Médecine Moléculaire / Molecular Medicine

Colloque organisé par Fotis Kafatos and Spyros Artavanis-Tsakonas du 23 au 27 septembre 1992

Participants

Spyros Artavanis-Tsakonas (Yale University), Hugues Blaudin de Thé (Hôpital Saint-Louis), Jenefer Blackwell (Cambridge University, UK), Laurent Degos (Hôpital Saint-Louis), Raymond L. Erikson (Harvard University, Cambridge), Elaine Fuchs (University of Chicago), Benjamin Geiger (Weizmann Institute of Science, Rehovot, Israel), François Jacob (Institut Pasteur), Fotis Kafatos (Harvard University, Cambridge and IMBB, Research Center of Crete), Jean-Pierre Kraehenbuhl (Université de Lausanne et Institut Suisse de recherche sur le cancer), Daniel Louvard (Institut Pasteur), Christine Petit (Institut Pasteur), Christian Pinset (Institut Pasteur), Eric Pringault (Institut Pasteur), David D. Sabatini (New York University School of Medicine), Ben Zion Shilo (Weizmann Institute of Science, Rehovot, Israel).

Résumé

Ce sixième colloque de biologie a exploré les incidences réciproques de la biologie moléculaire et de la médecine ; comment la biologie moléculaire peut-elle aider à résoudre des problèmes médicaux et comment de son côté la médecine peut-elle faire avancer les études de problèmes biologiques encore inexplorés?
Ont, entre autres, été abordées les questions suivantes : Quelles sont les occasions et les limites pour passer de la biologie d’une mouche ou d’une souris à celle de l’homme ? La thérapie génique est-elle un but réaliste ? Comment favoriser le dialogue nécessaire entre la biologie fondamentale et la médecine ?
Durant cette rencontre, les participants ont présenté des découvertes clefs dans leur discipline et ont tenté de la rapprocher de la biologie humaine.

Compte-rendu (en anglais)

In the last decade cell biology and molecular genetics have made impressive advances leading to considerable improvements on our understanding of how cells of unicellular and multicellular organisms reproduce, interact with each other and exchange information through signalling circuits.

These fundamental principles provide a conceptual and practical framework readily useful for biomedical research and development of diagnostic and/or therapeutical tools necessary to investigate a wide variety of required or genetically inherited human diseases.

The goal of this meeting, entitled “Molecular Medicine”, was to unravel some important topics along these lines of research. In September 1992, al “Les Treilles” sixteen leading cell and molecular biologists involved in biomedically relevant basic biological research presented the work carried out in their laboratories and discussed future directions of research avenues.

The topics discussed were broad and included the most recent research discoveries related to human diseases, cellular architecture, signal transduction, cell adhesion and cellular communication(s). Biologists working in these area are able to provide the information required to carry out innovative biomedical research in the fields of parasitology, human cancers, developmental defects and immunological defence mechanisms.

Included were presentations by Daniel Louvard and Eric Pringault who discussed the role of an actin binding protein named villin. Villin is a key player during the assembly of intestinal microvilli when these epithelial cells differentiate. These authors showed that villin is necessary and sufficient to induce morphogenetic events leading to the final shape of intestinal cells.

In addition, they discussed the use of the promoter of the gene encoding for villin (only expressed in a few epithelial cells) as a tool to target oncogenes in transgenic mice. It is hoped that this strategy could lead the immortalization of intestinal cells. These cells may ultimately be put into cell culture for in vitro studies.

Alternatively, the transgenic animals expressing the relevant oncogene(s) ma y be used as animal models for further studies on human colorectal cancers and for pharmacological studies aimed at development of reagents and procedures with applications in the treatment of this cancer.

Laurent Degos and Hugues de The discussed the action of transretinoïc acid (RA) on a rare myelogeneous malignant proliferation called acute promyelocytic leukemia (APL) characterized by a severe bleeding diasthesis and a poor prognosis. Eradication of malignant clones can be accomplished when patients are treated orally with trans-RA. This is due to a progressive differentiation of blast cells into granulocytes that eventually die. The beneficial effect of the “differentiation therapy” is probably due to the induction of the natural death of the malignant cell. A molecular basis of this leukemia may be explained by the fact that an allele of the retinoic acid receptor (RAR) is translocated and fused with a new gene, PML, found in every patient tested. This chimeric protein displays structural and functional features of a transcription factor, it may interfere with either normal RAR or PML function. A functionally altered receptor could thus antagonize cell maturation and lead to transformation.

