Inhibition of Transforming Growth Factor β-enhanced Serum Response Factor-dependent Transcription by SMAD7

Abstract

Transforming growth factor (TGF)-beta is present in large amounts in the airways of patients with asthma and with other diseases of the lung. We show here that TGFbeta treatment increased transcriptional activation of SM22alpha, a smooth muscle-specific promoter, in airway smooth muscle cells, and we demonstrate that this effect stems in part from TGFbeta-induced enhancement of serum response factor (SRF) DNA binding and transcription promoting activity. Overexpression of Smad7 inhibited TGFbeta-induced stimulation of SRF-dependent promoter function, and chromatin immunoprecipitation as well as co-immunoprecipitation assays established that endogenous or recombinant SRF interacts with Smad7 within the nucleus. The SRF binding domain of Smad7 mapped to the C-terminal half of the Smad7 molecule. TGFbeta treatment weakened Smad7 association with SRF, and conversely the Smad7-SRF interaction was increased by inhibition of the TGFbeta pathway through overexpression of a dominant negative mutant of TGFbeta receptor I or of Smad3 phosphorylation-deficient mutant. Our findings thus reveal that SRF-Smad7 interactions in part mediate TGFbeta regulation of gene transcription in airway smooth muscle. This offers potential targets for interventions in treating lung inflammation and asthma.

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

“…1A, right). Baseline effects have been seen following other manipulations of TGF-␤ signaling components (7,12,20,47,55,57,59) and typically follow the same trend as responses to added TGF-␤. This likely reflects normal receptor signaling caused by low levels of TGF-␤ in the serum-supplemented medium (reference 22 and references within) and/or a TGF-␤ autocrine response (56; reviewed in reference 15).…”

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

“…10 The EGFP-SRF fusion protein construct (pCMV-EGFP-SRF) and the dominant-negative fusion construct (pCMV-EGFP-mNLS-SRF), where amino acids 95R and 96R within the nuclear localization signal (95-RRGLKR-100) were mutated to 95E and 96E, were the kind gift of Julian Solway (University of Chicago, Chicago, IL, USA). 40 The bacterial expression construct for His-SRF (pET-SRF) and GST-SRF (pGEX-SRF) were a gift from Warren Zimmer (Texas A&M University) and the GST-Nkx3.2-HA bacterial expression construct (pGEX-Nkx3.2-HA) was a gift from Andrew Lassar (Harvard University, Boston, MA, USA). 22,23 The bacterial expression construct for GST-mNLS-SRF was created by excising the mutant NLS sequence from pCMV-EGFPmNLS-SRF using flanking XmaI sites and ligating the fragment into the corresponding region of pGEX-SRF where the wild-type NLS had been excised.…”

Regulating the stability of TGFβ receptors and Smads

“…It is important to clarify the actual location in the cell where these protein complexes form, and exactly where Smad7 is acetylated, de-acetylated and finally degraded, that is, during receptor trafficking in the cytoplasm, in the nucleus or in both places. The emerging evidence that nuclear Smad7 directly antagonizes R-Smad/Co-Smad binding to DNA and inhibits transcriptional signaling by the latter proteins [77,78] suggests it is highly possible that nuclear Smurfs or Arkadia may target chromatinbound Smad7, thus releasing negative regulation from the Smad complex and permitting productive transcription, as we discuss below for the Ski/SnoN corepressors (Figure 3).…”

“…Possibly by studying the specific timing of pathway regulation by each one of these enzymes and their adaptor proteins like Smad7. Smad7, which regulates both nuclear Smad signaling [77,78] and TGFb receptor signaling in the cytoplasm [23,24,74,75], is a key factor. Whether or not Smad7 exists in nuclear complexes together with Ski and SnoN remains currently unknown.…”