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Abstract: SA-PO251

FRMD3/Protein 4.1O Splice Variants Regulate Hippo Signaling and Cell Migration

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Koenigshausen, Eva, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
  • Reisewitz, Timo, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
  • Matten, Larissa, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
  • Vienken, Theresia, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
  • Wiech, Thorsten, University Hospital Hamburg-Eppendorf Department of Pathology, Hamburg, Germany
  • Rump, Lars C., Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
  • Sellin, Lorenz, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Nordrhein-Westfalen, Germany
Background

FRMD3 has been identified as a candidate gene for diabetic nephropathy and encodes for protein 4.1O. Different protein 4.1O splice variants have been identified.
In diabetic kidney disease Hippo signaling (key effectors Yes-associated kinase (YAP) and its paralogue TAZ) is increased. Unphosphorylated YAP/TAZ activates target genes via different transcription factors (e. g. c-myc). Phosphorylated YAP/TAZ remains within the cytoplasm and is degraded. Activation of YAP/TAZ is increased by Myosin light chain kinase (MLCK). C-myc, YAP/TAZ and growth factors, e. g. EGF, have been shown to promote cell migration.

Methods

Human kidney biopsy samples from healthy and diabetic patients were stained for protein 4.1O. HEK293T cells were stimulated with low (5 mMl), high (25 mM) glucose concentrations or an osmotic control (5mM glucose + 19,5 mM mannitol). RNA was isolated and PCR performed. A c-myc reporter assay was analyzed. HEK293T cells expressed protein 4.1O splice variants or the control and were stimulated with low, high glucose or mannitol. After cell lysis, western blot was performed for phospho-YAP, myosin light chain (MLC), phospho-MLC and actin. Human podocytes were stably transduced with 4.1O splice variants. For migration assays, human podocytes were stimulated with FCS or EGF.

Results

Protein 4.1O expression is detected in healthy human glomeruli. In diabetic patients with CKD stage 3b to 5 and gross proteinuria protein 4.1O expression seems to be increased in podocytes. High glucose leads to enhanced transcription of FRMD3. Under high glucose condition protein 4.1O significantly increases YAP phosphorylation. This effect is abrogated if a splice variant lacking a c-terminal domain is expressed. In line with this finding, protein 4.1O decreases c-myc transactivation. MLC phosphorylation is not altered by 4.1O splice variants in high, low glucose or mannitol. Protein 4.1O (lacking the FERM domain and a c-terminal domain) reduces podocyte migration.

Conclusion

Expression of protein 4.1O is increased in human diabetic kidney disease and under high glucose conditions. Protein 4.1O splice variants have differential effects on c-myc transactivation, YAP/TAZ activation and migration. This improved understanding of protein 4.1O splice variant function helps to better understand its role in diabetic nephropathy.