TGF‐β1‐induced Smad 3 binding to the Smad 7 gene: Knockout of Smad 7 gene transcription by sense phosphorothioate oligos, autoregulation, and effect on TGF‐β1 secretion: Bleomycin acts through TGF‐β1

Bleomycin produces its fibrogenic effect, at least in part, by TGF‐β1 secretion. Treatment of IMR‐90 human embryonic lung fibroblasts with bleomycin at 0.5 μg/ml results in a 1.6‐fold increase of TGF‐β1 as determined by a specific ELISA assay for TGF‐β1 after acidification of the conditioned media. This elevation of TGF‐β1 secretion is furthermore enhanced in vivo by TGF‐β1 autoinduction of the TGF‐β1 gene. To demonstrate TGF‐β1 autoinduction, the fibroblasts were pretreated with 12.5 ng/ml TGF‐β1, washed extensively to remove any residual TGF‐β1, and then allowed to incubate for 24 h in AIM V synthetic serum‐free media. The media when assayed using the ELISA assay contained a 1.6‐fold increase of TGF‐β1. The distal promoter of the human TGF‐β1 gene contains a Smad 3 element (CAGGACA), which is homologous to the Smad 3 binding element motif (CAGA). The nuclear extracts of human embryonic lung fibroblasts treated for either 15 min or 24 h with TGF‐β1 did not demonstrate specificity of binding of a protein(s) to the homologous Smad 3 element as determined by cold wild‐type oligodeoxynucleotide competition experiments. However, specific Smad 3 binding to the Smad 3 element (GTCTAGAC) found in proximal promoter of the Smad 7 gene was observed by cold oligo competition and supershift assays using a goat polyclonal Smad 3 antibody in the presence and absence of an N‐terminal Smad 3 peptide. To determine the functionality of this Smad 3 binding to the Smad 3 element in the proximal promoter of the Smad 7 inhibitory gene to TGF‐β1 secretion, fibroblasts were transiently pretransfected with double‐stranded phosphorothioate oligo “decoys” containing the Smad 7/Smad 3 element in the presence of plasmin to convert latent TGF‐β1 to active TGF‐β1. Under these conditions, which simulate the in vivo situation of 2.2‐fold increase of total active TGF‐β1 was observed. Fibroblasts were also pretransfected with these double‐stranded oligo “decoys,” washed, then treated with TGF‐β1, washed and incubated in AIM V for an additional 24 h. In this latter experiment, a superinduction of TGF‐β1 secretion was observed. We propose that these oligo “decoys” bind Smad 3 preventing this initiation factor from binding to the Smad 7/Smad 3 element thereby decreasing the transcription of the Smad 7 gene. The decrease of the inhibitory Smad 7 would result in less binding of this Smad inhibitor to the Type I TGF‐β receptor and less antagonism of active TGF‐β1, more autoinduction of the TGF‐β1 gene, and more of the fibrogenic effects of TGF‐β1. J. Cell. Biochem. 89: 474–483, 2003. © 2003 Wiley‐Liss, Inc.