1. The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes.
- Author
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Hall G, Lane BM, Khan K, Pediaditakis I, Xiao J, Wu G, Wang L, Kovalik ME, Chryst-Stangl M, Davis EE, Spurney RF, and Gbadegesin RA
- Subjects
- Animals, Apoptosis genetics, Cell Movement genetics, Cells, Cultured, Gene Expression Regulation, Glomerulosclerosis, Focal Segmental pathology, Glomerulosclerosis, Focal Segmental physiopathology, Humans, Mutation, Missense, Podocytes metabolism, Sensitivity and Specificity, Signal Transduction, Zebrafish, rac1 GTP-Binding Protein genetics, Glomerulosclerosis, Focal Segmental genetics, Microfilament Proteins genetics, Phosphatidylinositol 3-Kinases genetics, Proto-Oncogene Proteins c-akt genetics, TOR Serine-Threonine Kinases metabolism
- Abstract
Background: We previously reported that mutations in the anillin ( ANLN ) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP., Methods: We conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLN
R431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN ( ANLNWT ) or ANLNR431C ., Results: Experiments in anln -deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C -expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C -expressing podocytes. Inhibition of mTOR, GSK-3 β , Rac1, or calcineurin ameliorated the effects of ANLNR431C . Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR., Conclusions: The ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes., (Copyright © 2018 by the American Society of Nephrology.)- Published
- 2018
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