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The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/Mtor/Rac1 Signaling in Podocytes

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The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/Mtor/Rac1 Signaling in Podocytes BASIC RESEARCH www.jasn.org The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes Gentzon Hall,1,2,3 Brandon M. Lane,1,2 Kamal Khan ,4 Igor Pediaditakis,4 Jianqiu Xiao,4 Guanghong Wu,1,3 Liming Wang,3 Maria E. Kovalik,1,2,3 Megan Chryst-Stangl,1,2 Erica E. Davis,1,4 Robert F. Spurney,3 and Rasheed A. Gbadegesin 1,2,3 Departments of 1Pediatrics and 3Medicine, Duke University School of Medicine, Durham, North Carolina; 2Duke Molecular Physiology Institute, Durham, North Carolina; and 4Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina 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 hypoth- esized 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 pre- viously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT)orANLNR431C. 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-3b, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expres- sion of endogenous ANLN and mTOR. Conclusions The ANLNR431C mutation causes multiple derangements in podocyte function through hyper- activation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calci- neurin 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. J Am Soc Nephrol 29: 2110–2122, 2018. doi: https://doi.org/10.1681/ASN.2017121338 Received December 29, 2017. Accepted May 31, 2018. FSGS is the most common primary glomerular G.H. and B.M.L. contributed equally to this work. 1 disorder causing ESKD in the United States. De- Published online ahead of print. Publication date available at spite current therapies, approximately 50% of pa- www.jasn.org. tients with nephrotic FSGS develop ESKD within Correspondence: Dr. Rasheed A. Gbadegesin, Department of 2 10 years of initial diagnosis. It is, therefore, critical Pediatrics, Duke University Medical Center, Duke University, T- to improve our understanding of the pathobiology Level RM0909 CHC, BOX 3959, Durham, NC 27710. Email: of the disease to devise more effective treatment [email protected] strategies. Copyright © 2018 by the American Society of Nephrology 2110 ISSN : 1046-6673/2908-2110 JAmSocNephrol29: 2110–2122, 2018 www.jasn.org BASIC RESEARCH Studies of familial forms of FSGS suggest that disease results Significance Statement from injury or loss of glomerular epithelial cells (i.e.,podo- cytes).3 Podocytes are terminally differentiated cells that The authors previously reported that mutations in ANLN can cause maintain the structural integrity of the glomerular filtration familial FSGS. Anillin is an F-actin binding protein that modulates barrier. This function is highly dependent on the dynamic cell motility and signaling through the phosphoinositide 3-kinase (PI3K) pathway. This study examines its signaling through the PI3K regulation of the podocyte actin cytoskeleton. The phosphoi- pathway in podocytes to understand its role in the pathobiology nositide 3-kinase (PI3K)/AKTsignaling pathway is an essen- of FSGS. Mutant anillin induced hypermotility and apoptosis, tial regulator of podocyte actin cytoskeletal dynamics that has enhanced cellular proliferation, and activated PI3K/AKT/mTOR/ been shown to participate in slit diaphragm signaling through Rac1 signaling. Aberrant podocyte phenotypes induced by the interactions with proteins, such as CD2-associated protein mutation were ameliorated by inhibition of downstream effectors of PI3K and calcineurin phosphatase, and calcineurin inhibition 4 (CD2AP),nephrin,andpodocin. Pathogenic mutations in ameliorates ANLNR431C-induced podocyte apoptosis and down- CD2AP cause FSGS through mechanisms that involve the dis- regulates endogenous mTOR and ANLN expression. Drugs tar- ruption of PI3K/AKT signaling, highlighting the importance geting these pathways may be useful in the treatment of some of this pathway in the pathobiology of podocyte dysfunction forms of FSGS. in FSGS.5 fi We previously identi ed disease-causing muta- suppression experiment as described in Supplemental Mate- . tions (c.1852G T: p.Gly618Cys[G618C] and c.1291C T: rial. We