Kidney Week

Abstract: SA-PO485

Human and Mouse FPC-CTD Activate the MYC/Myc P1 Promoter: Implications for Renal Cystogenesis in ARPKD

Session Information

• Cystic Kidney Diseases: Basic/Translational
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Yang, Chaozhe, Children's National Medical Center, Washington, District of Columbia, United States

Chaozhe Yang,

• Harafuji, Naoe, Children's National Medical Center, Washington, District of Columbia, United States

Naoe Harafuji,

• Cuevas, Santiago, Children's National Medical Center, Washington, District of Columbia, United States

Santiago Cuevas,

• Odinakachukwu, Maryanne, Children's National Medical Center, Washington, District of Columbia, United States

Maryanne Odinakachukwu,

• Caldovic, Ljubica, Children's National Medical Center, Washington, District of Columbia, United States

Ljubica Caldovic,

• Bell, Phillip Darwin, University of Alabama, Birmingham, Alabama, United States

Phillip Darwin Bell,

• Trudel, Marie, Institut de recherches cliniques de Montreal, Montreal, Quebec, Canada

Marie Trudel,

• Guay-Woodford, Lisa M., Children's National Medical Center, Washington, District of Columbia, United States

Lisa M. Guay-Woodford,

Background

Human ARPKD (MIM 263200) results from mutations in PKHD1, but mouse Pkhd1 models express limited or no renal cystic disease. The protein product, FPC, undergoes Notch-like processing with regulated membrane-release and nuclear translocation of the intracellular C-terminal domain (FPC-CTD) (Kaimori, 2007). We have previously shown that c-Myc is overexpressed in human ARPKD and mouse Cys1^cpk cystic kidneys, but not in Pkhd1^cyli, Pkhd1^del67, or Pkhd1^del3-67 kidneys (ASN 2018). Trudel (2019) has demonstrated that c-Myc plays a dual role in Pkd1-induced pathogenesis, through Myc-mediated mechanisms and a feed-forward regulatory Pkd1–Myc loop. The current in vitro study was designed to compare MYC/Myc regulation by human and mouse FPC-CTD in collecting duct cell lines.

Methods

We generated immortalized normal human (hTERT) and mouse (mTERT) kidney collecting duct cell lines; V5-tagged hFPC-CTD and V5-tagged mFPC-CTD constructs; and luciferase constructs of the P1 promoter for human MYC and mouse Myc. Luciferase reporter assays were performed as previously described (Wu, 2013).

Results

Sequence analysis revealed 74% identity between the human MYC and mouse Myc P1 promoters and 55% identity between hFPC-CTD and mFPC-CTD. Combinatorial luciferase assays demonstrated that the hFPC-CTD and mFPC-CTD constructs enhanced MYC and Myc P1 promoter activity in both human and mouse collecting duct cell lines.

Conclusion

These in vitro data demonstrate that FPC-CTD activates MYC/Myc expression in both human and mouse collecting duct cells. Paradoxically, we have previously shown that loss of FPC-CTD leads to cystogenesis and c-Myc overexpression in human ARPKD kidneys, but not in mouse Pkhd1 models. We speculate that in renal collecting duct cells, FPC-CTD regulation of MYC/Myc transcription is differentially modulated by species-specific mechanisms. In the mouse, we propose that in normal collecting duct cells, FPC-CTD function at the P1 promoter is constrained by mouse-specific co-factors and overexpression of mFPC-CTD would stimulate Myc-induced cystogenesis. Current efforts are focused on ex vivo studies in mouse metanephroi to test this model.

Funding

• Private Foundation Support