Sphingomyelin nanosystems decorated with TSP-1 derived peptide targeting senescent cells.

Senescent cells accumulation can contribute to the development of several age-related diseases, including cancer. Targeting and eliminating senescence cells, would allow the development of new therapeutic approaches for the treatment of different diseases. The 4N1Ks peptide, a 10 amino acid peptide derived from TSP1 protein, combines both features by targeting the CD47 receptor present in the surface of senescent cells and demonstrating senolytic activity, thereby representing a new strategy to take into account. Nonetheless, peptide drugs are known for their biopharmaceutical issues, such as low short half-life and tendency to aggregate, which reduces their bioavailability and limits their therapeutic potential. In order to overcome this problem, herein we propose the use of biodegradable and biocompatible sphingomyelin nanosystems (SNs), decorated with this peptide for the targeting of senescent cells. In order to efficiently associate the 4N1Ks peptide to the nanosystems while exposing it on their surface for an effective targeting of senescent cells, the 4N1Ks peptide was chemically conjugated to a PEGylated hydrophobic chain. The resulting SNs-4N1Ks (SNs-Ks), were extensively characterized for their physicochemical properties, by dynamic light scattering, multiple-angle dynamic light scattering, nanoparticle tracking analysis and atomic force microscopy. The SNs-Ks demonstrated suitable features in terms of size (∼100 nm), association efficiency (87.2 ± 6.9%) and stability in different biorelevant media. Cell toxicity experiments in MCF7 cancer cells indicated an improved cytotoxic effect of SNs-Ks, decreasing cancer cells capacity to form colonies, with respect to free peptide, and an improved hemocompatibility. Lastly, senescence escape preliminary experiments demonstrated the improvement of SNs-Ks senolytic activity of in chemotherapy-induced senescence model of breast cancer cells. Therefore, these results demonstrate for the first time the potential of the combination of SNs with 4N1Ks peptide for the development of innovative senolytic therapies to battle cancer.

Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy­induced senescence escape.

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy­induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH­MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH­MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

O-GlcNAcylation Links Nutrition to the Epigenetic Downregulation of UNC5A during Colon Carcinogenesis.

While it is now accepted that nutrition can influence the epigenetic modifications occurring in colorectal cancer (CRC), the underlying mechanisms are not fully understood. Among the tumor suppressor genes frequently epigenetically downregulated in CRC, the four related genes of the UNC5 family: UNC5A, UNC5B, UNC5C and UNC5D encode dependence receptors that regulate the apoptosis/survival balance. Herein, in a mouse model of CRC, we found that the expression of UNC5A, UNC5B and UNC5C was diminished in tumors but only in mice subjected to a High Carbohydrate Diet (HCD) thus linking nutrition to their repression in CRC. O-GlcNAcylation is a nutritional sensor which has enhanced levels in CRC and regulates many cellular processes amongst epigenetics. We then investigated the putative involvement of O-GlcNAcylation in the epigenetic downregulation of the UNC5 family members. By a combination of pharmacological inhibition and RNA interference approaches coupled to RT-qPCR (Reverse Transcription-quantitative Polymerase Chain Reaction) analyses, promoter luciferase assay and CUT&RUN (Cleavage Under Target & Release Using Nuclease) experiments, we demonstrated that the O-GlcNAcylated form of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2) represses the transcription of UNC5A in human colon cancer cells. Collectively, our data support the hypothesis that O-GlcNAcylation could represent one link between nutrition and epigenetic downregulation of key tumor suppressor genes governing colon carcinogenesis including UNC5A.

Chemotherapy-induced senescence, an adaptive mechanism driving resistance and tumor heterogeneity.

Senescence is activated in response to chemotherapy to prevent the propagation of cancer cells. In transformed cells, recent studies have shown that this response is not always definitive and that persistent populations can use senescence as an adaptive pathway to restart proliferation and become more aggressive. Here we discuss the results showing that an incomplete and heterogeneous senescence response plays a key role in chemotherapy resistance. Surviving to successive chemotherapy regimens, chronically existing senescent cells can create a survival niche through paracrine cooperations with neighboring cells. This favors chemotherapy escape of premalignant clones but might also allow the survival of adjacent clones presenting a lower fitness. A better characterization of senescence heterogeneity in transformed cells is therefore necessary. This will help us to understand this incomplete response to therapy and how it could generate clones with increased tumor capacity leading to disease relapse.

