Year: 2015
Type: dissertation
Abstract: In this study the role of the chondrocyte cytoskeleton in regulating cellular mRNA levels in chondrocytes was examined. Passaging of chondrocytes in traditional 2D monolayer culture results in chondrocyte dedifferentiation. As compared to primary chondrocytes, passaged cells exhibited a lower g-/f-actin ratio. However tubulin polymerization was not significantly altered by passaging. Pharmacological actin depolymerization partially redifferentiated passaged chondrocytes whereas modulation of tubulin did not. To delineate the mechanism by which actin polymerization regulates gene expression, myocardin related transcription factor A (MRTF) signaling was investigated. Passaging resulted in an increased amount of MRTFa as well as a predominantly nuclear localization of MRTF; these changes occurred within 5 days of culture and were sustained in passaged chondrocytes. Plating passaged cells in high density 3D or in suspension on top of agarose or exposure of cells in monolayer culture to latrunculin B resulted in increased g-/f-actin, cytoplasmic localization of MRTFa, and fibroblast matrix (col1 and tnc) mRNA levels. Exposure of chondrocytes to MRTFa inhibitor (CCG1423) resulted in only small changed to cell shape did not significantly affect gene expression but did result in reduced fibroblast matrix gene expression. siRNA mediated MRTF knockdown mimicked some but not all effects on gene expression as actin depolymerization. In addition SRF knockdown did not completely mimic MRTF knockdown. This demonstrated that actin could utilize several different signaling mechanisms to regulate gene expression. Although actin depolymerization did not completely redifferentiate passaged chondrocytes, it appears to be a potent regulator passaged chondrocytes gene expression and initiates the redifferentiation process.
Source: TSpace (University of Toronto)
Author: Justin Parreno
Language: English
Subfield: Rheumatology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2804730516