Abstract: TH-PO577

Phosphorylation Insensitive 4E-BP1 Reduces Hyperproliferative Phenotype In Vitro

Session Information

Category: Genetic Diseases of the Kidney

  • 801 Cystic Kidney Diseases

Authors

  • Holditch, Sara, UC Denver Anschutz Medical Campus, Aurora, Colorado, United States
  • Brown, Carolyn Nicole, UC Denver Anschutz Medical Campus, Aurora, Colorado, United States
  • Ravichandran, Kameswaran, UC Denver Anschutz Medical Campus, Aurora, Colorado, United States
  • Edelstein, Charles L., University of Colorado Denver, Aurora, Colorado, United States
Background

Unchecked proliferation of cystic epithelial cells is a major contributor to cyst growth in PKD. The 4E-BP1 pathway is a crucial checkpoint in protein translation initiation and cellular proliferation. Evidence from oncology supports the malignant potential of 4E-BP1. A recognized oncotarget, 4E-BP1 is associated with worsening progression, metastasis, and morbidity in oncology. The aim of this study was to determine 1) whether PKD patient and animal model kidney tissues have dysregulated phospho 4E-BP1 species and 2) the effect of a phosphorylation insensitive 4E-BP1 (F113A) on phospho 4E-BP1 species distribution, cap dependent protein translation, and proliferation in renal epithelial cells.

Methods

Immunofluorescence staining of phospho 4E-BP1 species (T70, T37/47, S65) was performed on human ADPKD and Han:SPRD rat (Cy) kidneys. Western blot analysis, Cyquant cellular proliferation, and Firefly-renilla assays were performed on human primary epithelial cells from normal renal cortical tubular epithelium (PKD1+/+) and ADPKD cyst-lining epithelium (PKD1-/-) transfected with control or pCAG-F113A or transduced with control or F113A lentivectors.

Results

Phospho 4E-BP1 species were present in cyst lining cells of human ADPKD and Cy renal tissues. F113A resulted in substantially reduced phospho 4E-BP1 T37/46(0.89±0.08 vs 0.012±0.004DU, p<0.01) and S65 (0.63±0.04 vs 0.003±0.001DU, p<0.01), reduced cap-dependent protein translation (37%, p<0.01), and reduced 72hr proliferation (250±4 vs 180±5 480/528nm O.D, p<0.0001) in PKD1-/- cells. Surprisingly, in PKD1+/+ cells, F113A resulted in no phospho 4E-BP1 reduction, reduced cap-dependent protein translation (32%, p<0.01), and marginally reduced proliferation (375±5 vs 314±4 480/528nm O.D, p<0.0001). Acute stimulation with insulin resulted in maintained S65 suppression with F113A transfection in PKD1-/- (2.1±0.3 vs 0.2±0.1AU, *p<0.0001).

Conclusion

F113A results in a shift towards hypophosphorylated 4E-BP1 species, reduced cap dependent protein translation, and reduced proliferation, with more aggressive effects in PKD1-/- vs PKD1+/+ . Utilizing F113A gene therapy to counter the loss of the translationally repressive 4E-BP1 pathway in a murine model of PKD, is the next step in addressing a pathway seemingly integral to the pathobiology of PKD.

Funding

  • Other U.S. Government Support