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Abstract: FR-PO552

Cytoplasmic Domain of Fibrocystin/Polyductin Suppresses cAMP-Induced Cyst Formation of Pkhd1 Knockout (KO) Cells

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Ziegler, Wolfgang H., Medizinische Hochschule Hannover Zentrum fur Kinderheilkunde und Jugendmedizin, Hannover, Niedersachsen, Germany
  • Hassan, Fatima, Medizinische Hochschule Hannover Zentrum fur Kinderheilkunde und Jugendmedizin, Hannover, Niedersachsen, Germany
  • Hahnenstein, Susanne, Medizinische Hochschule Hannover Zentrum fur Kinderheilkunde und Jugendmedizin, Hannover, Niedersachsen, Germany
  • Haffner, Dieter, Medizinische Hochschule Hannover Zentrum fur Kinderheilkunde und Jugendmedizin, Hannover, Niedersachsen, Germany
Background

Loss of ciliary protein function leads to defective control of epithelial homeostasis in hereditary polycystic kidney diseases. To address molecular aspects of epithelial function, monolayered epithelial spheroids can be used to analyze consequences of protein expression and pharmacological intervention. Here, we employed epithelial cell clones deficient for the protein fibrocystin/polyductin (FPC), the cause of ARPKD, to study the impact of FPC cytoplasmic domain expression on cAMP/Src-induced cyst formation.

Methods

We used pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout (KO) of Pkhd1 / FPC, and corresponding controls. Cells were grown in matrigel to allow formation of epithelial spheroids within 3 days. Forskolin (Fsk) treatment was employed to induce cAMP-mediated cyst growth mimicking disease conditions. Proportional lumen, i.e. the ratio of lumen to spheroid area, provided the measure to detect the enhanced water / ion transport across the barrier that is characteristic for cystic epithelia. Cellular signals known to stimulate cyst formation were modulated by viral expression of the FPC C-terminal domain and/or interventional treatment.

Results

In Pkhd1-KO cell lines, enhanced cAMP levels resulted in massive lumen expansion of epithelial spheroids with no increase in cell number. Cyst induction was sensitive to inhibition of Src kinase and led to activation and increased Y705 phosphorylation of STAT3. To address the contribution of FPC to cyst signaling, expression of a membrane-bound FPC C-terminal protein domain was studied, and its processing and intracellular localization determined. Controlled expression of the FPC cytoplasmic domain in Pkhd1-KO cell lines suppressed the Fsk-induced increase of proportional lumen / cyst formation, and furthermore, reduced activation of Src and STAT3 as well as STAT3-dependent transcription.

Conclusion

In FPC-deficient pl-MDCK cells, cyst formation stimulated by high cAMP levels is associated with enhanced Src/STAT3 signaling. Expression of the FPC cytoplasmic domain can correct control of the epithelial barrier in Pkhd1-KO and inhibit cyst growth in vitro. Therefore, expression of FPC domain constructs can lead to a gain-of-function and limit cyst promoting signals, a proposed cellular function of FPC protein.

Funding

  • Government Support – Non-U.S.