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Efficient TGF-β Induction of the Smad7 Gene Requires Cooperation between AP-1, Sp1, and Smad Proteins on the Mouse Smad7 Promoter
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
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2000 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 37, 29023-29030 p.Article in journal (Refereed) Published
Abstract [en]

Sma- and Mad-related protein 7 (Smad7) is an antagonist of transforming growth factor-beta (TGF-beta) signaling, which has been shown to be induced by TGF-beta itself and also by other stimuli. In an effort to understand the molecular mechanisms underlying the transcriptional regulation of the Smad7 gene by TGF-beta, we cloned and functionally characterized a mouse genomic DNA fragment encompassing the mouse Smad7 proximal promoter. This region was found to contain a CpG island and to be devoid of a classical TATA box. Cloned upstream of a promoter-lacking luciferase reporter gene, this region conferred robust TGF-beta-induced transcription. Point mutations in a palindromic Smad binding element, abolished TGF-beta inducibility completely. Through the use of electrophoretic mobility shift assays, we showed the presence of Smad2, Smad3, and Smad4 in complexes binding to the Smad binding element. Interestingly, we also found that point mutation and/or deletion of binding sites for the transcription factors activator protein-1 and Sp1 led to an attenuation of the basal promoter activity, as well as of the TGF-beta-mediated induction of Smad7. Taken together, our data imply that Smads, together with activator protein-1 and Sp1 transcription factors, are essential for efficient Smad7 promoter activity.

Place, publisher, year, edition, pages
2000. Vol. 275, no 37, 29023-29030 p.
Identifiers
URN: urn:nbn:se:uu:diva-89786PubMedID: 10843994OAI: oai:DiVA.org:uu-89786DiVA: diva2:161532
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Smad7 in TGF-β Signalling
Open this publication in new window or tab >>Smad7 in TGF-β Signalling
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors regulate a vast array of biological functions in the adult, and are of great importance in governing cell fate determination and patterning in the developing embryo. The TGF-β signal is propagated intracellularly by Smad proteins resulting in transcriptional responses. Smad6 and Smad7 are inhibitory Smads known to downregulate the TGF-β signal and thereby possibly modulating the biological response. This thesis describes a functional analysis of the inhibitory Smad7 from an in vitro and in vivo perspective.

The prostate gland is dependent on androgens for its growth and differentiation. Androgen withdrawal can cause regression and apoptosis in normal and malignant prostate. Previous studies suggest a role for TGF-β in the apoptotic mechanism. We investigated the expression levels of Smad proteins in the rat ventral prostate as well as in an androgen sensitive prostate tumor model (Dunning R3327 PAP) by immunohistochemistry. We observed an increased immunoreactivity for Smad3, Smad4 and phosphorylated Smad2 in the rat ventral prostate epithelial cells after castration, as well as in the prostate tumor cells. Expression of inhibitory Smad6 and Smad7 were also increased in both normal and malignant prostate in response to castration.

Several studies have shown that Smad7 is upregulated in response to TGF-β stimuli, suggesting a role in a negative feedback loop attenuating the TGF-β response. We investigated the molecular mechanism behind that response by studying the transcriptional regulation of the Smad7 gene. We identified a palindromic Smad binding element (SBE) in the promoter. Point mutations introduced into the SBE abolished transcriptional activation via TGF-β. We also observed that mutating or deleting binding motifs for Sp1 and AP-1, led to an attenuation of the TGF-β mediated transcriptional induction as well as the basal promoter activity.

Gene ablation of Smad proteins has revealed specific physiological and developmental roles. We analysed mice targeted on the Smad7 locus. The mice appeared viable and fertile with a slight reduction in litter size, suggesting a perinatal loss. Biochemical analysis of mouse embryonic fibroblasts (MEFs) showed no major difference between wild type and mutant MEFs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 55 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1135
Keyword
Oncology, TGF-β, Smad7, Prostate, Transcription, Gene targeting, Onkologi
National Category
Cancer and Oncology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-1949 (URN)91-554-5271-X (ISBN)
Public defence
2002-04-26, Room B41, Biomedical Centre, Uppsala, 09:00
Opponent
Available from: 2002-04-05 Created: 2002-04-05Bibliographically approved

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