Christine Petit talked about a gene responsible for Kallmann syndrome (Ks) which is linked to the X chromosome (KA1 gene). Ks is defined by the association of hypogonadotropic hypogonadism with anosmia (deficiency of the sense of smell). Its incidence is about one in 10000 males. Hypogonadism in Kallmann patients is due to a deficiency of hypothalamic gonadotropin releasing hormone (GnRH), while anosmia has been described to agenesis or hypoplasia of the olfactory bulbs. The expression of the gene responsible for this disease correlates with the defects observed in the reproductive and the olfactory systems. A complete cDNA has been isolated. Detection of mutations within this gene in KS patients has provided convincing evidence that the isolated gene is the actual KAL gene. Based on physiopathological data, a role for the KAL protein has been proposed in the navigation of the olfactory axons towards the brain and also in the migration of GnRH neurons from the olfactory epithelium to the developing forebrain. The KAL protein, as predicted from the deduced amino acid sequence, is likely to be an extracellular matrix component with antiprotease and adhesion functions. Such functions are known to be involved in axonal outgrowth and neuronal migration as well as in synaptogenesis. The spatiotemporal expression of the gene has been studied and led this group to propose that the KAL protein could be involved in targeting of some neuronal connections or in consolidation of synapses.

Elaine Fuchs presented data demonstrating that several human genetic diseases characterized by severe defects of skin (epidermolysis bulbeous or hypokeratolysis) are due to mutations in conserved structural proteins found in keratinocytes, the keratins. In vitro studies took advantage of cultured keratinocytes into which a defective keratin gene was introduced. Moreover, in vivo experiments have been carried out with transgenic mice. These studies led to the direct demonstration that truncated keratins or keratin mutants can exert a dominant negative effects on the normal function of keratin(s), as the building blocks of intermediate filaments, and produce defects mimicking the naturally occurring physiopathological states. The cellular defects can be attributed to improper assembly of intermediate filaments when a variant keratin protein is synthesized. Cells expressing the mutant demonstrate a lack of mechanical resistance which can account for the physiopathological defects observed. When patients with such diseases are studied, the genes encoding for keratins 5, 10 and 14 displayed conserved mutations at key positions that can affect severely the function of the encoded protein. Thus, the origin of several skin diseases can be explained by a mutation or mutations found in genes encoding for structural proteins such as the keratin genes.

Jenefer Blackwell talked about genetic resistance to Leishmania leprosium and tuberculosis. Much of their work on the macrophage resistance gene, first identified in mice, attempts to elucidate its role in determining resistance to Leishmania donovani and Salmonella typhimurium, and as a regulator of mycobacterial infections. The gene is known to regulate the priming and activation of macrophages for enhanced TNF a release and killing via nitrogen intermediates. TNF α mediates changes in early gene expression within minutes of macrophage stimulation via integrin receptors. The gene product has not been yet identified. Work is now in progress to clone the gene on the basis of its map location using yeast artificial chromosomes. Additional studies have been undertaken to identify an LSH gene homologue and in determining resistance and susceptibility to leishmania and mycobacterial infections in man using linkage analysis.

Fotis Kafatos presented the first trials carried out by his group toward obtaining a physical map of genetic markers for the mosquito genome. He stressed the fact that very little has been done to understand the genomic organisation of these animals. This is in sharp contrast with the fly, Drosophila, for which more than 90 % of the genome has been mapped. Mosquitoes are responsible for the contamination of about 300 million individuals and the death of 2 million children per year. Mosquitoes are found all over the world, they are very diverse and can adapt rapidly to adverse external stresses. For instance, mosquitoes have become highly resistant to DTT and (Chloroquine within a few decades after the first use of these chemicals.
Clearly a systematic analysis of the molecular basis of their resistance and adaptability is urgently required.

Christian Pinset presented evidence that supports the notion that myogenesis results from the convergence of several processes involving cell cycle control, signalling occurring through growth factors and hormones and activation of the muscle regulatory factors of the MyoD Family. Further characterization of these processes, the search for possible connections and the elucidation of the function of each member of the MyoD family should contribute to our understanding of commitment to cell fate and cellular differentiation.
IGFs are known to exert a positive influence on differentiation of determined muscle cells. A mouse myoblasts cell-line which is not autonomous for differentiation has been developed. These inducible cells fail to synthesize and secrete IGFs and do not express MyoD at the myoblast stage. These cells require exogenous IGFs to undergo terminal differentiation. Inhibition of autocrine production in permissive myoblasts using a vector producing anti-sense complementary to IGF 1 and IGF 2 mRNAs, leads to a decreased expression of MyoD and the loss of autonomous differentiation.
Expression patterns observed in embryos and in tissue culture, favor the hypothesis that the four myogenic regulatory factors of the myoD family fulfill distinct roles and do not simply serve redundant functions.

