α-actinin 4, an F-actin crosslinking protein and a transcription co-activator of the glucocorticoid receptor. Vasopressin induced F-actin depolymerization and α-actinin 4 nuclear translocation in the mpkCCD collecting duct cell model. Co-immunoprecipitation followed by immunoblotting showed increased interaction between α-actinin 4 and glucocorticoid receptor in response to vasopressin. ChIP-PCR showed results consistent with α-actinin 4 and glucocorticoid receptor binding to the aquaporin-2 promoter. α-actinin 4 knockdown reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. α-actinin 4 knockdown did not affect vasopressin-induced glucocorticoid receptor nuclear translocation, suggesting independent mechanisms of vasopressin-induced nuclear translocation of α-actinin 4 and glucocorticoid receptor. Glucocorticoid receptor knockdown profoundly reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. In the absence of glucocorticoid analog dexamethasone, vasopressin-induced increases in glucocorticoid receptor nuclear translocation and aquaporin-2 mRNA were greatly reduced. α-actinin 4 knockdown further reduced vasopressin-induced increase in aquaporin-2 mRNA in the absence of dexamethasone. We conclude that glucocorticoid receptor plays a major role in vasopressin-induced aquaporin-2 gene expression that can be enhanced by α-actinin 4. In the absence of vasopressin, α-actinin 4 crosslinks F-actin underneath the apical plasma membrane, impeding aquaporin-2 membrane insertion. Vasopressin-induced F-actin depolymerization in one hand facilitates aquaporin-2 apical membrane insertion and in the other hand frees α-actinin 4 to enter the nucleus where it binds glucocorticoid receptor to enhance aquaporin-2 gene expression.
Highlights
Vasopressin is a peptide hormone that regulates osmotic water reabsorption by the kidney collecting ducts (Knepper et al, 2015)
Vasopressin-induced F-actin depolymerization was recorded in live mpkCCD cells (Loo et al, 2013), a collecting duct principal cell model that expresses all necessary molecular components required for the vasopressin actions (Yu et al, 2009)
About 60.3% α-actinin 4 was found colocalized with F-actin, which had much pronounced staining at the cell periphery (Figures 1A,B)
Summary
Vasopressin is a peptide hormone that regulates osmotic water reabsorption by the kidney collecting ducts (Knepper et al, 2015) It does so by elevating intracellular cAMP and Ca2+ concentrations that control the molecular water channel protein aquaporin-2 (AQP2) in two modes (Judith Radin et al, 2012; Pearce et al, 2015). Vasopressin-induced F-actin depolymerization was recorded in live mpkCCD cells (Loo et al, 2013), a collecting duct principal cell model that expresses all necessary molecular components required for the vasopressin actions (Yu et al, 2009) To add another layer of complexity, AQP2 constantly exocytoses to and endocytoses from the plasma membrane (Brown, 2003; Tajika et al, 2005; Wang et al, 2020). Trafficking of AQP2 to and from the plasma membrane is tightly coupled with F-actin dynamics
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