Differential effects of TGF-β on expression of prepro-endothelin-1 and phenotype markers in human alveolar type lung (A 549) and bronchial (H 292) epithelial cells

Mareille Warnken1, Ahmedat Ahmedat1, Ulrich Reitzenstein1, Stefan M. Holtin1, Peter Mayer3, Uwe R Juergens2, Kurt Racké1. 1Depart. Pharmacol, Univ. Bonn, Bonn, Germany, 2Univ. Hospital, Med. Clinic II, Bonn, Germany, 3Federal Inst. for Drugs and Medical Devices, Bonn, Germany.

There is increasing evidence that endothelin is involved in the pathogenesis of pulmonary hypertension and fibrotic remodelling processes associated with chronic inflammatory airway diseases. TGF-β is a cytokine of particular importance for the initiation and maintenance of fibrotic process. In the lung TGF-β can trigger epithelial-mesenchymal transition (EMT), a process which may result in the generation of activated pulmonary fibroblasts. The present study aimed to explore the effect of TGF-β on the expression of endothelin in alveolar and bronchial epithelial cells.

Human alveolar type lung (A 549) and bronchial (H 292) epithelial cells were cultured for 1 week in presence of 1% FCS and absence or presence of 5 ng/ml TGF-β. Expression of mRNA encoding E-cadherin (an epithelial marker), N-cadherin (a mesenchymal marker) and prepro-endothelin-1 was determined by quantitative real time PCR.

After one week exposure to TGF-β, A 549 as well as H 292 cells had lost their typical epithelial shape and showed a fibroblast-like appearance, changes associated with an increase in N-cadherin mRNA expression by 875 ± 85% (n=5) and 1085 ± 220% (n=13) (means± s.e.mean), respectively. However, after exposure to TGF-β, expression of E-cadherin mRNA was reduced by 98 ± 1% in A 549 cells, but was increased by 150 ± 28% in H 292 cells. Although A 549 and H 292 cells showed similar basal prepro-endothelin-1 mRNA levels (ΔCT over GAPDH, 11.00 ± 0.21 and 11.03 ± 0.18, respectively), TGF-β caused an increase in prepro-endothelin-1 mRNA by 1258 ± 90% in H 292 cells, but a decrease by 76 ± 28% in A 549. Most interestingly, insulin (1 μM) present together with TGF-β markedly opposed the TGF-β-induced up-regulation of prepro-endothelin-1 and N-cadherin mRNA expression in H 292 cells, but augmented the TGF-β-induced down-regulation of prepro-endothelin-1 mRNA in A 549 cells.

In human (alveolar type) lung and bronchial epithelial cells TGF-β induces differential phenotype alterations. The marked up-regulation of endothelin-1 in bronchial epithelial cells by TGF-β suggests a role of endothelin in fibrotic airway remodelling processes. The mechanisms by which insulin opposes the TGF-β mediated induction of mesenchymal properties and the up-regulation of endothelin-1 in bronchial epithelial cells remain to be illuminated.