Mechanisms of Regulation of the Cell Cycle Inhibitor p21Waf1/Cip1 in TGF-β-Mediated Cell Growth Inhibition

Abstract: TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes.The cell cycle inhibitor p21Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect.The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal p21 promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as Id2 that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene Meox2, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur.This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.