Date of Award
Doctor of Philosophy (PhD)
William F. Glass
Chronic renal diseases show increased deposition of extracellular matrix (ECM) in the glomerulus (glomerulosclerosis). Glomerulosclerosis is associated with activation of normally quiescent glomerular mesangial cells into myofibroblast-like cells. The overall objective of this study is to delineate cellular mechanism/s of myofibroblast-differentiation in disease states. In cultured mesangial cells certain characteristics of myofibroblast differentiation (α-smooth muscle actin (α-SMA) and hypertrophy) are associated with an increase in polymeric actin microfilaments (stress fibers). It is likely that other genes are also regulated in an actin cytoskeleton-dependent manner during myofibroblast differentiation. In these studies, we therefore examined the hypothesis that changes in the actin cytoskeleton regulate myofibroblast differentiation of mesangial cells.
In vivo, myofibroblasts play a major role in ECM accumulation and tissue scarring. TGFβ increases ECM deposition by increasing plasminogen actuator inhibitor type-1 (PAI-1; expression. PAI-1 inhibits ECM degradation by tissue- (tPA) and urokinase-type (uPA) plasminogen activators. Additionally, the Rho family of GTPases is required for TGFβ-induced PAI-1 expression. Since, regulation of actin cytoskeletal organization is a major function of Rho, the hypothesis that Rho-mediated changes in actin cytoskeleton modulate PAI-1 expression in glomerular cells was proposed The effects of modulating the actin cytoskeleton on TGFβ-induced PAI-1 expression were examined in cultured human glomerular mesangial cells. Inhibitors of Rho signaling decreased basal and TGFβ induced PAI-1 mRNA and protein expression. These effects were mimicked by direct inhibition of actin polymerization by Cytochalasin B (CytB) suggesting that the effects of Rho on PAI-1 expression are mediated through the actin cytoskeleton. CytB also inhibited the activity of a PAI-1 promoter. Conversely, stabilization of actin microfïlaments with jaspiakinolide (JAS) had the opposite effect on TCFβ-induced PAI-1 expression. These results indicate a bi-directional regulation of PAI-1 by the actin cytoskeleton depending on the state of actin polymerization. In contrast to inhibition of PAI-1, actin-depolymerizing agents increased tPA mRNA expression.
In summary, the changes in the actin cytoskeleton mediate the effects of Rho-GTPases in modulation of PAI-1 and tPA expression in response to TGFβ. Thereby, they may control FCM degradation. Overall, the results suggest that changes in organization of the actin cytoskeleton regulate myofibroblast differentiation and ECM accumulation by coordinately modulating expression of multiple genes.
"Modulation of TGFβ-Induced PAI -1 Expression by Changes in Actin Polymerization in Human Mesangial Cells"
(2006). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/x7yh-d092