Smad pathway-specific transcriptional regulation of the cell cycle inhibitor p21WAF1/Cip1

Transforming growth factor-beta (TGF-beta) inhibits epithelial cell growth, in part via transcriptional induction of the cell cycle inhibitor p21(WAF1/Cip1) (p21). We show that bone morphogenetic protein (BMP)-7 induces higher p21 expression than TGF-beta 1 in various epithelial cells. Despite this, BMP-7 only weakly suppresses epithelial cell proliferation, as ld2, a cell cycle-promoting factor, becomes concomitantly induced by BMP-7. Signaling studies with all type I receptors of the TGF-beta superfamily show that BMP receptors induce higher p21 expression than TGF-beta/activin receptors. Smad4 is essential for p21 regulation by all receptor pathways. Based on the previously known ability of c-Myc to block p21 expression and epithelial growth arrest in response to TGF-beta 1, we demonstrate that ectopic c-Myc expression can abrogate Smad-mediated p21 induction by all TGF-beta and BMP receptors. Furthermore, p21 induction by all receptor pathways can be blocked by the natural inhibitors of the TGF-beta superfamily. Smad7 inhibits all pathways whereas Smad6 selectively inhibits the BMP pathways. The observed pathway specificity reflects the efficiency by which BMP Smads, compared to TGF-beta Smads, transactivate the p21 promoter. In addition, BMP-specific Smads, Smad1, Smad5, and especially Smad8, induce enclogenous p21 rnRNA and protein levels, while they fail to induce epithelial growth inhibition when compared to TGF-beta receptor-phosphorylated Smads (R-Smads), Smad2 and Smad3. Thus, p21 is a common target of all TGF-beta superfamily pathways. However, the ability of TGF-beta superfamily members to induce cell growth arrest depends on the regulation of additional gene targets.