[Biomedical research: the debate on the reduction and emergence concepts]. / Recherche biomédicale: le débat autour des notions de réduction et d'émergence.

The theoretical bases of medical knowledge exert a strong influence on both clinical practice and representations of living and health. In this perspective, reduction and emergence notions play a major role. Microreduction is the predominant analytical strategy used today in biology, as it is usually considered that essential life mechanisms can be reduced to molecular processes. Likewise, macroreduction proposes that parts can be defined in terms of their belonging to wholes, as it is usually assumed, for instance, in genetic epidemiology. With regard to emergence, this notion, which focuses on properties of a whole that cannot be deduced from properties of its parts, is consistent with both nature of living and evolution theory. The apparent success of reduction like analytical modality has generated in scientific community and public opinion an ideological reductionism, which corresponds, ontologically, to both physicalism (things can be entirely understood in terms of their parts), and atomism (things go their own way, independently of other things). Genetic reductionism has generated new cosmological representations of living, where past, present and future of living beings could potentially be deduced from fallacious, simple views of genome sequences. These views may lead to quantitative or qualitative definitions of standard patterns and hierarchies. In practical terms, research activity should integrate limits, strains as well as reductionism advantages. Biologists should also consider risks associated with an ideological, unrestricted reductionism, applied to any existence aspect, a notion with questionable legitimacy and with potential ethical, philosophical, and political involvements that go beyond the simple selection of a research strategy.

cRel induces mitochondrial alterations in correlation with proliferation arrest.

We have previously shown that overexpressing cRel, a transcription factor of the Rel/NF-kappa B family, concomitantly inhibits proliferation of HeLa cells and makes them resistant against TNF alpha-induced apoptosis. Both effects rely on the upregulation of the manganese superoxide dismutase (MnSOD), a mitochondrial enzyme that converts O(2)(*-) in H(2)O(2). Here we describe additional alterations induced by cRel, namely mitochondrial clustering and accumulation of dense dark granules near the nucleus. These changes preferentially occur in cells that display a sustained cRel expression in the nucleus and that are cell-cycle arrested. As the cell-cycle arrest, these changes are reproduced by directly overexpressing MnSOD or by treating cells with H(2)O(2), suggesting they are due to MnSOD induction and ensuing H(2)O(2) accumulation. We propose that mitochondria cluster because they are damaged by the H(2)O(2) they overproduce. They would then be autophagocytosed and degraded in secondary lysosomes. In support of this scenario, we documented the occurrence of oxidative damage and the presence of lysosomes in the area of mitochondrial clustering. In addition, we identified the dense dark granules as lipofuscin, based on their autofluorescence. Lipofuscin could directly originate from the mitochondrial degradation products that would aggregate and become indigestible because of the presence of H(2)O(2) in the secondary lysosomes. Altogether, our findings show that cRel overexpression in HeLa cells creates, via the induction of MnSOD, an oxidative injury that culminates in mitochondrial degeneration, proliferation blockage, and resistance against TNF alpha-induced apoptosis.

Rel/NF-kappaB transcription factors protect against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by up-regulating the TRAIL decoy receptor DcR1.

Rel/nuclear factor (NF)-kappaB transcription factors play a major role in the regulation of programmed cell death. A few anti-apoptotic Rel/NF-kappaB target genes have been characterized; they act either downstream in the apoptotic pathway or upstream, for example at the tumor necrosis factor (TNF) receptor level. We found using DNA arrays, reverse transcription-polymerase chain reaction, and immunofluorescence that Rel/NF-kappaB factors up-regulate DcR1, a receptor for TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family that induces apoptosis in tumor cells. Four related receptors bind TRAIL, two death receptors (DR4 and DR5) that signal apoptosis and two decoy receptors (DcR1 and DcR2) that act as dominant negative inhibitors of TRAIL-mediated apoptosis. DcR1 is devoid of an intracellular domain and is anchored at the cell surface membrane by a glycophospholipid. Our results indicate that overexpression of cRel or activation of endogenous Rel/NF-kappaB factors by TNFalpha in HeLa cells up-regulates DcR1 without changing the expression of DcR2, DR4, and DR5 and makes cells resistant against TRAIL-induced apoptosis. This resistance is a consequence of DcR1 up-regulation, because it was abolished when DcR1 was removed from the cell surface by a phosphatidylinositol phospholipase C. Therefore, Rel/NF-kappaB transcription factors could regulate the sensitivity of cells to TRAIL, by controlling the ratio of TRAIL-decoy to -death receptors.

