Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Abstract

BackgroundTransforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-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 1α1 (COL1A1), CTGF and MMP-2 mRNA.MethodsSerum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-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.ResultsTGFβ-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 TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.ConclusionsRGZ and CGZ inhibited profibrotic changes in TGF-β1-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 PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.

Simvastatin Attenuates TGF-�1-Induced Epithelial-Mesenchymal Transition in Human Alveolar Epithelial Cells

“…A549 cells demonstrated many features of AECII cells [32], and most studies investigating EMT have used A549 cells as a model of AECII [30,31,[33][34][35]. In addition, a few studies have used both A549 cells and primary AECIIs to study EMT and showed that A549 cells presented similar features as primary AECIIs during the EMT process [36,37].…”

Runx2/Smad3 Complex Negatively Regulates TGF-β-Induced Connective Tissue Growth Factor gene Expression in Vascular Smooth Muscle Cells

“…Interfering with Smad3, PPAR and its ligands suppress CTGF expression in response to TGF- 46,47) . Our study demonstrates that overexpression of Runx2 blunted the TGF-induction of CTGF expression.…”

Inhibitory Effect of Safflor Yellow on Pulmonary Fibrosis

“…2) This cytokine also drive EMT. 20,21) Besides, persistence of TGF-b1 expression counters the apoptosis of fibroblast by inducing activation of the adhesion-dependent focal adhesion kinase pathway and the soluble growth factor-mediated AKT pathway. 22,23) Other studies found that the aqueous extract of safflower (40 mg/ml) had anti-proliferative and pro-apoptotic effects on hepatic stellate cells that play a central role in hepatic fibrosis, 13) and it (150 mg/3 ml/0.25 kg/d) also could reduce serum TGF-b1 of unilateral ureteral obstruction-induced renal interstitial fibrosis in rats.…”