Patrick Mehlen

DCC association with lipid rafts is required for netrin-1-mediated axon guidance

During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues.

Caspase cleavage of the transcription factor FLI-1 during preB leukemic cell death

Programmed cell death (apoptosis) is a complex phenomenon that is mediated in mammals mainly via the selective cleavage of intracellular proteins by the large family of cysteine aspartate protease caspases. Apoptosis is tightly regulated by the competitive effect of numerous proteins displaying either pro-apoptotic or anti-apoptotic activity. The ETS-family transcription factor FLI-1, frequently associated with malignant transformation, has been shown to display anti-apoptotic activity in several cell types including avian erythroblasts, mouse fibroblasts or lymphoid cells. We show here that apoptosis of murine preB leukemic cells is accompanied with the specific cleavage of FLI-1 by a caspase-like activity. We also demonstrate that the two isoforms of FLI-1 are indeed cleaved at three conserved sites by caspase 3 in vitro. The conservation of these cleavage sites among species suggests that the caspase cleavage of the anti-apoptotic transcription factor FLI-1 may represent a critical step to ensure irreversible cell death.

Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response

Background & Aims Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1s implication in cell survival on UPR in this context is unknown. Methods Isolation of translational complexes, determination of RNA secondary structures by selective 2’-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro–grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. Results HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA–like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5 untranslated region–driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C–mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. Conclusions UPR-resistant, internal ribosome entry site–driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1–mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.

Caprine arthritis-encephalitis virus induces apoptosis in infected cells in vitro through the intrinsic pathway

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus that causes natural inflammatory disease in goats, with chronic lesions in several different organs. CAEV infection of in vitro cultured cells is accompanied by apoptosis, but the involvement of the intrinsic and extrinsic pathways has not previously been elucidated. We have studied the activation of caspases-3, -8 and -9 by fluorescent assays in various goat cells infected in vitro by CAEV, and the effects of transfected dominant negative variants of theses caspases, to show that CAEV-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of mitochondrial pathway.

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway.

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.

Les récepteurs à dépendance DCC et UNC5H : rôle de l'apoptose dans le contrôle de la tumorigenèse

Des etudes recentes ont mis en relief un nouvel aspect des recepteurs cellulaires. Linteraction ligand-recepteur etait jusqua present consideree comme indispensable pour permettre lactivation du recepteur. Il a cependant ete suggere que certains de ces recepteurs, appeles « recepteurs a dependance », peuvent egalement etre actives en labsence de ligand et induire un signal specifique de mort cellulaire. Par consequent, lexpression dun ou plusieurs de ces recepteurs rend les cellules dependantes de la presence du ligand pour leur survie. Nous avons emis lhypothese que ce mecanisme permet dinhiber la croissance tumorale en conduisant a lapoptose les cellules «anormales» qui, en labsence de ligand, seraient normalement amenees a proliferer - croissance cellulaire locale ou proliferation a distance du site tumoral. Dans le meme ordre didee, au debut des annees 90, le groupe de Vogelstein a suggere quun gene, le gene DCC (pour deleted in colorectal cancer), pourrait jouer un role de suppresseur de tumeur car il etait retrouve delete dans plus de 70 % des cancers colorectaux et de nombreux autres cancers. Les donnees recueillies au cours des quinze dernieres annees nont pas permis dapaiser la controverse concernant limplication du gene DCC dans la suppression tumorale. Toutefois, notre equipe a pu montrer que DCC agit comme un recepteur a dependance qui induit la mort de la cellule lorsque son ligand, la netrine-1, est absent, et que des souris modifiees pour surexprimer la netrine-1 et bloquer ainsi lapoptose induite par le gene DCC etaient predisposees au developpement de cancers colorectaux. Ces donnees renforcent lhypothese selon laquelle les recepteurs a dependance auraient un role de suppresseurs de tumeurs. La presente revue decrit le role de linteraction netrine-1/recepteur comme nouveau mecanisme de controle du developpement tumoral.

Netrin PIPes up

Guidance molecules ensure that nerve cells grow in the correct direction during brain development. However, very little is known about the signals transmitted by these molecules. It now seems that netrin-1 — the first guidance molecule discovered — achieves this, in part, through activation of PITPα, a protein involved in lipid transfer.