Antiproliferative and antiapoptotic effects of crel may occur within the same cells via the up-regulation of manganese superoxide dismutase.

Rel/nuclear factor kappaB transcription factors were shown to have either pro- or antiapoptotic as well as pro- or antiproliferative functions, and it is often assumed that the outcome of their activation depends on the cell type or cellular context. Inconsistent with this assumption, we show here that cRel is able in one cell type to inhibit proliferation, protect against apoptosis induced by tumor necrosis factor alpha (TNF-alpha) + cycloheximide (CHX), and increase the basal rate of apoptosis. Both the effects of proliferation inhibition and protection against TNF-alpha + CHX-induced apoptosis are massive and occur in the same cells. Using reverse transcription-PCR, Western blot and immunofluorescence, and transactivation assays, we found that the manganese superoxide dismutase (MnSOD), an enzyme that converts O2*- in H2O2, is up-regulated by cRel through a kappaB site in intron 2. Inhibition of MnSOD induction by antisense oligonucleotides and overexpression of MnSOD respectively reverts and mimics both the antiproliferative and antiapoptotic effects of cRel, suggesting that they both occur via the induction of this gene. On one hand, MnSOD could improve the efficiency of cRel-overexpressing cells in eliminating toxic O2*- produced on TNF-alpha treatment, explaining why they escape TNF-alpha-induced apoptosis. On the other hand, cRel-overexpressing cells should accumulate H2O2. We present evidence linking this H2O2 accumulation to the proliferation arrest induced by cRel. Therefore, different effects on proliferation and apoptosis could arise from the induction of MnSOD and thus coexist in cRel-overexpressing cells.

[Genomics in the understanding of the mechanisms of transcriptional regulation]. / Apports de la génomique à la compréhension des mécanismes de régulation transcriptionnelle.

Whole genome" expression profiles can be used as molecular signatures or portraits characteristic of a tumour or of the physiological state of a cell. This global description also provides a new approach for the study of the transcriptional regulation, and this review is dedicated to this fundamental aspect of the transcriptome. Gene expression studies will be first considered at the single cell level. In situ analyses reveal that transcription is a stochastic phenomenon, indicating that the transcriptome contains fluctuating regions. The transcriptional response of a cell population to an external stimulus has been used to evaluate the contribution of the various signalling pathways and transcription factors. One of the emerging themes is the co-ordinate expression of genes associated with the same physiological process. In the spirit of this analysis of the transcriptional regulatory networks, it is becoming possible to design artificial biological networks to implement desired functions, paving the way to new therapeutic approaches.

The Ets family contains transcriptional activators and repressors involved in angiogenesis.

