Rationale Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with unknown aetiology and no effective therapy. Myofibroblasts are the primary effector cells in the pathogenesis of IPF and differentiation from fibroblasts is a major source of myofibroblasts. Prostaglandin E2 (PGE2) inhibits fibroblast to myofibroblast differentiation via the E Prostanoid 2 (EP2) receptor and cAMP, suggesting cAMP is a key regulator of myofibroblast differentiation. The aim of the present study was to evaluate the effect of different cAMP elevating agents on myofibroblast differentiation.
Methods Fibroblasts from lungs of patients with IPF (F-IPF) and from non-fibrotic lungs (F-NL) were used. TGF-β1 (2ng/ml 3d) was used to induce myofibroblast differentiation. The effect of PGE2, β2-agonists Salmeterol and Formoterol, the direct adenylyl cyclase activator Forskolin and the phosphodiesterase 4 (PDE4) inhibitor Roflumilast (all 1µm, 3d) was evaluated. IL-1β (2ng/ml, 24hr) was used for cyclooxygenase 2 (COX-2) induction. α-smooth muscle actin (α-SMA, a myofibroblast marker), COX-2, EP2, EP4 and β2-receptor expression was analysed by Western blotting and immunocytochemistry, respectively. Adenylyl cyclase mRNA was measured by qPCR and cAMP was measured by radioimmunoassay.
Results F-IPF showed increased α-SMA and collagen expression and repressed COX-2 expression compared to F-NL. PGE2 treatment prevented TGF-β1-induced α-SMA expression and COX-2 repression in F-NL, which was mimicked by β2-agonists and Forskolin. PGE2 also reduced α-SMA expression and increased COX-2 expression in F-IPF despite that it induced significantly less cAMP than in F-NL But this effect on F-IPF was not mimicked by β2-agonists and Forskolin as they induced even less cAMP than PGE2 in these cells. TGF-β-treated F-NL also produced less cAMP than untreated cells in response to these cAMP. stimulants. However, the expression of EP2, EP4, β2-adrenoceptors and adenylyl cyclase isoforms was similar in F-NL and F-IPF. Furthermore, combination of PGE2 with Roflumilast showed greater effect than PGE2 alone on α-SMA reduction and COX-2 expression in F-IPF and F-NL, whereas Roflumilast alone had no effect.
Conclusions cAMP is a key anti-fibrotic regulator of myofibroblast differentiation. However, cAMP production in myofibroblasts is defective, probably due to increased degradation by PDE4.