Differential regulation of TGF-β signaling through Smad2, Smad3 and Smad4

Abstract

Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-b (TGF-b) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-b-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-b-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiationcompetent cells by using improved antisense molecules. We found that TGF-b mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-b-induced cell cycle arrest and to induce or suppress endogenous cell cycle regulators. Inhibition of Smad4 expression exhibited a partial effect, whereas inhibition of Smad2 expression had no effect. By gene expression profiling, we identified TGF-b-dependent genes that are differentially regulated by Smad2 and Smad3 under regular growth conditions on a genome-wide scale. We show that Smad2, Smad3 and Smad4 contribute to the regulation of TGF-b responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.

Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

“…This antisense RNA:DNA hybrid oligonucleotides recruit RNaseH to the Smad2 or Smad3 target mRNA, causing its degradation. Several lines of evidence have indicated already that this RNaseH-mediated antisense strategy is a valid and alternative approach for siRNA-mediated depletion of gene products (15,25). Moreover, we have validated some of our results obtained with GBs also with the siRNA-based approach and the same results were obtained using either tool, except for the effect of Smad2 depletion on MMP-9 gene induction by TGFβ.…”

“…In accordance with our results obtained in the Nme cells used here, the data collected from these other cell types point to Smad3 as an essential mediator of responses to TGFβ. Three reports from studies in human epithelial cell lines, including keratinocytes or hepatocytes, showed that Smad3 also has a prominent role in TGFβ-mediated cell cycle arrest and apoptosis (25)(26)(27).…”

“…The anti-Smad GBs and their corresponding MM control oligonucleotides (Atugen) were used as previously described (25) and the GBs used for this study contained the following sequences: Smad2 GB (no. 73263), 5′-aacccuGGTTGACAGacugag-3′; Smad2&3 GB (no.…”

Id proteins: Novel targets of activin action, which regulate epidermal homeostasis

“…Thus, in addition to TGF-b (Ling et al, 2002;Kretschmer et al, 2003;Siegel et al, 2003;Kowanetz et al, 2004), activin was identified as a novel regulator of Id gene expression. Similar to TGF-b, activin has opposite effects on keratinocytes and fibroblasts.…”

Smad3 Knockout Mice Exhibit a Resistance to Skin Chemical Carcinogenesis

“…In vitro studies have shown that TGF-␤-induced growth inhibition in epithelial cells is preferentially mediated by Smad3 (10,11) and that cyclin-dependent kinase (cdk) 2 and cdk4 can phosphorylate Smad3, thus inactivating its ability to instigate cell cycle arrest (12). Therefore, Smad3 may mediate TGF-␤1-induced growth inhibition during cancer development.…”