The Ets family contains a growing number of transcriptional activators and inhibitors, which activity is regulated by phosphorylation and protein-protein interactions. Among these factors, Ets1, Erg1 and Fli1 are expressed in endothelial cells during angiogenesis in normal and pathological development. The expression of these transcription factors is regulated by angiogenic factors in cultured endothelial cells, as well as by various stresses occurring during angiogenesis. Transfection experiments and transgenic mice analysis revealed that Ets family members are involved in the transcriptional regulation of endothelial specific genes such as those encoding Tie1 and -2, VEGFR1 and -2 and VE-Cadherin. In vitro studies plead for a role of Ets family members in endothelial cell adhesion, spreading and motility. Gene inactivation experiments show that Ets1 is dispensable for embryonic development. The phenotype of knocked-out embryos indicates that Tel is required for maintenance of the developing vascular network in the yolk sac. Altogether, we suggest that Ets family members act both positively and negatively during the different steps of the angiogenic process. The regulation of the initiation of gene transcription arises from the combined activity of different transcriptional regulators. Therefore very few transcription factors are specific for a physiological process, or a given cell type. The transcriptional network that regulates blood vessel formation involves transcription factors which are expressed in a variety of situations. The Lung Kruppel Like Factor (LKLF) which is required for blood vessel stabilisation during murine development is also expressed in the primitive vertebrae and in the lung of the adult (C.T. Kuo, M.L. Veselits, K.P. Barton, M.M. Lu, C. Clendenin, J.M. Leiden, The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilisation during murine embryogenesis, Genes Dev. 11 (22) (1997) 2996-3006). Scl/Tal1 which is essential for angiogenic remodelling of the yolk sac capillary network (J.E. Visvader, Y. Fujiwara, S.H. Orkin, Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development, Genes Dev. 12 (4) (1998) 473-479), is involved in blood cell development and is also expressed in the developing brain. The EPAS transcription factor which was thought to be endothelial cell specific in the mouse embryo (H. Tian, S.L. McKnight, D.W. Russell, Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells, Genes Dev. 11 (1) (1997) 72-82) is also expressed in the liver, kidney and cells of the sympathetic nervous system of the chick embryo (J. Favier, H. Kempf, P. Corvol, J.M. Gasc, Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase, FEBS Lett. 462 (1-2) (1999) 19-24). Ets1, which expression was originally detected in lymphoid cells of adult tissues, has been the first transcription factor to be identified in endothelial cells during angiogenesis in the embryo (B. Vandenbunder, L. Pardanaud, T. Jaffredo, M.A. Mirabel, D. Stehelin, Complementary patterns of expression of c-etsl, c-myb and c-myc in the blood-forming system of the chick embryo, Development 107 (1989) 265-274 [5]) and in tumours (N. Wernert, M.B. Raes, P. Lassalle, M.P. Dehouck, B. Gosselin, B. Vandenbunder, D. Stehelin, The c-ets 1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularisation and other forms of angiogenesis in man, Am. J. Path. 140 (1992) 119-127 [6]). Since then, the Ets family has extended and this review will emphasise the relationships between these factors and angiogenesis.

Sequential activation of ERK and repression of JNK by scatter factor/hepatocyte growth factor in madin-darby canine kidney epithelial cells.

The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.

The conserved redox-sensitive cysteine residue of the DNA-binding region in the c-Rel protein is involved in the regulation of the phosphorylation of the protein.

The DNA-binding activity of the transcription nuclear factor kappaB (NF-kappaB) is regulated by a redox-control mechanism involving the reduction of a disulphide bond from a specific cysteine residue conserved in all members of the NF-kappaB family. Thioredoxin is involved in this redox control. DNA binding and transactivating capacity of NF-kappaB are up-regulated by inducible phosphorylation. Here we demonstrate that the conserved redox cysteine in the c-Rel protein is involved in the phosphorylation regulation of the protein. When this cysteine residue is mutated to an aspartic acid residue, the mutant protein loses its capacity to be phosphorylated and its DNA-binding activity. In addition, our results suggest that, when the conserved redox cysteine is chemically modified by N-ethylmaleimide and 2-chloro-1,3-dinitrobenzene, the protein c-Rel cannot be phosphorylated. In contrast, the protein in which the cysteine residue was replaced by a serine residue, creating a potential phosphorylation site, is highly phosphorylated and binds kappaB sequences. The protein could loose the redox regulation of the phosphorylation when the residue replacing the cysteine can be itself phosphorylated. We also show that specific inhibitors of thioredoxin reductases impair the phosphorylation of the c-Rel protein, suggesting that the redox regulation of the protein controls its phosphorylation.

ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin.

