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

Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-beta (TGF-beta) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-beta-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-beta-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiation-competent cells by using improved antisense molecules. We found that TGF-beta mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-beta-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 pro. ling, we identified TGF-beta-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-beta responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.