Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation

B Hu, Z Wu, T Liu, MR Ullenbruch, H Jin… - American journal of …, 2007 - atsjournals.org
B Hu, Z Wu, T Liu, MR Ullenbruch, H Jin, SH Phan
American journal of respiratory cell and molecular biology, 2007atsjournals.org
Gut-enriched Krüppel-like factor (GKLF) has been reported to partially inhibit α-smooth
muscle actin (α-SMA) gene transcription by competing for binding to the TGF-β 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 α-SMA promoter mutated in the TCE remained
susceptible to inhibition by GKLF in rat lung fibroblasts consistent with the existence of an …
Gut-enriched Krüppel-like factor (GKLF) has been reported to partially inhibit α-smooth muscle actin (α-SMA) gene transcription by competing for binding to the TGF-β 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 α-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-β– induced α-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 α-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 α-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-β signaling, which should lead to a clearer understanding of the mechanism of how GKLF regulates TGF-β–induced gene expression.
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