Glycogen synthase kinase-3 (GSK-3) regulates TGF-ß1-induced differentiation of pulmonary fibroblasts.

ABSTRACT

BACKGROUND: Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-ß is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-ß1-induced myofibroblast differentiation is currently largely unknown. PURPOSE: To determine the contribution of GSK-3 signalling in TGF-ß1-induced myofibroblast differentiation. EXPERIMENTAL APPROACH: We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively. RESULTS: Stimulation of MRC5 and primary human lung fibroblasts with TGF-ß1 resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-ß1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-ß1-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-ß/smad signalling. CONCLUSION AND IMPLICATION We demonstrate that GSK-3 signalling regulates TGF-ß1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.