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Abstract: FR-PO580

The Immune Checkpoint Protein PD-L1 Interacts with BBS5 to Regulate Ciliogenesis and Hedgehog Signaling

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Agborbesong, Ewud, Mayo Clinic Department of Internal Medicine, Rochester, Minnesota, United States
  • Li, Xiaoyan, Mayo Clinic Department of Internal Medicine, Rochester, Minnesota, United States
  • Zhou, Xia, Mayo Clinic Department of Internal Medicine, Rochester, Minnesota, United States
  • Calvet, James P., University of Kansas Medical Center Department of Biochemistry and Molecular Biology, Kansas City, Kansas, United States
  • Harris, Peter C., Mayo Clinic Department of Internal Medicine, Rochester, Minnesota, United States
  • Li, Xiaogang, Mayo Clinic Department of Internal Medicine, Rochester, Minnesota, United States
Background

Programmed cell death 1 ligand 1 (PD-L1) is an immune checkpoint protein regulates the immune synapse, a structure formed between immune cells and share structural and functional homology to primary cilium. However, whether and how PD-L1 regulates ciliogenesis remains elusive.

Methods

To investigate the role of PD-L1 on ciliogenesis, we performed immunostaining, Western blot, and qRT-PCR analysis in mouse NIH3T3 fibroblasts and human retinal pigment epithelium (RPE) cells. We also performed co-immunoprecipitation analysis to examine the interaction between PD-L1 and cilia associated proteins.

Results

We found that PD-L1 is located at the basal body and the Golgi apparatus as seen by its co-localization with centrosome marker γ-tubulin, Golgi associated protein Ift20, and Golgi marker Giantin. Knockdown of PD-L1 resulted in an increase in percentage of ciliated cells and average cilia length in 3T3 and RPE cells, whereas that overexpression of GFP-PD-L1 inhibited ciliogenesis in those cells. Consistent with this result, we found that serum starvation, which induces ciliogenesis, resulted in the downregulation of PD-L1 levels. Knockdown of PD-L1 increased the accumulation of Rab8a, BBS5 and polycystin 2 along the cilia axoneme in 3T3 cells but decreased the accumulation of Ift140 along the cilia in 3T3 cells and RPE cells. In addition, we found that the protein levels of BBS5, IFT20 and Rab8a were upregulated in PD-L1 knockdown 3T3 and RPE cells. We also found that PD-L1 formed a complex with BBS5 by co-immunoprecipitation analysis, and that knockout of BBS5 in PD-L1 knockdown 3T3 cells decreased the accumulation of polycystin 2 along the cilia. However, knockdown of BBS5 did not affect the localization, or protein level of PD-L1. We further found that knockdown of PD-L1 resulted in 1) the accumulation of the components of the hedgehog signaling pathway, including Smoothened (Smo) and Gli3, on cilia axoneme in 3T3 cells, 2) the upregulation of mRNA and protein levels of Gli1 and Gli3, and 3) the downregulation of Smo, Gli2 and Ptch1 proteins but not their mRNAs.

Conclusion

This study shows that PD-L1 is a novel basal body/Golgi component to regulate ciliogenesis and polycystin 2 ciliary trafficking through BBS5 and connects for the first time an immune checkpoint protein with ciliopathies, and possibly immune response.

Funding

  • NIDDK Support