Jean-Pierre Kraehenbuhl presented original work on a project aiming to design a mucosal vaccine against retroviruses. Immune protection of mucosal surfaces is, in part, mediated by secretory IgA (slgA) antibodies which crosslink microorganisms and prevent invasion and infection through epithelial barriers. The goal is to determine whether slgA alone can protect against a retro virus that infects the host via mucosal surfaces. Mouse mammary tumor virus (MMTV) is shed into the mille of infected mothers, enters the intestine of suckling animals and is transcytosed by M cells into the Peyer’s patch mucosa. The virus then infects the lymphocytes in mucosal lymphoid tissue causing a rapid proliferation of CD4+T lymphocytes that express T cell receptors with distinct VIS elements. Stimulation is followed by the deletion of this T cell subset from the repertoire, and the systemic spread and infection of the mammary gland epithelial cells. Tumor formation is due to insertional mutagenesis and typical adenocarcinomas appear between 6 and 10 months of age depending of the virus strain. Recently, an MMTV variant (MMTV SW) which exhibits strong superantigen activity when administrated orally to newborn mice has been characterized. An adult infectivity model is now developed by infection of 2 months old females via the rectal or the genital route. The viral life cycle allows intervention at three discrete steps: 1) prevention of uptake in the gut, 2) inhibition of superantigen function, 3) prevention of infection to mammary gland. If the rectal and/or genital infection can easily be monitored it will be possible to rectally administer live vaccines expressing the relevant epitopes.

David Sabatini discussed some structural and functional features of the establishment and maintenance of epithelial cell polarity.
Polarized epithelial cells are capable of incorporating distinct sets of membrane proteins into their apical and basolateral plasma membrane domains. In particular, in virally infected kidney cells (the MDCK cell-line), glycoproteins destined to opposite plasma membrane domains are sorted in the trans-Golgi network (TGN) to be incorporated into different vesicles which are directly delivered to the cell surface domain where each protein accumulates. The process of vesicle formation in the TGN and of vesicle targeting to the cell surface has been studied using several different experimental Systems. Firstly, the toxin Streptolysin O was used to selectively permeabilize either the apical or basolateral membrane of MDCK cells thus permitting the examination of the effects of impermeant reagents on membrane protein transport to the intact surface. Studies with this system indicate that GTP-binding proteins are involved in a constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged with GTP early in this process, probably during vesicle formation. Vesicle formation in an in vitro system using a Golgi fraction from VSV or Influenza virus- infected cells bas been achieved. Delivery from the Golgi apparatus to the plasma membrane was studied employing a purified immobilized basolateral plasma membrane fraction from MDCK monolayers.

Ben Zion Shilo presented experiments illustrating the research of his group which has been involved, in particular, on the role of receptor tyrosine kinases during Drosophila embryogenesis. Cell fates during embryogenesis are determined, in part, by dynamic cell-cell interactions. Receptor tyrosine kinases were shown to play a pivotal role in these processes. The role of two receptors were dissected by genetic analysis. DER (the EGF receptor homolog) appears to be essential for establishment of ventral polarity in the ectoderm. Future experiments will test whether the receptor has the capacity to induce ventral identity or whether it is only necessary for blocking the expansion of the dorsalizing pathway. A second receptor (DFGF-RI) which was discussed is an FGF receptor homolog. This receptor is essential for the migration of the tracheal cells and specific midline glial cells, but not for determination of cell identity. Experiments to test the precise role of the receptor in the migration process were described. Tracheal migration may provide a useful model system with possible implications for such processes as vasculogenesis in vertebrate organisms.

Benjamin Geiger talked about the molecular mechanism of cell adhesion. Cell adhesion to the underlying extracellular matrix as well as to neighboring cells, occurs through specialized structures known as cell junctions. Of particular importance to morphogenesis are the adherens-type junctions (AJ) which are associated with the actin microfilament system.
Studies were presented indicating adhesion to both cellular and non-cellular surfaces. This adhesion involves in a specialized set of proteins found in membrane systems (cadherins or integrins respectively), in actin and actin-associated proteins as well as interconnecting membrane associated molecules such as vinculin. In addition, adherens junctions contain regulatory molecules which control the extent of cytoskeletal anchorage and its level of adhesion. In the lecture the kinetic properties of adherens junctions were discussed as well as the cellular mechanisms responsible for its modulation. These included controlled expression of the various junctional constituents and the phosphorylation on tyrosyl residues.

