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

Abstract

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.

Figure 1.

Inhibition property of α-SMA promoter activity by GKLF. Wild-type and TCE-mutated α-SMA promoters mutant constructs were co-transfected with GKLF expression plasmid or empty expression vectors into rat lung fibroblasts as indicated. Cells were then treated with TGF-β and analyzed for firefly and Renilla luciferase activities, respectively. After normalization to the Renilla luciferase activity, the data were expressed as fold over promoterless control and shown as means ± SE. Experiments with each combination were repeated two to four times and yielded similar results.

Figure 2.

Co-immunoprecipitition analysis of GKLF–Smad3 interaction. Fibroblasts were treated with either TGF-β or buffer only (None) for 48 h. The total proteins were then extracted and, after preclearing with protein A beads, were incubated with anti-Smad3 (upper panel) or anti-GKLF antibodies (middle and lower panels) (IP ANTIBODY) or their corresponding IgG control. After stringent washing, the proteins bound on the respective beads were eluted and equal volume of the elution was subjected to Western blot analysis using the indicated IB ANTIBODY.

Figure 3.

Effect of GKLF on Smad3 binding to SBE. ³²P-labeled double-stranded oligonuleotide probe corresponding to the sequence of the SBE in the rat α-SMA promoter was incubated with either recombinant Smad3 protein or GST protein only, and then analyzed by gel electrophoresis. Selected samples were pretreated with the indicated substances before addition of radiolabeled probe. A shifted band indicative of DNA–protein complex formation was indicated with an arrow. Addition of a 100-fold excess of unlabeled probe, as indicated, was used to document the specificity of the binding. A film was shown from a representative experiment, which was repeated twice, yielding similar results.

Figure 4.

ChIP analysis of Smad3 binding to the α-SMA promoter. Rat lung fibroblasts were transfected with GKLF expression plasmid or the empty vector only and then treated with TGF-β. After fixation with formaldehyde and sonication, the lysates were incubated with anti-Smad3 antibody (Anti-Smad3), nonimmune rabbit IgG (Rabbit IgG), or PBS as indicated. These precipitated DNA samples, along with untreated whole samples (Input DNA) were analyzed by PCR using primers spanning the SBE region of the α-SMA promoter. The PCR products and a 100-bp DNA ladder were analyzed by agarose gel electrophoresis. A representative gel is shown.

Figure 5.

TCE-independent inhibition by GKLF depended on Smad3. TCE-mutated α-SMA promoters were co-transfected with GKLF expression plasmid or empty expression vectors into either smad3^(+/+) or smad3^(−/−) fibroblasts as indicated. Cells were then treated with TGF-β, and then analyzed for firefly and Renilla luciferase activities. After normalization to the Renilla luciferase activity, the data were expressed as fold over promoterless control and shown as means ± SE. Experiments with each combination were repeated two to four times and yielded similar results.

Figure 6.

GKLF inhibited Smad3-regulated artificial promoter. Artificial pBV-luc luciferase fusion construct (pBV) and the same vector with four tandem SBE repeats (pSBE4) were transfected into NIH3T3 cells, and co-transfected with the indicated expression plasmid(s) or empty vector control (C2). After treatment with TGF-β or buffer only (None), the cells were analyzed for firefly and Renilla luciferase activities. After normalization to the Renilla luciferase activity, the results were expressed as fold increase over the pBV control promoter activity and shown as means ± SE. Experiments with each combination were repeated two to four times, with similar results.