Abstract: FR-PO709
Mutation in Transient Receptor Potential Cation Channel Subfamily C Member 6 (TRPC6) Regulates Yes-Associated Protein 1(YAP1) Phosphorylation
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1304 Glomerular Diseases: Podocyte Biology
Authors
- Chou, Chia An, Bristol Renal, Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom
- Saleem, Moin, Bristol Renal, Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom
- Welsh, Gavin Iain, Bristol Renal, Department of Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom
Background
Transient receptor potential cation channel, subfamily C member 6 (TRPC6), is a cation channel associated with hereditary focal segmental glomerulosclerosis. Angiotensin II(Ang II) activates TRPC6 and its downstream signalling. We investigated signalling regulated by TRPC6 in podocytes to understand the disease pathogenesis.
Methods
We developed conditionally immortalized wild-type and TRPC6-knockout (T6K) podocytes. Lentiviral transduction was used to express GFP-tagged TRPC6 in T6K cells. Phosphoproteomic analysis was used to identify changes in signalling pathways mediated by knocking out TRPC6. Immunoblotting and quantitative reverse transcription PCR (RT-qPCR) were applied for validation.
Results
T6K podocytes showed increased phosphorylation of Yes-associated protein (YAP) compared with the control. Ang II treatment increased YAP phosphorylation in control podocytes but not in T6K. RT-qPCR showed decreased expression of connective tissue growth factor (CTGF) and cellular communication Network Factor 1 (CCN1), YAP downstream transcriptional targets, which returned to control levels upon expression of TRPC6GFP in T6K.
Conclusion
TRPC6 regulates the YAP phosphorylation and its downstream targets' level in podocytes. YAP is a transcriptional factor controlling cell proliferation. Our experiments suggest that knocking-out TRPC6 increases YAP phosphorylation and inhibits YAP-induced transcription.
Immunoblotting of p-YAP in T6K and control podocytes.
Cells were treated by 0.1 uM ANG II with various duration.
RT-qPCR of CTGF and CCN1 levels in WT, T6K and T6K+TRPC6GFP cell lines. Graphs represent mean±SEM. WT: wild-type; T6K: TRPC6 Knockout, T6K+TRPC6GFP:T6K transduced with GFP-tagged TRPC6.
Funding
- Private Foundation Support