DOI
https://doi.org/10.25772/HC1Z-YW06
Defense Date
2015
Document Type
Thesis
Degree Name
Master of Science
Department
Pharmacology & Toxicology
First Advisor
Pin-Lan Li
Abstract
Autophagy is of importance in the regulation of cell differentiation and senescence in podocytes, the highly differentiated glomerular epithelial cells. It is possible that derangement of autophagy under different pathological conditions activates or enhances Epithelial-to-Mesenchymal Transition (EMT) in podocytes, resulting in glomerular sclerosis. To test this hypothesis, the present study produced lysosome dysfunction by inhibition of vacuolar- type H+-ATPase (V-ATPase) to test whether deficiency of autophagic flux enhances EMT in podocytes. By Western blot analysis, inhibition of lysosome function by V-ATPase inhibitor or its siRNA was found to induce a significantly enhanced EMT in cultured podocytes, as shown by marked decreases in P-cadherin (P-cad) and zonula occludens-1 (ZO-1) as epithelial markers and simultaneous increases in the mesenchymal markers, fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These changes in EMT markers were confirmed by confocal microcopy. This enhancement was accompanied by deficient autophagic flux, as demonstrated by remarkable increases in LC3B-II levels and accumulation of p62/Sequestosome 1 (p62) regardless of whether the autophagosome formation was stimulated or not. However, inhibition of the autophagosome formation using spautin-1 (Sp-1) significantly attenuated both enhancement of EMT and deficiency of autophagic flux. To explore the mechanisms by which deficient autophagic flux enhances EMT, we tested the role of accumulated p62 as a signal hub in this process. Neither the nuclear factor erythroid 2–related factor 2 (Nrf2) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κBs) pathway of p62 regulation contributed to enhanced EMT. However, inhibition of cyclin-dependent kinase 1 (CDK1) activity reduced phosphorylation of p62 and enhanced EMT in podocytes similar to lysosome dysfunction. Given that the lack of phosphorylated p62 leads to a faster exit from cell mitosis, enhanced EMT associated with lysosome dysfunction may be attributed to accumulation of p62 and associated reduction of p62 phosphorylation.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
4-7-2015