Jean-Luc Baert

Expression of the PEA3 group of ETS-related transcription factors in human breast-cancer cells

The PEA3 group of transcription factors belongs to the ets family and is composed of 3 known members, PEA3, ERM and ER81, which are more than 95% identical within the DNA‐binding ETS domain and exhibit 50% aa identity overall. Recently, transgenic mice bearing the c‐erbB‐2/neu oncogene have been shown to over‐express PEA3 mRNA in mammary adenocarcinomas, suggesting a role for this gene family in mammary tumorigenesis. In the present work we characterized the mRNA expression levels of PEA3‐group genes in a series of human epithelial breast cell lines. Each of the 3 genes was highly expressed in normal human HMEC 1001‐7 and HMEC 219‐4 cells. In breast‐cancer cell lines, the 3 genes were highly expressed in the ER−MDA‐MB‐436, MDA‐MB‐330, MDA‐MB‐231 and BT‐20 cell lines, but not in the ER+MDA‐MB‐134‐VI and ZR‐75‐1 cells. In an attempt to characterize the PEA3‐group proteins in breast‐cancer cells, we first produced and characterized specific antibodies against each of these 3 proteins. The anti‐ERM and anti‐ER81 antibodies recognized specific strong bands at approximately 72 kDa and 62 kDa, corresponding to ERM and ER81, respectively, in MDA‐MB‐231 and Hs‐578T cells expressing significant levels of the 3 mRNAs. No protein was detected in MCF‐7 cells expressing low levels of mRNA for PEA3‐group‐family genes, or in ZR‐75‐1 cells, where mRNA was undetectable by Northern blot. Although in vitro‐translated PEA3 is specifically immunoprecipitated by anti‐PEA3 anti‐serum, we were unable to immunoprecipitate PEA3 protein from MDA‐MB‐231 and Hs‐578T cells. In order to study the transcription factor activity of ERM, PEA3 and ER81 proteins in mammary‐cancer cells, we tested their ability to transactivate a reporter plasmid containing 3 Ets‐binding sites, and were able to show that, in all the breast‐cancer cells tested, transfected ERM, PEA3 and ER81 are able to transactivate. Although the target genes of the PEA3 group of transcription factors in breast‐cancer cells have yet to be determined, these genes have a potential role in the regulation of growth and the progression of human breast cancer.Int. J. Cancer 70:590–597.

The PEA3 Group of ETS-related Transcription Factors

The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor

The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.

The transcription of the intercellular adhesion molecule-1 is regulated by Ets transcription factors

The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions −158 and −138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.

Molecular characterization of mouse 17β-hydroxysteroid dehydrogenase IV

17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17 beta-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17 beta-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal beta-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17 beta-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17 beta-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17 beta-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17 beta-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17 beta-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.

Theromin, a novel leech thrombin inhibitor.

We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-β-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.

Biochemical evidence of angiotensin II-like peptides and proteins in the brain of the rhynchobdellid leech Theromyzon tessulatum

The peptides contained in neurons localized in the brain of the leech Theromyzon tessulatum (Hirudinae, Rhynchobdellida) and showing an immunopositive reaction with an antibody directed against angiotensin II (AII), were purified by reversed-phase HPLC. Three AII-like peptides (P1, P2 and P3) which exhibited the same retention times and chromatographic behaviors as synthetic AVII (fragment 6-8 of AII), AIV (fragment 3-8 of AII) and AII, respectively, were resolved in brain extracts. An identification of the proteins immunoreactive to an anti-AII was performed at the level of both brain extracts and in vitro brain-translated RNA products. The protein detected at the level of the brain extracts (of a molecular mass of approximately 18 kDa) is multipeptidic as it is also recognized by two other antisera, a polyclonal one directed against gamma-MSH and a monoclonal one (Tt159) raised against a leech brain epitope. It could be the pro-AII-like precursor. The protein detected at the level of in vitro brain-translated RNA products (of a molecular mass of approximately 19 kDa) could be the prepro-AII-like precursor.

