ESPN 54th Annual Meeting

ESPN 2022


 
SUPPRESSION OF ENHANCED SRC/STAT3 SIGNALING INHIBITS CYST FORMATION IN AN ARPKD EPITHELIAL CELL MODEL
FATEMA HASAN 1 HANNA BURMESTER 1 JENS H. WESTHOFF 2 THOMAS POKRANT 3 JULIA SCHOLZ 4 BJOERN BUCHHOLZ 4 JAN FAIX 3 DIETER HAFFNER 1 WOLFGANG H. ZIEGLER 1

1- DEPARTMENT OF PEDIATRIC KIDNEY, LIVER AND METABOLIC DISEASES, HANNOVER MEDICAL SCHOOL, GERMANY
2- DEPARTMENT OF PEDIATRICS I, UNIVERSITY CHILDREN’S HOSPITAL, HEIDELBERG, GERMANY
3- INSTITUTE FOR BIOPHYSICAL CHEMISTRY, HANNOVER MEDICAL SCHOOL, GERMANY
4- DEPARTMENT OF NEPHROLOGY AND HYPERTENSION, FRIEDRICH-ALEXANDER-UNIVERSITY ERLANGEN-NUERNBERG, GERMANY
 
Introduction:

Hereditary polycystic kidney diseases are characterized by defective epithelial morphogenesis and/or homeostasis. To study molecular aspects of epithelial defects, genetically modified canine renal tubular epithelial cells, MDCK, provide a well-established model. In 3D culture, monolayered epithelial spheroids with apicobasal polarity and controlled water and ion transport can be employed to analyze consequences of pharmacological intervention. Here, we study epithelial cells with fibrocystin deficiency, the cause of ARPKD, to address the relevance of STAT3 signaling and its inhibition on cyst formation.

Material and methods:

We employed pl-MDCK, sub-cloned principal-like cell lines, with CRISPR/Cas9-based genetic knockout of Pkhd1 and corresponding controls. Cells were grown in matrigel to allow spheroid formation, within 4 days, and treated with forskolin (Fsk) to stimulate cAMP-induced cystic growth, as reported for disease conditions. Lumen and cyst size were determined based on apical and basolateral markers using ImageJ/FIJI. Cyst formation was related to induction of STAT3 signaling.

Results:

Enhanced cAMP levels led to massive lumen expansion in epithelial spheroids of Pkhd1-KO cell lines as compared to wildtype. While Fsk treatment did not increase numbers of epithelial cells in cysts, KO cells showed moderately up-regulated proliferation in 2D culture. Cyst formation was accompanied by a rise in pSTAT3 in Pkhd1-KO cells. In addition, induction of STAT3 target genes, SOCS3 and c-myc, was observed in Fsk stimulated cells, and inhibition of Src kinase reduced pSTAT3 levels. Application of different approved drugs affecting STAT3 signaling strongly reduced lumen size of Pkhd1-KO cysts in a dose-dependent manner.

Conclusions:

In fibrocystin deficient pI-MDCK cells, induction of cyst formation by enhanced cAMP levels is associated with enhanced Src/STAT3 signaling. Its suppression by approved drugs resulted in inhibition of cystic growth in vitro. Thus, targeting enhanced Src/STAT3 signaling is a promising measure to ameliorate disease progression in ARPKD rendering further studies.