Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members

Abstract

The cell cycle inhibitor p21͞WAF1͞Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions ؊2,300͞؊210) and a proximal (positions ؊124 to ؊61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the Smad family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor ␤, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of Smad4 that was shown previously to block signaling through Smads. Smad3͞4 and to a much lesser extent Smad2͞4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N-or C-terminally truncated forms of Smad3. By using Gal4-Sp1 fusion proteins, we show that Smad proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that Smad proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.

Schnurri interacts with Mad in a Dpp‐dependent manner

“…TGF-b induces the expression of the plasminogen inhibitor-1 (PAI-1) gene, and AP-1 ) and TFE3 (Hua et al 1998) have been implicated in the induction. Functional interaction of Smads with Sp1 has also been reported (Moustakas & Kardassis 1998). An important indication deduced from these observations is that the DNA binding af®nity of Smads alone may be relatively low.…”

SRF is a nuclear repressor of Smad3-mediated TGF-β signaling

“…The proximal regions of the p21 Cip1 or p15 INKb promoters are known to mediate the transcriptional activation of the p21 Cip1 or p15 INKb promoters by members of the Smad family of proteins, which play an important role in the transduction of extracellular signals such as TGF-b, activin, etc. (Moustakas and Kardassis, 1998;Feng et al, 2002). As shown in Figure 9, Smad complex binding induced by TGF-b1 treatment was markedly decreased in SRF expressing SNU620 cells.…”

ELAC2, a putative prostate cancer susceptibility gene product, potentiates TGF-β/Smad-induced growth arrest of prostate cells

“…It is well known that TGF-b activates the transcription of p21 gene, followed by growth inhibition of most cell types (Massague´et al, 2000;Siegel and Massague´, 2003). As Sp1 which binds to proximal region of p21 promoter (Datto et al, 1995) is a key regulator to activate p21 gene with Smads (Moustakas and Kardassis, 1998;Pardali et al, 2000), we examined whether ELAC2 can potentiate the interaction of Smad2 with Sp1 upon ALK5 activation. Thus, COS7 cells were transfected with Gal4-Sp1, 6xMyc-Smad2, ALK5ca/V5 in the absence or the presence of ELAC2.…”