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Abstract: PO0613

Autophagy Deficiency in Urothelial Cells Activates Progressive NF-κB Signaling

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Ortega, Michael, The Queen's Medical Center, Honolulu, Hawaii, United States
  • Villiger, Ross, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Harrison-Chau, Malia H., University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Kepler, Joshua, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Patwardhan, Geetika Yi, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Tamashiro, Kadee-Kalia, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Lee, Amanda J., University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Fogelgren, Ben, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
Background

The urothelium is a specialized epithelium that functions as a urine permeability barrier along the upper urinary tract and bladder. We have shown that conditional knockout (CKO) of exocyst gene Exoc5 in ureteric bud cells disrupts the urothelial stratification process during ureter development, which subsequently triggers cell death and ureter obstructions. This Exoc5 CKO mouse is a novel model of congenital obstructive uropathy (COU) and may be useful for elucidating the underlying pathological mechanisms of COU. Here, we investigated the role of exocyst-mediated autophagy in the stress responses of urothelial cells.

Methods

Cre/loxP Exoc5 urothelial ablation was accomplished with Ksp-Cre and Upk3-CreERT2 mouse driver strains for both embryonic and adult urothelial knockout. An immortalized human urothelial cell line (SV-HUC-1) was used for cellular assays. Autophagic flux and cell stress signaling were measured by immunofluorescence and western blotting.

Results

We report that urothelial Exoc5 ablation disrupted autophagy and promoted non-canonical NF-κB signaling during ureter development in Ksp-Cre mice. Adult urothelial Exoc5-knockout mice also showed disrupted autophagy, with an accumulation of lysosomes in the bladder urothelium. In SV-HUC-1 cells, EXOC4 co-immunoprecipitated with ATG7, and silencing of Exoc5 led to an accumulation of LC3II/I and p62, indicating poor autophagic flux. Direct inhibition of autophagy with BafA1 or VPS34i induced an early canonical RelA NF-κB response followed by a delayed p52 non-canonical NF-κB response and eventual cell death.

Conclusion

Here, we report that Exoc5 contributes to autophagy in urothelial cells, and impaired autophagy triggers progressive NF-κB signaling. The initial stress response activates canonical RelA NF-κB signaling, which is associated with survival mechanisms and inflammation. However, when the injury is not resolved, a delayed p52 non-canonical NF-κB signaling follows. Under these conditions, non-canonical NF-κB mediators TWEAK and its receptor Fn14 were highly responsive. Further investigation of this progressive NF-κB signaling series in urothelial cells may be critical for understanding the etiology of COU and any lingering chronic response after COU is resolved.

Funding

  • NIDDK Support