TGF- ß Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the α -Fetoprotein Promoter.

α-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-ß signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-ß signaling. To further understand how TGF-ß suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-ß. We found that the TGF-ß signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs). Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-ß stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-ß signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-ß signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.

Identification of CCAAT enhancer binding protein alpha binding sites on the human alpha-fetoprotein gene.

Development- and tissue-specific alpha-fetoprotein (AFP) gene expression is controlled by various transcription factors including hepatocyte nuclear factors (HNFs), and a number of cis-acting elements. We recently identified multiple CCAAT/enhancer binding protein (C/EBP) binding sites in the enhancer of the human AFP gene. In this study, we have identified and functionally characterized seven C/EBPalpha-binding sites in the promoter and enhancer regions. An electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis identified two and five C/EBPalpha-binding sites located in the promoter and enhancer regions, respectively. Chromatin immunoprecipitation analyses showed that C/EBPalpha binds both enhancer and promoter regions of the AFP gene in human AFP-producing hepatoma and stomach cancer cells, but not in non-AFP-producing cells. Reporter transfection assays showed that transcription was stimulated by C/EBPalpha binding to each of the elements. These results indicate that C/EBPalpha regulates AFP gene expression through direct binding to multiple sites in the human AFP gene in cultured human cells.

Interleukin-6 and mevastatin regulate plasminogen activator inhibitor-1 through CCAAT/enhancer-binding protein-delta.

OBJECTIVE: We sought to determine the etiologic mechanism of proinflammatory cytokine, interleukin-6 (IL-6), and statin as regulators of synthesis of plasminogen activator inhibitor-1 (PAI-1), the physiological fibrinolysis inhibitor and an acute-phase reactant. METHODS AND RESULTS: Transient transfection and luciferase assay in HepG2 human hepatoma-derived cells demonstrated that IL-6 increased PAI-1 promoter activity and mevastatin decreased IL-6-inducible response. Systematic deletion assay of the promoter demonstrated that the region (-239 to -210 bp) containing a putative CCAAT/enhancer-binding protein (C/EBP) binding site was necessary. Point mutation in this site abolished the IL-6-inducible response. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta were involved in protein-DNA complex formation in intact cells. Deoxyribonuclease (DNase) I footprinting analysis revealed that 5' flanking region (-232 to -210 bp) is acute-phase response protein-binding site. C/EBPdelta binding activity was increased by IL-6 and attenuated by mevastatin. Mevastatin attenuated IL-6-mediated increase of C/EBPdelta protein in the nuclear extracts. IL-6 also increased PAI-1 and C/EBPdelta mRNA in mouse primary hepatocytes. CONCLUSIONS: IL-6 increases hepatic PAI-1 expression mediated by the -232- to -210-bp region of the promoter containing a C/EBPdelta binding site. Vascular protection by statins may be partly mediated through regulation of CEBPdelta and consequent modulation of PAI-1 expression.

Functional mapping of tissue-specific elements of the human alpha-fetoprotein gene enhancer.

Serum alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC) patients and expression of the protein in cultured HCC cell lines are highly variable. These observations may arise from features correlated with tissue-specific expression of the gene. Extremely strong and potent liver-specific enhancer activity is confined from -4.1 to -3.3 kb upstream to the human AFP gene in contrast with that of the rodent which exists in three widely separated regions. To understand the tissue-specific expression of AFP, we examined cis-acting elements in the enhancer. Results revealed binding sites for selected liver-enriched transcription factors (LETFs) in both domains A (-4120 to -3756 bp) and B (-3492 to -3300 bp) of the gene. These sites included: one hepatocyte nuclear factor (HNF)-1 and HNF-4, two HNF-3, and two C/EBP binding sites in domain A. An adjacent domain B contained one HNF-3 site and three C/EBP sites plus a previously identified HNF-1 site. Each of these elements alone has the ability to stimulate heterogeneous promoter activity in a dose-dependent manner when transfected into AFP producing cells. A comparative study showed that the presence of two HNF-1 and one HNF-4 site is a characteristic feature of human but not rodent AFP enhancer. The mRNA levels of the liver-enriched transcription factors (LETFs) were variable in individual HCC cell lines and together with silencer activities may underlie differential expression of the AFP gene.

ING1 represses transcription by direct DNA binding and through effects on p53.

The ING family of proteins is involved in the regulation of diverse processes ranging from cell cycle and cellular senescence to apoptosis. These effects are most likely through activation of acetylation-dependent pathways that ultimately alter gene expression. Despite reports linking ING to p53 activation, the molecular basis of how ING activates p53 function has not been elucidated. In this study, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) up-regulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein.