Kidney Week

Abstract: TH-PO1089

Reciprocal Regulation Between ANLN and AKT Modulates Podocyte Proliferation

Session Information

• Glomerular Diseases: Podocyte Biology - I
  November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1204 Podocyte Biology

Authors

• Hall, Gentzon, Duke University Medical Center, Durham, North Carolina, United States

Gentzon Hall,

• Wu, Guanghong, Duke university, Durham, North Carolina, United States

Guanghong Wu,

• Strickland, Shelby, Duke University, Durham, North Carolina, United States

Shelby Strickland,

• Kovalik, Maria Eugenia, Duke University, Durham, North Carolina, United States

Maria Eugenia Kovalik,

• Sempowski, Benjamin Allen, Duke University Medical Center, Durham, North Carolina, United States

Benjamin Allen Sempowski,

• Lane, Brandon M., Duke University, Durham, North Carolina, United States

Brandon M. Lane,

• Chryst-Stangl, Megan, Duke Molecular Physiology Institute, Durham, North Carolina, United States

Megan Chryst-Stangl,

• Spurney, Robert F., Duke University Medical Center, Durham, North Carolina, United States

Robert F. Spurney,

• Gbadegesin, Rasheed A., Duke University Medical Center, Durham, North Carolina, United States

Rasheed A. Gbadegesin,

Background

We previously reported that mutations in anillin (ANLN) cause familial FSGS; however, the physiologic functions of ANLN in podocytes remain unknown. ANLN is widely recognized as a driver of cell survival signaling and proliferation through its CD2AP-mediated interactions with the PI-3K/p85/AKT signaling module. We previously showed that ANLN expression is upregulated in proliferating podocytes and described a dynamic modulation of AKT activation with alterations in ANLN expression. Here, we evaluated the reciprocal influence of AKT on ANLN activity and expression in podocytes.

Methods

We established stably expressing ANLN_WT-, ANLN_S659E-, ANLN_S659A, ANLN siRNA, AKT1 siRNA, STAT3 siRNA, β-Catenin siRNA and scrambled siRNA podocyte lines. We evaluated the lines in proliferation and apoptosis assays and in complimentary biochemical pathway analyses.

Results

ANLN Expression - ANLN expression was markedly reduced with AKT inhibitors and in AKT1 knockdown (KD) podocytes. We screened the ANLN promoter to search for transcription factors regulated by AKT and identified multiple candidate binding sequences for β-Catenin and STAT3. ANLN expression was significantly reduced in β-Catenin and STAT3 KD podocyte lines and phosphorylation/activation of β-Catenin was downregulated in AKT1 KD podocytes. Conversely, STAT3 phosphorylation was upregulated in AKT1 KD podocytes suggesting a compensatory function of STAT3 activation in AKT1 KD podocytes. ANLN Phosphorylation - We identified a highly conserved AKT phosphorylation motif (i.e. R-X-X-S₆₅₈) in the ANLN F-actin binding domain. We generated a phospho-specific ANLN_S658 antibody and observed that phosphorylation of ANLN at Ser658 was abrogated in ANLN_S658A-expressing podocytes demonstrating the specificity of the antibody. Podocytes expressing ANLN_S658E exhibited significantly enhanced proliferation relative to ANLN_WT- and ANLN_S658A-expressing podocytes suggesting that AKT may enhance the activity/stability of ANLN via direct phosphorylation of Ser658.

Conclusion

ANLN and AKT dynamically regulate one another to modulate podocyte proliferation. This study delineates the mechanisms of this reciprocal regulation and highlights potential therapeutic targets for the treatment of ANLN-induced podocyte dysfunction.

Funding

• NIDDK Support