Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.