Identification of Novel TGF-β/Smad Gene Targets in Dermal Fibroblasts using a Combined cDNA Microarray/Promoter Transactivation Approach*

By F. Verrecchia, M. Chu, A. Mauviel. Journal of Biological Chemistry, 2001

696 citations recorded by the source corpus.

Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-β (TGF-β) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-β in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-β is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-β stimulation, (2) activation of the promoter by both exogenous TGF-β and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-β blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-β not possible inSmad3 −/− mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-β/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-β target genes in fibroblasts.

Subfields: Immunology and Allergy, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/d9cb58b5f75de8230584b11e1bf0d98392984852

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Citations in the corpus

TGF-β signaling in fibrosis (2011, background): “Activation of the Smad3 signaling pathway appears to be important in mediating TGF-b-induced ECM protein synthesis and TIMP upregulation (Verrecchia et al. 2001).”
TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. (2005, background): “transcriptomic screens of the TGF-β pathway in various cell types (Perou et al., 1999; Akiyoshi et al., 2001; Verrecchia et al., 2001; Zavadil et al., 2001; Ota et al., 2002; Chambers et al., 2003; Coyle et al., 2003; Jechlinger et al., 2003; Kang et al., 2003a; Xie et al., 2003; Kowanetz et al., 20”
Making sense of latent TGFβ activation (2003, background): “The ability of these enzymes to activate the latent TGF β complex couples matrix turnover with the production of a molecule, TGFβ, that has a primary role in maintaining matrix integrity and stability (Ignotz and Massague, 1986; Verrecchia et al., 2001).”
Smad3 as a mediator of the fibrotic response (2004, background): “The induction of the tissue inhibitor of metalloprotease-1 (TIMP-1) by TGF-b also appears to be Smad3 dependent (Verrecchia et al. 2001).”
Roles of TGF-beta in hepatic fibrosis. (2002, methodology): “A direct link between TGF-beta signal transduction and TIMP-1 was found in dermal fibroblasts, where a combined cDNA microarray/promoter transactivation approach for the identification of direct TGF-beta target genes was used (85).”
TGF‐β signaling and the fibrotic response (2004, background): “Indeed, it has been shown that Smad 3 is essential for the induction of matrix, as induction of profibrotic genes such as type I collagen and connective tissue growth factor (CTGF) is prevented in fibroblasts lacking the Smad3 gene (64, 65).”
Diverse Roles of TGF-β/Smads in Renal Fibrosis and Inflammation (2011, background): “Many fibrogenic genes, such as (ColIa1, ColIa2, ColIIIa1, ColVa2, ColVIa1, and ColVIa3) and tissue inhibitor of MMP-1 (TIMP-1), are the downstream targets of TGF-β/Smad3 signaling [41], suggesting that Smad3 may be a critical mediator of TGF-β/Smad signaling in fibrosis.”
The role of TGF-β Signaling in Myocardial Infarction and Cardiac Remodeling (2007, background): “Activation of the Smad3 signaling pathway appears to be important in mediating TGF-βinduced extracellular matrix protein synthesis and TIMP upregulation [ 31 ].”
Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis (2011, background): “, we investigated the expression of collagen III, a known TGF-β and SMAD3 targe”
A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2 (2005, result): “As multiple collagens normally expressed in the aorta are derived from early-induced TGFβ target genes (including COL1A1 and COL3A1 ), these data are consistent with increased (rather than decreased) TGFβ signalin”