Transforming growth factor-β1 induces interleukin-6 expression via activating protein-1 consisting of JunD homodimers in primary human lung fibroblasts

Transforming growth factor (TGF)-beta 1 induces extracellular matrix deposition and proliferation of mesenchymal cells. We recently reported that interleukin (IL)-6 is an essential mediator of growth factor-induced proliferation of lung fibroblasts. Here, we demonstrate by reverse transcriptase polymerase chain reaction and enzyme-linked immunoassay that TGF-beta 1 is a potent inducer of IL-6 mRNA and protein in primary human lung fibroblasts, Transient transfections of fibroblasts with a luciferase reporter gene construct containing nucleotides -651 to +1 of the human IL-6 promoter revealed that TGF-beta 1 also potently activated IL-6 promoter activity. Progressive 5'-deletions and site-directed mutagenesis of the parental construct located the TGF-pl-responsive cis-regulatory element to a known activating protein-1 (AP-1) sequence (nucleotides -284 to -276). Gel shift analyses revealed that AP-1 DNA binding activity in nuclear extracts was increased 30 min after stimulation with TGF-beta 1. In contrast, neither CCAAT enhancer-binding protein-beta, NF-kappa B, nor Spl were activated by TGF-beta 1, Supershift analyses demonstrated that the AP-1 complex induced by TGF-beta 1 was composed of Jun isoforms and absent of Fos isoforms, Moreover, this complex was found to be a JunD homodimer, Our data thus demonstrate that TGF-beta 1 is a potent inducer of IL-6 in primary human lung fibroblasts, The TGF-beta 1-activated JunD homodimer may be essential for a majority of the biological effects induced by TGF-beta 1 in this cell type, such as proliferation and extracellular matrix synthesis.