Transcriptional Activation of p21waf1/cip1 by Alkylphospholipids

Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C 2 H 2 -type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GCbox recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.Transcriptional regulation of the eukaryotic gene is a complicated process, involving a series of complex molecular interactions among regulatory and transcription factors. Specificity protein 1 (Sp1) 1 is probably one of the best characterized sequence-specific transcription factors, and has numerous functions in the transcription of many cellular and viral genes harboring GC boxes in their promoters (1, 2). Sp1 and its family of proteins have been implicated in a host of essential biological processes, and have been proven important in apoptosis, cell growth inhibition, differentiation, and carcinogenesis (Refs. 3-5 and references therein).Sp1 is a member of the Sp-multigene family, which also includes Sp2, Sp3, Sp4, Sp5, Sp6, Sp7, and Sp8 (3-5). The Sp-family proteins exhibit similar domain structures and are evolutionarily closely related. All of these proteins possess highly conserved C 2 H 2 -type zinc finger DNA binding domains at their C termini, and all belong to the Krü ppel-like zinc finger superfamily (3-5). In addition, the Sp-family proteins have been demonstrated to undergo post-translational modifications as the result of diverse mechanisms. For example, Sp1 is glycosylated and phosphorylated by Erk2, protein kinase C, casein kinase II, and cAMP-dependent protein kinase; Sp3 is acetylated and SUMOylated by the protein inhibitor of activated STAT (PIAS1, Ref. 3 and references therein, Refs. 6 -10).The Sp1 protein co...

Section: Discussionmentioning

confidence: 99%

“…It has been suggested that MEK, from the MAP kinase pathway, phosphorylates Sp1 at amino acids 453 and 739 (36,38,40). The phosphorylation of these two sites results in an increase in Sp1 binding to the target sites and in increased transcription activation.…”

Section: Waf/cip1mentioning

confidence: 99%

Transcriptional Activity of Sp1 Is Regulated by Molecular Interactions between the Zinc Finger DNA Binding Domain and the Inhibitory Domain with Corepressors, and This Interaction Is Modulated by MEK

Lee

Suh

Kang

et al. 2005

“…Cells were cotransfected with 10 ng/well of wild-type mouse ER␣ or mutant nonclassical ER E207A/G208A (AA) expression vector and 500 ng/well of the indicated reporter constructs (AP-1-luciferase or p21-luciferase) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The 2.4-kb full-length human p21 reporter was kindly provided by Dr. Xiao-Fan Wang (Duke University) (21,22). The ER expression and AP-1 reporter constructs have been previously described (11,15).…”

Section: Methodsmentioning

confidence: 99%

Estrogen-induced Proliferation of Uterine Epithelial Cells Is Independent of Estrogen Receptor α Binding to Classical Estrogen Response Elements

O’Brien

Peterson

Tong

et al. 2006

113

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/ c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ER␣, a mutant ER␣ (E207A/G208A) that selectively lacks ERE binding, or ER␣ null. In the compound heterozygote (AA/؊) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17␤-estradiol (E 2 ). The uterine epithelial proliferative response to E 2 and 4-hydroxytamoxifen was retained in the AA/؊ females, and uterine luminal epithelial height increased commensurate with the extent of ER␣ signaling. This proliferative response was confirmed by 5-bromo-2-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathwayresponsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ER␣ (E207A/G208A) mutant. These results indicate that nonclassical ER␣ signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ER␣ plays a critical physiologic role in the uterine response to estrogen.The uterus responds to cyclical changes in estrogen and progesterone levels in preparation for embryo implantation. Estrogen mediates the principal proliferative response of the uterus through two related but distinct estrogen receptors (ER␣ 2 and ER␤) (1-3). ER␣ is the predominant form in the murine uterus (4), but ER␤ transcripts are also detected at very low levels (4). After estrogens bind to the ligand domain of the ER, it undergoes conformational changes that allow interactions with coactivator molecules (5-7). In the classical pathway of estrogen action, the ER binds as a dimer to EREs in the promoter regions of target genes (8). However, it is now clear that the ER can regulate genes that lack a canonical ERE, suggesting additional pathways for estrogen action (9 -11). For example, the ER may activate genes via protein-protein tethering with c-Fos/c-Jun B (AP-1) and Sp1 (12, 13) or suppress expression mediated through NF-B (14). This alternate response mechanism has been referred to as the tethered or nonclassical pathway.We previously generated an ER␣ mutant that selectively functions via the nonclassical pathway. Substitution of two amino acids in the DNA recognition sequence of the fir...

“…The transcription factor SP1 plays a role in ERK-mediated p21 transcription in various cell types, including nerve growth factor-treated PC12 cells (60), Ras-transformed NIH3T3 cells (61), alkylphospholipid-treated HaCaT cells (33), and arsenic trioxide-exposed A431 epidermoid carcinoma cells (62). ETS and C/EBP␤ (63) are also involved in ERK-dependent, p53-independent expression of p21 expression in primary hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

“…For example, ERK contributes to the induction of neuronal differentiation by nerve growth factor (29) and to growth arrest and the induction of apoptosis through phosphoactivation of p53 (30,31). Elsewhere, several studies have demonstrated that ERK signaling is associated with up-regulation of p21 expression in a variety of cell types (32)(33)(34)(35)(36)(37)(38). However, despite the emerging recognition that MAPKs inhibit cell proliferation by affecting p21 expression, little is yet known about the mechanisms by which these kinases regulate p21 transcription.…”

mentioning

confidence: 99%

The ETS Family Transcription Factor ELK-1 Regulates Induction of the Cell Cycle-regulatory Gene p21 and the BAX Gene in Sodium Arsenite-exposed Human Keratinocyte HaCaT Cells

Shin

Kim

Lim

et al. 2011

Cyclin-dependent kinase inhibitor (CDKN1A), often referred to as p21Waf1/Cip1 (p21), is induced by a variety of environmental stresses. Transcription factor ELK-1 is a member of the ETS oncogene superfamily. Here, we show that ELK-1 directly transactivates the p21 gene, independently of p53 and EGR-1, in sodium arsenite (NaASO 2 )-exposed HaCaT cells. Promoter deletion analysis and site-directed mutagenesis identified the presence of an ELK-1-binding core motif between ؊190 and ؊170 bp of the p21 promoter that confers inducibility by NaASO 2 . Chromatin immunoprecipitation and electrophoretic mobility shift analyses confirmed the specific binding of ELK-1 to its putative binding sequence within the p21 promoter. In addition, NaASO 2 -induced p21 promoter activity was enhanced by exogenous expression of ELK-1 and reduced by expression of siRNA targeted to ELK-1 mRNA. The importance of ELK-1 in response to NaASO 2 was further confirmed by the observation that stable expression of ELK-1 siRNA in HaCaT cells resulted in the attenuation of NaASO 2 -induced p21 expression. Although ELK-1 was activated by ERK, JNK, and p38 MAPK in response to NaASO 2 , ELK-1-mediated activation of the p21 promoter was largely dependent on ERK. In addition, EGR-1 induced by ELK-1 seemed to be involved in NaASO 2 -induced expression of BAX. This supports the view that the ERK/ELK-1 cascade is involved in p53-independent induction of p21 and BAX gene expression.