Progesterone Regulates Transcription of the p21 Cyclindependent Kinase Inhibitor Gene through Sp1 and CBP/p300

Abstract

Progesterone has biphasic effects on proliferation of breast cancer cells; it stimulates growth in the first cell cycle, then arrests cells at G 1 /S of the second cycle accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21. We now show that progesterone regulates transcription of the p21 promoter by an unusual mechanism. This promoter lacks a canonical progesterone response element. Instead, progesterone receptors (PRs) interact with the promoter through the transcription factor Sp1 at the third and fourth of six Sp1 binding sites located downstream of nucleotide 154. Mutation of Sp1 site 3 eliminates basal transcription, and mutation of sites 3 and 4 eliminates transcriptional up-regulation by progesterone. Progesterone-mediated transcription is further prevented by overexpression of E1A, suggesting that CBP/p300 is required. Indeed, in HeLa cells, Sp1 and CBP/p300 associate with stably integrated flagtagged PRs in a multiprotein complex. Since many signals converge on p21, cross-talk between PRs and other factors co-localized on the p21 promoter, may explain how progesterone can be either proliferative or differentiative in different target cells.Progesterone is a paradoxical hormone having either growth stimulatory effects or growth inhibitory and differentiative effects, depending on the tissue in question and the dose and treatment regimen (1, 2). In the uterus for example, progesterone inhibits epithelial growth and has differentiative effects (3). It is therefore used to counteract the proliferative and carcinogenic effects of unopposed estrogens in women prescribed hormone replacement therapy (4). In the breast, the role of progesterone is more complex. The hormone is required for terminal growth and differentiation of the mammary gland (2). Therefore, mice lacking progesterone receptors (PRs) 1 exhibit incomplete mammary gland ductal branching and failure of lobulo-alveolar development (5). In animal models of mammary carcinogenesis, progesterone, depending on the regimen used, can either inhibit or promote tumor formation (2). On the other hand, in animals with established PR-positive mammary tumors, progesterone is usually proliferative, and progesterone antagonists inhibit tumor growth (6). Despite this, in humans, second-line high dose progestin therapy effectively suppresses the growth of hormone-dependent PR-and estrogen receptorpositive breast cancers that have acquired resistance to the antiestrogen tamoxifen (6).How can these contradictory effects of progesterone be reconciled? Recent studies have dealt with the effects of progesterone on mitosis and key cell cycle regulatory proteins in cultured human breast cancer cells (1, 7-9). Treatment of such cells with progestins produces biphasic effects. Studies focusing on the initial growth stimulatory component show that progestin-induced entry of cells into S-phase is accompanied by transient increases of cyclin D1 and cyclin-dependent kinase 4 activity (1, 7). Indeed, cyclin D1 is a critical component of the mitogenic respon...

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

“…Similarly, induction of p21 during keratinocyte di erentiation or by progesterone is dependent on spacing of the promoter elements. Insertions between responsive elements or placing of the responsive elements too close to the transcription initiation site abolish the transcriptional activation of p21 (Owen et al, 1998;Prowse et al, 1997). Here, a mutant construct a ecting the TbRE but not eliminating any of the Sp1-binding sites (mut 2.3) (Datto et al, 1995b), showed a weaker induction of luciferase activity by Ras similarly to the mutants a ecting the Sp1-binding site 2 and TbRE simultaneously (mut 2 and mut 2.2).…”

“…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 .…”

“…The transactivation is mediated by speci®c p53-binding sites in the p21 promoter (ElDeiry et al, 1993). Other transcriptional activators of p21 include tumor suppressor BRCA-1 , transforming growth factor-b (Datto et al, 1995b), activin A (Zauberman et al, 1997), cytokines acting through STAT-pathway (Chin et al, 1996), nerve growth factor (Yan and Zi , 1997), tumor promoters (Zeng and El-Deiry, 1996), vitamin D 3 (Liu et al, 1996b), retinoic acid receptor (Liu et al, 1996a), glucocorticoids , progesterone (Owen et al, 1998), Raf (Sewing et al, 1997), and MAPKK (Liu et al, 1996c), while Ras-mediated induction of p21 is negatively regulated by Rho (Olson et al, 1998). Furthermore, growth factors PDGF, FGF, and EGF (Michieli et al, 1994), as well as tumor suppressor gene WT1 (Englert et al, 1997) induce p21 mRNA levels.…”

Bmf is a possible mediator in histone deacetylase inhibitors FK228 and CBHA-induced apoptosis

“…Consistent with this idea, the promoter region of the Bmf gene possesses . [31][32][33] Like most promoter activities, this may reflect the fact that FK228-mediated Bmf transcription can be activated depending upon these multiple promoter activities. This may at least partially explain why FK228 or CBHA activates Bmf expression differently in different cells, since these promoter activity may differ depending upon cell types.…”