Effects of PPAR gamma ligands on TGF-beta1-induced epithelial-mesenchymal transition in alveolar epithelial cells.
BACKGROUND
Transforming growth factor beta1 (TGF-beta1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPAR gamma ligands have been shown to inhibit fibroblast activation by TGF-beta1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-beta1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1 alpha 1 (COL1A1), CTGF and MMP-2 mRNA.


METHODS
Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 microM) in the absence or presence of the PPAR gamma antagonist GW9662 (10 microM) before TGFbeta-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.


RESULTS
TGFbeta-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGF beta 1-induced changes in cell morphology, and PPAR gamma-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-beta1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-beta1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-beta1 was not inhibited by RGZ or CGZ.


CONCLUSIONS
RGZ and CGZ inhibited profibrotic changes in TGF-beta1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPAR gamma-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-beta1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.
Tan
Xiahui
X
Department of Pharmacology, University of Melbourne, Victoria, Australia.
Dagher
Hayat
H
Hutton
Craig A
CA
Bourke
Jane E
JE
D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000378:metabolism*; D047495:PPAR gamma; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000008:administration & dosage* / Q000037:antagonists & inhibitors
D047495:PPAR gamma; D053773:Transforming Growth Factor beta1