Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Dec 8;95(25):14769-74.
doi: 10.1073/pnas.95.25.14769.

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta

L Vindevoghel  1 , R J Lechleider, A Kon, M P de Caestecker, J Uitto, A B Roberts, A Mauviel
Affiliations

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta

L Vindevoghel et al. Proc Natl Acad Sci U S A. .

Abstract

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

PubMed Disclaimer

Figures

Figure 1
Figure 1
SMAD4 is required for efficient transcription of the COL7A1 promoter and its responsiveness to TGF-β. COS-1 cells were transfected with either the −524COL7A1/CAT or the −456COL7A1/CAT construct of the human COL7A1 promoter, in the absence (−) or in the presence (+) of TGF-β, without (−) or with (+) a Smad4 expression vector. TGF-β (10 ng/ml) was added to the medium 4 h later, and the incubation continued for 40 h. In this and the subsequent figures, cell extracts were assayed for CAT activity with [14C]chloramphenicol as a substrate by using identical amounts of β-galactosidase activity. The relative CAT activity (mean ± SD) of at least two experiments performed in duplicate is shown in the form of a bar graph.
Figure 2
Figure 2
A COL7A1 SBS tandem repeat confers TGF-β responsiveness to a heterologous promoter. Confluent fibroblast cultures were transfected with several constructs derived from pBLCAT5 by the calcium phosphate/DNA coprecipitation procedure. These constructs contained one copy of the COL7A1 SBS (SBS-TK/CAT), two copies of the SBS [(SBS)2-TK/CAT], or two copies of a mutated SBS [(SBSmut)2-TK/CAT]. After glycerol shock, the cultures were incubated in fresh medium containing 1% fetal calf serum, without (−) or with (+) TGF-β (10 ng/ml) added to the medium 4 h later. After 40 h of incubation, cell extracts were assayed for CAT activity. Results are the mean ± SD of three independent experiments.
Figure 3
Figure 3
Flag-SMAD3 binds the COL7A1 SBS. COS-1 cells were transfected with expression vectors encoding various SMADs tagged with the Flag epitope as indicated, and nuclear extracts were prepared 40 h later. (A) Western blot of each of the nuclear extract preparations, using a Flag antibody. Note that each Flag-SMAD protein is present at high levels in their respective extracts. (B) EMSA using the COL7A1 SBS as a probe. Note the strong binding present in nuclear extracts containing Flag-SMAD3 (arrow). (C) Supershift assay using 3 μg/lane of anti-Flag antibody. Note that the faint band (∗) is likely to represent endogenous Smad3-containing binding activity.
Figure 4
Figure 4
(A) Recombinant GST-SMAD3ΔC and GST-SMAD4 bind the COL7A1 SBS. EMSAs were performed by using 100 ng of E. coli-derived GST-fusion proteins of SMAD2, SMAD2ΔC, SMAD3, SMAD3ΔC, and SMAD4, using the COL7A1 SBS as a probe and 10 μg/lane of BSA. Note that only SMAD3ΔC and SMAD4 could bind the probe. (B) Binding of TGF-β-induced SMAD-containing complex to the SBS requires phosphorylation. EMSAs were performed by using either the COL7A1 SBS promoter fragment (left) or a consensus Sp1 oligonucleotide (right) as probes. Nuclear extracts from either control (−) or TGF-β-treated (+) fibroblast cultures were digested (+) or not (−) with calf intestine phosphatase (CIP, 0.1 unit/ml, 10 min), and their ability to bind either probe was tested.
Figure 5
Figure 5
SMAD3 and SMAD4 synergize to activate −524COL7A1/CAT and (SBS)2-TK/CAT. (A) Human dermal fibroblasts were cotransfected with (SBS)2-TK/CAT and either SMAD2, SMAD3, and/or SMAD4 expression vectors. Cell extracts were prepared and assayed for CAT activity 40 h later. (B) SMAD4−/− MDA-MB-468 cells were transfected with −524COL7A1/CAT (open bars) or (SBS)2-TK/CAT (closed bars) constructs, together with SMAD3 and/or SMAD4 expression vectors. After 40 h, cell extracts were prepared and assayed for CAT activity. Results are the mean ± SD of three independent experiments.
Figure 6
Figure 6
SMAD3 but not SMAD4 may participate in SBS/protein complex formation. (A) Nuclear extracts were prepared from dermal fibroblasts (DF) treated (+) or not (−) with TGF-β for 30 min or from Flag-SMAD3 transfected cells. Their ability to bind the COL7A1 SBS was compared by EMSA. Note the identical migratory pattern of the TGF-β-specific band with the Flag-SMAD3/DNA complex. (B) Nuclear extracts from Flag-SMAD3 and SMAD4-Myc-transfected COS-1 cells were used in EMSA and were supershifted with 3 μg of either anti-Flag or anti-Myc antibodies. Note that the anti-Myc antibody does not supershift complex 1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Massagué J, Hata A, Liu F. Trends Cell Biol. 1997;7:187–192. - PubMed
    1. Heldin C H, Miyazono K, ten Dijke P. Nature (London) 1997;390:465–471. - PubMed
    1. Macias-Silva M, Abdollah S, Hoodless P A, Pirone R, Attisano L, Wrana J L. Cell. 1996;87:1215–1224. - PubMed
    1. Zhang Y, Feng X-H, Wu R-Y, Derynck R. Nature (London) 1996;383:168–172. - PubMed
    1. Liu F, Pouponnot C, Massagué J. Genes Dev. 1997;11:3157–3167. - PMC - PubMed

Publication types

LinkOut - more resources

  NODES
Note 6
twitter 2