Ets1 is a transcription factor expressed in endothelial cells during angiogenesis but its target genes and function in blood vessel formation are still unknown. We have over-expressed Ets1 as a tagged protein in brain capillary endothelial cells and in 3T3 fibroblasts using a retroviral vector. Over-expression of Ets1 reduced by nearly half cell density at confluence of endothelials but not of fibroblasts. As density at confluence is controlled in part by cadherins, this growth arrest could be due to the up-regulation of these cell contact molecules. Indeed, Ets1 increased the expression of the endothelial-specific VE-cadherin without affecting N-cadherin expression levels. In parallel, both a dominant negative mutant of Ets members and an Ets1 anti-sense oligonucleotide inhibited VE-cadherin expression in endothelial cells. Ets1 bound to two Ets-binding sites located in the proximal region of the VE-cadherin promoter. Mutation of these sites abolished Ets1-induced transactivation of the promoter. The present work is the first demonstration of a function of Ets1 in the regulation of a specific endothelial marker based on its endogenous gene and protein expression.

[Experimental angiogenesis : strategy for the functional study of the transcription factors of the Ets family during morphogenesis of the vascular tree]. / Angiogenèse expérimentale : stratégies pour l'étude fonctionnelle des facteurs de transcription de la famille Ets dans la morphogenèse du réseau vasculaire.

During morphogenesis of the vascular tree, the massive outgrowth of primitive capillaries is followed by the development and the maturation of some capillary branches whereas others regress. The direct observation and the manipulation of in vivo models, including a series of recent knock-out experiments, allow to delineate the mechanisms controlling this process, and to identify factors involved in the formation of a mature capillary, surrounded with a basal lamina and pericytes. The expression of several members of the Ets family of transcription factors, Ets1, Erg and Fli, correlates with the occurrence of invasive processes, such as angiogenesis during normal and pathological development. The description of the phenotype of cultured endothelial cells expressing the DNA binding domain of Ets1 suggests that members of the Ets family take part in the morphogenesis of the -vascular tree. Although transient transfection experiments allowed the identification of putative targets genes for Ets1 during angiogenesis, deciphering the Ets1 regulation networks remains a major goal for the future.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Constitutive expression of the DNA-binding domain of Ets1 increases endothelial cell adhesion and stimulates their organization into capillary-like structures.

We previously reported that the Ets1 transcription factor is expressed in endothelial cells during angiogenesis both in normal and pathological development. We analyse here the effects of the stable expression of an Ets transdominant negative mutant (Ets1-DB), consisting in an Ets1 protein lacking its transactivation domain. A retrovirus containing the Ets1-DB sequence fused to an IRES-Neo sequence was designed and used to infect brain capillary (IBE) and aorta (MAE) mouse endothelial cell lines. Cells expressing this Ets1 mutant were examined for proliferation, migration and adhesion. Consistent changes were observed on cell morphology, with increased spreading and modifications in the organization of the cytoskeleton, and increased cell adhesion. We investigated the ability of endothelial cells to organise into capillary-like structures using three-dimensional gels. On Matrigel, all endothelial cell lines formed a cord-like network within 24 h, with an increased ability of Ets1-DB cells to spread on this substrate. In long term cultures, IBE cells expressing Ets1-DB showed a higher capacity to form branched structures; this effect was potentiated by FGF2. These results demonstrate a role of the Ets transcription factors in the regulation of the adhesive and morphogenetic properties of endothelial cells.

Neoplastic AIDS-associated Kaposi's sarcoma cell line KSY-1 cannot transdifferentiate into capillaries.

OBJECTIVE: Kaposi's sarcoma (KS) is an acquired immunodeficiency syndrome (AIDS)-defining neoplasm histologically characterized by proliferation of spindle cells, inflammatory cells, and abundant neovascularization. When the malignant cell line KSY-1 derived from an AIDS-KS tumor is transplanted subcutaneously into nude mice, prominent neovascular features develop. Using this mouse model of neoplastic KS, we set out to determine, using c-ets 1 markers specific for mouse or human tissues, whether vascular growth and inflammatory infiltrate induced by the transplanted KSY-1 cells is of host cell or transplant origin. STUDY DESIGN/METHODS: KS tumors were induced by subcutaneous inoculation of 5 x 10(6) KSY-1 cells/200 microL in immunodeficient mice, and species-specific mouse and human riboprobes of the c-ets 1 protooncogene were used for in situ hybridization to define cell of origin. RESULTS: Five different tumors were examined. Tissue sections from all cases were hybridized with radiolabeled riboprobes for the presence of both mouse and human c-ets 1 mRNA. Tumor cells were labeled with the human c-ets 1 probe, whereas neovascular and inflammatory tissues were of mouse origin. CONCLUSIONS: The finding that vascular but not tumor cells are of host origin supports the model of tumor-induced vascularization via a mechanism of tumor cell-derived cytokine-medicated pathogenesis.