Proteomics Approaches to Define Senescence Heterogeneity and Chemotherapy Response.

In primary cells, senescence induces a permanent proliferative arrest to prevent the propagation of malignant cells. However, the outcome of senescence is more complex in advanced cancer cells where senescent states are heterogeneous. Here, this heterogeneity is discussed and it is proposed that proteomic analysis should be used to identify specific signatures of cancer cells that use this pathway as an adaptive mechanism. Since senescent cells produce an inflammatory secretome, MRM approaches and quantification with internal standards might be particularly suited to follow the expression of the corresponding markers in body fluids. Used in combination with imaging medical technics, a better characterization of senescence heterogeneity should help to monitor the response to chemotherapy treatment.

Regulation of senescence escape by TSP1 and CD47 following chemotherapy treatment.

Senescence is a tumor-suppressive mechanism induced by telomere shortening, oncogenes, or chemotherapy treatment. Although it is clear that this suppressive pathway leads to a permanent arrest in primary cells, this might not be the case in cancer cells that have inactivated their suppressive pathways. We have recently shown that subpopulations of cells can escape chemotherapy-mediated senescence and emerge as more transformed cells that induce tumor formation, resist anoikis, and are more invasive. In this study, we characterized this emergence and showed that senescent cells favor tumor growth and metastasis, in vitro and in vivo. Senescence escape was regulated by secreted proteins produced during emergence. Among these, we identified thrombospondin-1 (TSP1), a protein produced by senescent cells that prevented senescence escape. Using SWATH quantitative proteomic analysis, we found that TSP1 can be detected in the serum of patients suffering from triple-negative breast cancer and that its low expression was associated with treatment failure. The results also indicate that senescence escape is explained by the emergence of CD47low cells that express a reduced level of CD47, the TSP1 receptor. The results show that CD47 expression is regulated by p21waf1. The cell cycle inhibitor was sufficient to maintain senescence since its downregulation in senescent cells increased cell emergence. This leads to the upregulation of Myc, which then binds to the CD47 promoter to repress its expression, allowing the generation of CD47low cells that escape the suppressive arrest. Altogether, these results uncovered a new function for TSP1 and CD47 in the control of chemotherapy-mediated senescence.

Regulation of senescence escape by the cdk4-EZH2-AP2M1 pathway in response to chemotherapy.

Senescence is a tumor suppressive mechanism that induces a permanent proliferative arrest in response to an oncogenic insult or to the genotoxic stress induced by chemotherapy. We have recently described that some cells can escape this arrest, either because senescence was incomplete or as a consequence of a phenotypic adaptation. Malignant cells which resisted senescence emerged as more transformed cells that resist anoikis and rely on survival pathways activated by Akt and Mcl-1. In this study, we further characterize senescence escape, investigating how emergent cells could reproliferate. During the initial step of chemotherapy-induced senescence (CIS), we found that cyclin D1 was upregulated and that cell emergence was prevented when its main partner cdk4 was inactivated. Results indicate that this kinase induced the upregulation of the EZH2 methylase, a component of the polycomb PRC2 complex. Downregulated during the early step of treatment, the methylase was reactivated in clones that escaped senescence. The inactivation of EZH2, either by siRNA or by specific inhibitors, led to a specific inhibition of cell emergence. We used quantitative proteomic analysis to identify new targets of the methylase involved in senescence escape. We identified proteins involved in receptor endocytosis and described new functions for the AP2M1 protein in the control of chemotherapy-mediated senescence. Our results indicate that AP2M1 is involved in the transmission of secreted signals produced by senescent cells, suggesting that this pathway might regulate specific receptors involved in the control of CIS escape. In light of these results, we therefore propose that the cdk4-EZH2-AP2M1 pathway plays an important role during chemotherapy resistance and senescence escape. Since targeted therapies are available against these proteins, we propose that they should be tested in the treatment of colorectal or breast cancers that become resistant to first-line genotoxic therapies.

The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production.