Raymond Erikson presented work on the conversion of extracellular signals to serine/threonine reversible phosphorylation signals. A number of events at the plasma membrane, including activation of growth function protein tyrosine kinases, stimulation of protein kinase C or the presence of oncogene products such as pp60V-src or p21ras, lead to increase of serine/threonine phosphorylation of diverse cellular proteins. These include abundant proteins such as histone H1 and ribosomal protein S6 as well as numerous less abundant enzymes or transcription factors. These phosphorylated proteins are likely to contribute to cell proliferation and differentiation.
Studies on the regulation of protein kinases that lead to the phosphorylation of the ribosomal protein S6 were discussed. Two S6 protein kinases have been cloned. One of these, denoted as pp90rsk, is activated by serine phosphorylation but inactivated by a serine-specific protein phosphatase. A protein kinase that is practically responsible for pp90rsk phosphorylation is encoded by the ERK gene product. The ERK gene encodes a protein kinase previously identified as the MAP kinase (also called mBP kinase). The MAP kinase is activated by threonine/tyrosine phosphorylation. A multifunctional threonine/tyrosine protein kinase that phosphorylates the ERK gene product on threonine and tyrosine resulting in activation of its MAP/MMBP kinase activity, has been fully characterized. This enzyme was named MEK (for MAPkinase ERK kinase). The sequence of the cDNA corresponding to the mRNA for MEK predicts a protein kinase very similar to a yeast gene product. Altogether, studies on animal cells and yeast suggest conserved signal transduction pathways throughout evolution.

Spyros Artavanis-Tsakonas talked about molecules, expressed by embryonic cells, that control their cell to cell interactions and differentiation pathways toward coherently-organized tissues.
The manner in which two initially equivalent neighboring cells choose different developmental fates is of fundamental importance to the developing organism, since such fine-tuning will provide the final repertoire of cell types and dictate the position of each cell in a developing organ. In Drosophila melanogaster, analysis of a broad spectrum of mutant phenotypes and molecular analysis of the corresponding genes, have been used to show that neighboring cells depend on the parallel and probably independent action of a general cell interaction mechanism which is shared by many different tissues and more specific cell interaction events which are tissue specific. The so-called « Notch group” of genes have been implicated in a more general mechanism which is crucial for the correct developmental choices in a wide variety of precursor cells in Drosophila. The accumulated genetic and molecular studies suggest that these genes encode elements of a cell communication mechanism which includes cell surface, cytoplasmic, and nuclear components. The central player of the Notch group is the Notch (N) locus which encodes a transmembrane protein containing EGF-like repeats in its extracellular domain. There is evidence to suggest that this protein may act as a multifunctional receptor which interacts molecularly and genetically with Serrate and Delta, two other transmembrane, EGF-containing proteins of the Notch group. The other members of the Notch group are Deltex, Enhancer of split E(spl) and Mastermind (mam). Deltex seems to code for a cytoplasmic protein while Mastermind and Enhancer of split encode nuclear proteins. Evidence has been gathered which indicate that the Notch-mediated mechanism is operating in humans. This is a particularly exciting development since it appears that the fly paradigm can be applied to vertebrate systems. Cell fate choice abnormalities are predicted to cause a very broad spectrum of pathological conditions in vertebrates including various forms of cancers.

François Jacob discussed fascinating findings made by molecular biologists all over the world. He stressed the notion that tinkering strategies that occured during evolution have led to families of proteins made of multiple functional domains. As a result, it is possible to identify a new protein as a new member of a given group of proteins whose function(s) may be already known. However, a given motive may also be scattered in numerous families. It emerges that the number of sequence motives is large but not infinite. Also functional diversity between proteins contributes to unique functions that are obtained by combinatory events and by critical association of protein domains.

Daniel Louvard

Communications / Talks

• Spyros Artavanis-Tsakonas: Choosing a cell fate
• Hugues Blaudin de Thé: Acute leukemia, the retinoic acid paradox
• Jenefer Blackwell: Lsh/Ity/Bcg – a gene controlling macrophage priming/activation
• Laurent Degos: Differentiation therapy of leukemia
• Raymond L. Erikson: Reversible protein phosphorylation and. the cell cycle
• Elaine Fuchs: Of mice and men: Genetic disorders of keratin
• Benjamin Geiger: Molecular architecture of cell adhesions
• François Jacob: Biology and development
• Fotis Kafatos: From Drosophila to mosquitoes: Molecular genetic studies of the malaria vector
• Jean-Pierre Kraehenbuhl: How to design mucosal vaccines against pathogens that invade the host via mucosal surfaces
• Daniel Louvard: Molecular basis of cellular morphogenesis
• Christine Petit: Isolation and characterization of the gene responsible for Kallmann Syndrome
• Christian Pinset: Role of the myogenic regulatory factors of the MyoD family in determination, differentiation and maturation of skeletal muscle cells
• Eric Pringault: Experimental colorectal carcinogenesis
• David Sabatini: The biogenesis of epithelial cell polarity
• Ben Zion Shilo: The roles of receptor tyrosine kinases in Drosophila embryogenesis

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