Abstract: FR-PO165
Megalin Shuttles Extracellular Angiotensin II and Stanniocalcin-1 to the Mitochondria via Retrograde Early Endosomes to the Golgi Pathway and Regulates Glycolytic and Respiratory Capacities
Session Information
- Mitochondriacs and More
November 03, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Cell Biology
- 201 Cell Signaling, Oxidative Stress
Authors
- Li, Qingtian, Baylor College of Medicine, Houston, Texas, United States
- Lei, Fan, Baylor College of Medcine, Houston, Texas, United States
- Tang, Yi, West China Hospital of Sichuan University, Chengdu, China
- Pan, Jenny S., Baylor College of Medicine, Houston, Texas, United States
- Tong, Qiang, Baylor College of Medicine, Houston, Texas, United States
- Sheikh-Hamad, David, Baylor College of Medicine, Houston, Texas, United States
Background
Some extracellular signaling molecules are detected in the mitochondria, including angiotensin II, insulin, stanniocalcin-1 (STC1), TGF-ß and erythropoietin; these are known as mitochondrial intracrines. The mechanism of mitochondrial intracrine targeting is unknown. Megalin/LRP2 is highly expressed on the apical surface of kidney proximal tubule cells, and is involved in the uptake of filtered vitamins and proteins. Megalin mutations are linked to the pathogenesis of Donnai-Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction. Megalin has not been shown to reside in the mitochondria.
Results
Our data suggest that megalin is present in kidney mitochondria in vivo, and mitochondria of cultured 293T, C2C12 and Raw267.4 cells, and associates with stanniocalcin-1 and SIRT3. Megalin shuttles extracellular angiotensin II, TGF-ß and STC1 to the mitochondria (detailed in separate abstract).
We sought to characterize the shuttling pathway for megalin and its cargo (angiotensin II and STC1) from the cell surface to the mitochondria. Using chemical inhibitors of vesicular trafficking, we identified the microtubules and Golgi as key mediators, consistent with involvement of the retrograde early endosome-to-Golgi pathway. This was confirmed using an inhibitor of PIKfyve phosphoinositide kinase, a critical kinase involved in early endosome fusion with the Golgi. These findings were validated using CellLight® Reagent BacMan 2 technology, to trace the intracellular movement of extracellularly-applied rSTC1-FITC or angiotensin II-FITC. CRISPR-Cas9 knockdown of megalin in C2C12 cells prevents the entry of rSTC1-FITC and angiotensin II-FITC to the early endosome compartment (identified by early endosome RFP), while PIKfyve inhibition prevents their entry to the Golgi (identified by Golgi-RFP). Moreover, seahorse analysis reveals diminished glycolytic and respiratory capacities in megalin knockout C2C12 cells.
Conclusion
Megalin shuttles extracellularly applied angiotensin II and STC1 to the mitochondria through retrograde early endosome-to-Golgi pathway. Megalin is critical for glycolysis and respiration. The data also suggest that Donnai-Barrow and Lowe syndromes may represent mitochondrial intracrine signaling defects.
Funding
- Veterans Affairs Support