Involvement of the Sp3 Transcription Factor in Induction of p21 in Keratinocyte Differentiation

Abstract

The cyclin-dependent kinase inhibitor p21 is induced in several in vitro terminal differentiation systems as well as in differentiating tissues in vivo. To determine the mechanism responsible for p21 induction during differentiation of mouse primary keratinocytes, we performed a deletion analysis of the p21 promoter. The minimal region of the p21 promoter required for its induction in keratinocyte differentiation consists of a contiguous stretch of 78 base pairs, which contains a GC-rich region as well as the TATA box. We determined that transcription factors Sp1 and Sp3, present in primary keratinocyte nuclear extracts, bind the GC region concomitantly. Expression studies established that both Sp1 and Sp3 activate the p21 promoter, but showed that only Sp3 overexpression enhances promoter inducibility during differentiation. Furthermore, disruption of the GC-rich region dramatically decreases transcription factor binding as well as promoter activity and inducibility upon differentiation. The overexpression of either Sp1 or Sp3 restores the basal activity of the disrupted promoter, but only Sp3 can restore its inducibility. These findings show that both Sp1 and Sp3 can contribute to the basal activity of the p21 promoter, and establish Sp3 as a specific transcription factor involved in the induction of p21 promoter during keratinocyte differentiation.Induction of terminal differentiation is a complex process involving the regulation of many genes, including those directly responsible for cell cycle withdrawal and growth arrest. The activity of the cyclin-dependent kinases (CDKs) 1 is tightly regulated during passage through the cell cycle by cyclin association, subunit phosphorylation, and interaction with CDK inhibitors (1, 2). Induction of the CDK inhibitor p21 occurs in several in vitro terminal differentiation systems (3), as well as in differentiating tissues in vivo (4, 5), suggesting that this molecule plays an important role in regulating terminal differentiation-associated growth arrest.Control of p21 induction upon differentiation is of great interest, as it may involve mechanisms common to most cell types and others that are cell type-specific. In all the differentiation systems analyzed so far the induction of p21 occurs by p53-independent mechanisms (5-9). During differentiation of myoblasts to myotubes, p21 up-regulation has been reported to be under the control of MyoD (6), although other myogenic regulatory factors may also be involved, at least in MyoD knockout mice (5). In differentiating primary keratinocytes, the induction of p21 depends on the function of the nuclear phosphoprotein p300 (9), a novel transcriptional coactivator, which is thought to act as a bridge between specific transcription factors and the basal transcription complex. Little else is known about the regulation of p21 expression during normal differentiation.Recent data indicate that the GC-rich region of the p21 promoter, located next to the TATA box, binds the Sp1 transcription factor and can exert important regulatory ...

Unique induction of p21WAF1/CIP1expression by vinorelbine in androgen-independent prostate cancer cells

“…Several important biological modifiers have been shown to activate p21 transcription through different Spl binding sites (Cartel and Tyner, 1999). For example, phorbol ester and okadaic acid induce p21 expression through Spl-1 and Spl-2 sites (Biggs et al, 1996), whereas the Spl-3 site in the promoter of p21 has been shown to be required for p21 induction by transforming growth factor-b, histone deacetylase inhibitors such as TSA and butyrate, lovastatin, nerve growth factor (NGF) as well as calcium (Datto et al, 1995;Nakano et al, 1997;Prowse et al, 1997;Sowa et al, 1997;Lee et al, 1998;Billon et al, 1999). In this report, we demonstrated that Spl-3 and Spl-4 in the promoter of human p21 gene are required for vinorelbinemediated transcriptional restoration of p21 in the p21-deficient Al cells, which may provide a new mechanism in drug-mediated p21 regulation.…”

Ras induces p21Cip1/Waf1 cyclin kinase inhibitor transcriptionally through Sp1-binding sites

“…Besides p53, transcription factor Ap-2 (Zeng et al, 1997), E2F-1/DP-1 complex (Hiyama et al, 1997), E2A (Prabhu et al, 1997), an ets-oncogene related E1AF (Funaoka et al, 1997), C/EBP , RAR (Liu et al, 1996a), VDR (Liu et al, 1996b), STATs (Chin et al, 1996), and Sp1 family (Biggs et al, 1996;Li et al, 1998;Owen et al, 1998;Prowse et al, 1997;Yan and Zi , 1997) are known to stimulate p21 promoter activity. Our results show that Ras induces p21 transcriptionally and that the Ras-responsive region in p21 promoter spans a short region downstream of the nucleotide 7110 relative to the transcription start site.…”

“…With the use of more detailed mutant constructs, we mapped the Ras-responsive region to the Sp1-binding sites 2 and 4 in the promoter area 783 to 754. This GC-rich region has been ascribed to contain the TGF-b regulatory region (TGF-b responsive element, TbRE) (Datto et al, 1995b), and it mediates also the regulation of p21 by NGF (Yan and Zi , 1997) and progesterone (Owen et al, 1998), and overlaps the region implicated in induction of p21 in keratinocytes by calcium (Prowse et al, 1997), and in U937 cells by okadaic acid and phorbol ester (Biggs et al, 1996). However, the site is distinct from that used in BRCA-1-mediated induction of p21 .…”

Role of p53 in aziridinylbenzoquinone-induced p21waf1 expression

“…The expression of p21 is stimulated by a variety of external stimuli, such as various growth factors, cytokines, tumor promoters, as well as agents that generate DNA damage or reactive oxygen species, such as g-irradiation or adriamycin (ElDeiry et al, 1994;Sheikh et al, 1994). Not all stimulatory pathways involve the p53 protein, however, as some of the agents are able to induce p21 expression in p53-negative cells as well (Alpan and Pardee, 1996;El-Deiry et al, 1995;Li et al, 1995;Liu et al, 1996;Michieli et al, 1994;Parker et al, 1995;Prowse et al, 1997;Zeng and El-Deiry, 1996). The induction of the p21 gene by p53 occurs via two sites in the p21 promoter which directly bind p53 protein (ElDeiry et al, 1995).…”

“…The induction of the p21 gene by p53 occurs via two sites in the p21 promoter which directly bind p53 protein (ElDeiry et al, 1995). In addition, the p21 gene has binding sites for other transcription factors that may play a role in the regulation of p21 expression by p53-independent pathways (Chin et al, 1996;Macleod et al, 1995;Prowse et al, 1997).…”