[Transcription factors of the Ets family and morphogenesis of the vascular tree]. / Les facteurs de transcription de la famille Ets et la morphogenèse du réseau vasculaire.

The expression of several members of the Ets family of transcription factors, Ets1, Erg and Fli, correlates with the occurrence of invasive processes such as angiogenesis during normal and pathological development. The description of the phenotype of cultured endothelial cells expressing the DNA binding domain of Ets1 suggests that members of the Ets family take part in the morphogenesis of the vascular tree. Although transient transfection experiments allowed the identification of putative targets genes for Ets1 during angiogenesis, deciphering the Ets1 regulation networks remains a major goal for the future.

[Transcription factors and angiogenesis]. / Facteurs de transcription et angiogenèse.

Various strategies led to the identification of transcription factors that take part to the control of different steps during the formation of new blood vessels: the description of the expression pattern of genes encoding these factors during embryonic development for ETS-1, ERG and FLI, SCL/TAL, GATA1 and 2, the description of the phenotype of embryos obtained after gene inactivation by homologous recombination for ARNT or LKLF, and the study of transcriptional regulation in cultured endothelial cells for EGR1 or HOX-D3. Altogether, these results showed that there is no transcription factor specific for endothelial cells or for one step in the formation of blood vessels. Rather, factors controlling gene expression induced by hypoxia, shear-stress or growth factors take part in the morphogenesis of the vascular tree. The study of these factors may allow to identify potential therapeutic targets for treatments aimed at inhibiting or stimulating the development of new blood vessels.

The multisubstrate docking site of the MET receptor is dispensable for MET-mediated RAS signaling and cell scattering.

The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association-kinase assay we found that the mutated receptor can still associate and phosphorylate a approximately 250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering.

Expression of transcription factor c-Rel and apoptosis occurrence in polydactylous and syndactylous limb buds of the talpid3 mutant chick embryo.

The chicken proto-oncogene c-rel encodes a transcription factor of the Rel/NF-kappa B family. We have previously shown that c-rel mRNAs accumulate in different types of apoptotic cells of the chick embryo, especially in mesenchymal cells within the four cell death areas of the limb bud: the anterior and posterior necrotic zones, the opaque patch and the interdigital necrotic zones. This study aimed to further establish the involvement of c-Rel in apoptosis of the developing limb by investigating its expression in the talpid3 mutant which was originally shown to be defective in apoptosis. However, our preliminary examinations highlighted the apparent presence of apoptotic cells in talpid3 embryos. Hence, we performed a systematic study of the occurrence of apoptosis in mutant and control embryos by the TUNEL method. The results revealed that apoptosis does occur in talpid3 embryos but with altered spatial and temporal patterns. This suggests that the talpid3 mutation does not affect a gene involved in apoptosis per se but rather in the determination of the pattern of apoptosis. Neither the expression of c-Rel nor that of its I kappa B alpha inhibitor are grossly modified in talpid3 limb buds, suggesting that the talpid3 mutation does not affect any of these genes. They are mostly expressed in epidermal, endodermal and striated muscle cells in control and in talpid3 limb buds as well. C-Rel was also detected in some scarce mesenchymal cells that could be apoptotic, in both control and mutant embryos. The only slight difference between control and talpid3 limbs lies in the perichondrium which is not fully differentiated in talpid3 embryos: c-Rel and I kappa B alpha are only faintly expressed in talpid3 perichondrial cells, whereas they are both detected in control perichondrial cells. Taken together, these results suggest that c-Rel could participate in several developmental processes, especially in the differentiation of perichondrial cells, besides its already documented involvement in apoptosis and haematopoeisis.