AIMS: The angiotensin II type 1 receptor (AT1R) through the activation of immune cells plays a key role in arterial inward remodelling and reduced blood flow in cardiovascular disorders. On the other side, flow (shear stress)-mediated outward remodelling (FMR), involved in collateral arteries growth in ischaemic diseases, allows revascularization. We hypothesized that the type 2 receptor (AT2R), described as opposing the effects of AT1R, could be involved in FMR. METHODS AND RESULTS: We studied FMR using a model of ligation of feed arteries supplying collateral pathways in the mouse mesenteric arterial bed in vivo. Seven days after ligation, diameter increased by 30% in high flow (HF) arteries compared with normal flow vessels. FMR was absent in mice lacking AT2R. At Day 2, T lymphocytes expressing AT2R were present preferentially around HF arteries. FMR did not occur in athymic (nude) mice lacking T cells and in mice treated with anti-CD3ε antibodies. AT2R activation induced interleukin-17 production by memory T cells. Treatment of nude mice or AT2R-deficient mice with interleukin-17 restored diameter enlargement in HF arteries. Interleukin-17 increased NO-dependent relaxation and matrix metalloproteinases activity, both important in FMR. Remodelling of feeding arteries in the skin flap model of ischaemia was also absent in AT2R-deficient mice and in anti-interleukin-17-treated mice. Finally, remodelling, absent in 12-month-old mice, was restored by a treatment with the AT2R non-peptidic agonist C21. CONCLUSION: AT2R-dependent interleukin-17 production by T lymphocyte is necessary for collateral artery growth and could represent a new therapeutic target in ischaemic disorders.

Long Lasting Microvascular Tone Alteration in Rat Offspring Exposed In Utero to Maternal Hyperglycaemia.

Epidemiologic studies have demonstrated that cardiovascular risk is not only determined by conventional risk factors in adulthood, but also by early life events which may reprogram vascular function. To evaluate the effect of maternal diabetes on fetal programming of vascular tone in offspring and its evolution during adulthood, we investigated vascular reactivity of third order mesenteric arteries from diabetic mother offspring (DMO) and control mother offspring (CMO) aged 3 and 18 months. In arteries isolated from DMO the relaxation induced by prostacyclin analogues was reduced in both 3- and 18-month old animals although endothelium (acetylcholine)-mediated relaxation was reduced in 18-month old DMO only. Endothelium-independent (sodium nitroprusside) relaxation was not affected. Pressure-induced myogenic tone, which controls local blood flow, was reduced in 18-month old CMO compared to 3-month old CMO. Interestingly, myogenic tone was maintained at a high level in 18-month old DMO even though agonist-induced vasoconstriction was not altered. These perturbations, in 18-months old DMO rats, were associated with an increased pMLC/MLC, pPKA/PKA ratio and an activated RhoA protein. Thus, we highlighted perturbations in the reactivity of resistance mesenteric arteries in DMO, at as early as 3 months of age, followed by the maintenance of high myogenic tone in older rats. These modifications are in favour of excessive vasoconstrictor tone. These results evidenced a fetal programming of vascular functions of resistance arteries in adult rats exposed in utero to maternal diabetes, which could explain a re-setting of vascular functions and, at least in part, the occurrence of hypertension later in life.

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco.

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.

Overexpression of a Medicago truncatula stress-associated protein gene (MtSAP1) leads to nitric oxide accumulation and confers osmotic and salt stress tolerance in transgenic tobacco.

The impact of Medicago truncatula stress-associated protein gene (MtSAP1) overexpression has been investigated in Nicotiana tabacum transgenic seedlings. Under optimal conditions, transgenic lines overexpressing MtSAP1 revealed better plant development and higher chlorophyll content as compared to wild type seedlings. Interestingly, transgenic lines showed a stronger accumulation of nitric oxide (NO), a signaling molecule involved in growth and development processes. This NO production seemed to be partially nitrate reductase dependent. Due to the fact that NO has been also reported to play a role in tolerance acquisition of plants to abiotic stresses, the responses of MtSAP1 overexpressors to osmotic and salt stress have been studied. Compared to the wild type, transgenic lines were less affected in their growth and development. Moreover, NO content in MtSAP1 overexpressors was always higher than that detected in wild seedlings under stress conditions. It seems that this better tolerance induced by MtSAP1 overexpression could be associated with this higher NO production that would enable seedlings to reach a high protection level to prepare them to cope with abiotic stresses.

