Gut-enriched Krüppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.
Gut-enriched Krüppel-like factor (GKLF) has been reported to partially inhibit alpha-smooth muscle actin (alpha-SMA) gene transcription by competing for binding to the TGF-beta control element (TCE) with known activators such as Sp1 and other Krüppel-like factors. This incomplete inhibition via the TCE suggests an additional mechanism, which was evaluated in this study. The results showed that an alpha-SMA promoter mutated in the TCE remained susceptible to inhibition by GKLF in rat lung fibroblasts consistent with the existence of an additional TCE-independent mechanism. Since TGF-beta- induced alpha-SMA expression is Smad3-dependent, potential interaction between GKLF and Smad3 was examined as a basis for this additional inhibitory mechanism. Co-immunoprecipitation and yeast two-hybrid assays revealed that GKLF could bind Smad3 through the Smad3 MH2 domain. Electrophoretic mobility shift assays and ChIP assay indicated that this GKLF-Smad3 interaction inhibited Smad3 binding to the Smad3-binding element (SBE) in the alpha-SMA promoter, and the activity of an SBE containing artificial promoter. Further analysis using smad3(-/-) fibroblasts confirmed that the TCE-independent inhibition by GKLF was dependent on Smad3. These data taken together suggest that in addition to inhibition via the TCE, GKLF represses alpha-SMA gene expression by interacting with Smad3 to prevent Smad3 binding to the SBE. It represents the first evidence to directly link GKLF with Smad3, a key intracellular mediator of TGF-beta signaling, which should lead to a clearer understanding of the mechanism of how GKLF regulates TGF-beta-induced gene expression.
Hu
Biao
B
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.
Wu
Zhe
Z
Liu
Tianju
T
Ullenbruch
Matthew R
MR
Jin
Hong
H
Phan
Sem H
SH
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D051379:Mice; D018345:Mice, Knockout; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011916:Rats, Inbred F344; D020218:Response Elements; D051900:Smad3 Protein / Q000378:metabolism*; D014158:Transcription, Genetic; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D000199:Actins; C000715468:Klf4 protein, mouse; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1