Vol. 301, Issue 3, 830-837, June 2002

Hypoxia Differentially Enhances the Effects of Transforming Growth Factor- Isoforms on the Synthesis and Secretion of Glycosaminoglycans by Human Lung Fibroblasts

Eleni Papakonstantinou, Michael Roth, Michael Tamm, Oliver Eickelberg, Andre P. Perruchoud and George Karakiulakis

Department of Pharmacology, School of Medicine, Aristotle University, Thessaloniki, Greece (E.P., G.K.); and Departments of Research and Internal Medicine, University Hospital Basel, Basel, Switzerland (M.R., M.T., O.E., A.P.P.)

Interstitial lung diseases associated with hypoxia, such as lung fibrosis, are characterized by enhanced production of transforming growth factor- (TGF-) and increased deposition of extracellular matrix (ECM) molecules, including glycosaminoglycans (GAGs). In this study, we investigated the effect of hypoxia (3% O₂) on TGF--induced GAG synthesis by primary human pulmonary fibroblasts, established from lung biopsies. Total GAG synthesis was assessed by the incorporation of [³H]glucosamine into GAGs associated with the cell layer (cells and ECM) or secreted in the medium. GAGs were isolated and purified by gel filtration, fractionated by electrophoresis on cellulose acetate membranes, and characterized using GAG-degrading enzymes. GAG molecules identified in the cell layer and the medium were: hyaluronic acid, and chondroitin, dermatan, and heparan sulfates. All TGF- isoforms time dependently induced [³H]glucosamine incorporation into GAGs of the cell layer or the medium. Characterization of individual GAG molecules indicated that this was attributed to dermatan and heparan sulfates in the cell layer and to hyaluronic acid and chondroitin and dermatan sulfates in the medium. Hypoxia enhanced the effect of all TGF- isoforms, particularly that of TGF-3, on the secretion of hyaluronic acid and chondroitin and dermatan sulfates. In the cell layer, hypoxia stimulated only the effect of TGF-2-induced [³H]glucosamine incorporation into GAGs. Our data indicate that hypoxia differentially enhances the effect of TGF- isoforms on the secretion and deposition of GAGs and may hasten ECM remodeling associated with the pathogenesis of lung fibrosis.