validated the transient suppression experiment using p.Arg431Cys[R431C]) in familial FSGS, the latter of which is CRISPR/Cas9 genome editing of the anln locus in zebrafish in the F-actin binding domain of anillin (ANLN). ANLNR431C (Supplemental Material). induced hypermotility in podocytes and disrupted the crit- ical interaction between ANLN and CD2AP. To gain a better Conditionally Immortalized Human Podocyte Culture understanding of the role of ANLNR431C and ANLNG618C in Conditionally immortalized human podocytes were cultured the development of FSGS, we examined the effects of this and harvested as described.6 mutation in vivo through mRNA rescue experiments in anln- fi fi de cient zebra sh embryos and established that both Lentiviral Constructs and Infection mutations cause a loss of function during development. In Standard molecular cloning methods were used for stable complimentary in vitro studies, we showed that ANLNR431C transfection of all cell lines (Supplemental Material).7 overexpression hyperactivates AKT, mTOR, p70S6 Kinase, and Rac1 signaling in podocytes and that pharmacologic in- Immunofluorescence hibition of mTOR and Rac1 attenuates ANLNR431C-induced Conditionally immortalized human podocytes were differen- hypermotility and hyperproliferation. Additionally, we tiated and stained using standard protocols (Supplemental showed that ANLNR431C-induced hyperactivation of mTOR Material). induces endoplasmic reticulum (ER) stress and podocyte apoptosis and that this effect was attenuated by pharmaco- Rac1 and RhoA Activity Assay logic inhibition of mTOR, glycogen synthase kinase 3b GTP-bound Rac1 and RhoA were analyzed using PAK and (GSK-3b), and calcineurin phosphatase (Cn). Finally, we Rhotekin-Rho binding domain bead assays (Supplemental determined that endogenous ANLN and mTOR protein Material). expressions are downregulated by the Cn/NFAT pathway. Together, these findings suggest that ANLNR431C disrupts Targeted Inhibition podocyte cytoskeletal dynamics and promotes podocyte ap- For inhibition experiments, stock inhibitors were made in optosis through dysregulation of the PI3K/AKT/mTOR/ DMSO, and dilution was carried out as described in Supple- p70S6/Rac1 pathway. Furthermore, we identify the Cn/ mental Material. NFAT signaling pathway as a regulator of endogenous mTOR and ANLN expression and provide novel mechanistic Migration Assay insights into the beneficial clinical effects of Cn inhibitors in Scratch wound assays were used as described in Supplemental some forms of FSGS. Material. Proliferation Assay METHODS A cell counting colorimetric assay was used for the cell pro- liferation studies as described in Supplemental Material. Transient Suppression of anln and In Vivo Complementation Assays in Zebrafish Apoptosis Assay We designed a splice blocking morpholino (MO) targeting Serum starvation method was used to induce apoptosis in the donor site of exon 5 of the anln ortholog in zebrafish experimental podocyte lines as described in Supplemental (Supplemental Figure 1A) and performed a transient Material. J Am Soc Nephrol 29: 2110–2122, 2018 ANLN R431C, mTOR, and Rac1 in FSGS 2111 BASIC RESEARCH www.jasn.org In Silico Modeling embryos (3, 6, and 9 ng); we observed disruption of mRNA The ANLNR431C variant wasassessedforitseffectsonthe splicing in anln e5i5 morphants (Supplemental Figure 1, B and secondary structure of the protein. The in silico prediction C). Furthermore, we observed a dose-dependent response of program I-TASSER (http://zhanglab.ccmb.med.umich.edu/ e5i5 MO; larval batches scored live at 4 days postfertilization I-TASSER/) was used to generate the ANLNWTand ANLNR431C displayed ocular and pericardial edema (P,0.001 versus con- images.8 trols; n=63–81 per batch, repeated) (Supplemental Figure 1D). Next, we coinjected 3 ng MO with 150 pg of human Actin Bundling/Polymerization Assay ANLNWT mRNA and scored larval batches for edema pheno- HEK 293T cellsweregrown in DMEM + 5% FBSin T75flasksat types in three phenotypic classes. Whereas 92% of e5i5 mor- 37°C until 85% confluency and transfected using Lipofecta- phant larvae displayed edema phenotypes, an equivalent dose mine 2000 with plasmids containing
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