Purification and characterization of a cadmium-binding protein from Nereis diversicolor (Annelida, Polychaeta)

Abstract 1. The results obtained in this study confirm the previous ones related to the presence in Nereis (both in control and contaminated animals) of a low molecular weight protein having the capacity to bind Cd (Dennai et al ., 1986). This component probably also binds Zn and Cu. 2. By SDS-PAGE and amino acid analysis, the molecular weight of the purified protein was estimated at about 10 kD. 3. However, results of molecular analysis using gel filtration on Superose 12 indicate that the Cd-binding protein called metalloprotein II (MP II) because of its occurrence in the second main protein peak after gel filtration on a Sephadex G-75 column has a molecular weight of about 20 kD in its native state. 4. It is suggested that the native protein is a dimer of two identical subunit polypeptides. 5. The present study establishes that this molecule which only contains 0.9% of Cys is clearly not related to metallothionein.

Heterosynthetic origin of the major yolk protein, vitellin, in a nereid, Perinereis cultrifera (Polychaete annelid)

Abstract 1. 1. An examination of proteins synthesized by Perinereis cultrifera oocytes incubated in vitro with [3H]leucine clearly shows that these cells are not capable of synthesizing the main yolk protein previously identified in this worm. 2. 2. In addition, the detection of radiolabelled vitellin in oocytes after in vitro incubation of an oocyte-coelomocyte cell mixture in presence of [3H]leucine strongly suggests that the coelomocytes, free cells in the coelomic cavity, synthesize and secrete a vitellin precursor, vitellogenin, that is subsequently taken up by the oocytes. 3. 3. Two native proteins differing in mol. wt but reacting with anti-vitellin antibodies have been identified in coelomocyte incubation medium. Also found in the coelomic fluid, they have been designated VG1 (Mr = 530,000) and VG2 (Mr = 320,000). 4. 4. The two vitellogenins consist of a single type of polypeptide of Mr = 176,000 and are incorporated in the oocytes where they are apparently observed under a single molecular form corresponding to VG1, the highest mol. wt protein similar in size to the initial form of vitellin (VI, 530,000). 5. 5. From these data, it seems likely that VG2 is a monomeric molecule that is taken up by the oocytes as a dimer of VG1. 6. 6. We conclude that P. cultrifera accumulates vitellin heterosynthetically and that vitellogenin is produced by the coelomocytes. Moreover, a single polypeptide similar in size to the polypeptidic component of secreted vitellogenin has been detected in the coelomocytes. 7. 7. Since this polypeptide has been identified previously as the single intraoocytic precursor of the four lower mol. wt products that make up the mature form of vitellin (V5), it appears that P. cultrifera exhibits for vitellogenin a processing pathway in which cleavage of the precursor occurs only after uptake by the oocyte.

The E3 ubiquitin ligase complex component COP1 regulates PEA3 group member stability and transcriptional activity

In this study, we report that the PEA3 group members interact with the mammalian really interesting new gene (RING) E3 ubiquitin ligase constitutive photomorphogenetic 1 (COP1), which mediates ubiquitylation and subsequent proteasome degradation of the p53 and c-Jun transcription factors. This interaction is mediated by the central region of COP1 including the coiled-coil domain and two COP1-interacting consensus motifs localized in the well-conserved N-terminal transactivation domain of the PEA3 group members. At the transcriptional level, COP1 reduces the transcriptional activity of ERM and the two other PEA3 group proteins on Ets-responsive reporter genes; this effect being dependent on the RING domain of COP1 and the two COP1-interacting motifs of ERM. Reduced transcriptional activity was, however, not related to COP1-induced changes in ERM stability. In fact, increased ubiquitylation and subsequent proteasome-mediated degradation of ERM is achieved only when COP1 is expressed with DET1, a key COP1 partner within the ubiquitylation complex. Conversely, we show that the depletion of COP1 or DET1 by small interference RNA (siRNA) in U2OS cells stabilizes endogenous ERM whereas only COP1 knockdown enhances expression of ICAM-1, a gene regulated by this transcription factor. These results indicate that COP1 is a complex regulator of ERM and the two other PEA3 group members.