A stress-associated protein containing A20/AN1 zing-finger domains expressed in Medicago truncatula seeds.

MtSAP1 (Medicago truncatula stress-associated protein 1) was revealed as a down-regulated gene by suppressive subtractive hybridization between two mRNA populations of embryo axes harvested before and after radicle emergence. MtSAP1 is the first gene encoding a SAP with A20 and AN1 zinc-finger domains characterized in M. truncatula. MtSAP1 protein shares 54% and 62% homology with AtSAP7 (Arabidopsis thaliana) and OsiSAP8 (Oryza sativa) respectively, with in particular a strong homology in the A20 and AN1 conserved domains. MtSAP1 gene expression increased in the embryos during the acquisition of tolerance to desiccation, reached its maximum in dry seed and decreased dramatically during the first hours of imbibition. Abiotic stresses (cold and hypoxia), abscisic acid and desiccation treatments induced MtSAP1 gene expression and protein accumulation in embryo axis, while mild drought stress did not affect significantly its expression. This profile of expression along with the presence of anaerobic response elements and ABRE sequences in the upstream region of the gene is consistent with a role of MtSAP1 in the tolerance of low oxygen availability and desiccation during late stages of seed maturation. Silencing of MtSAP1 by RNA interference (RNAi) showed that the function of the encoded protein is required for adequate accumulation of storage globulin proteins, vicilin and legumin, and for the development of embryos able to achieve successful germination.

ABA-mediated inhibition of germination is related to the inhibition of genes encoding cell-wall biosynthetic and architecture: modifying enzymes and structural proteins in Medicago truncatula embryo axis.

Radicle emergence and reserves mobilization are two distinct programmes that are thought to control germination. Both programs are influenced by abscissic acid (ABA) but how this hormone controls seed germination is still poorly known. Phenotypic and microscopic observations of the embryo axis of Medicago truncatula during germination in mitotic inhibition condition triggered by 10 microM oryzalin showed that cell division was not required to allow radicle emergence. A suppressive subtractive hybridization showed that more than 10% of up-regulated genes in the embryo axis encoded proteins related to cell-wall biosynthesis. The expression of alpha-expansins, pectin-esterase, xylogucan-endotransglycosidase, cellulose synthase, and extensins was monitored in the embryo axis of seeds germinated on water, constant and transitory ABA. These genes were overexpressed before completion of germination in the control and strongly inhibited by ABA. The expression was re-established in the ABA transitory-treatment after the seeds were transferred back on water and proceeded to germination. This proves these genes as contributors to the completion of germination and strengthen the idea that cell-wall loosening and remodeling in relation to cell expansion in the embryo axis is a determinant feature in germination. Our results also showed that ABA controls germination through the control of radicle emergence, namely by inhibiting cell-wall loosening and expansion.

A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination.

A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.

Recruitment of STAT3 for production of IL-10 by colon carcinoma cells induced by macrophage-derived IL-6.

The immunosuppressive cytokine IL-10 is associated with poor prognosis in colon cancer. Although macrophages are involved in antitumor defenses, production of IL-10 by tumor cells may permit malignant cells escape to cell-mediated immune defenses. To investigate interactions between macrophages and tumor cells in humans, we cultured macrophages isolated from patients and tested the effect of these macrophages on the production of IL-10 by several tumor cell lines. Macrophages were isolated from pleural effusions of patients with malignancy and from noncancer control patients. We demonstrated that culture supernatants of macrophages from both sources strongly stimulated IL-10 production by the three different human colon adenocarcinoma cell lines, Colo 205, Colo 320, and HT29. Recombinant IL-6, but not IL-10, TNF-alpha, and IFN-alpha, stimulated the secretion of IL-10 by colon tumor cells. mAbs against IL-6 and IL-6R prevented the effect of macrophage culture supernatants and of rIL-6, respectively, on the production of IL-10 by the three cell lines. Cocultures of macrophages and colon cancer cells showed that these tumor cells first stimulated macrophages to produce IL-6, which was then followed by IL-6-induced IL-10 production by colon cancer cells. Finally, we showed that IL-10 gene regulation was mediated by STAT3, which was phosphorylated after the binding of IL-6 to IL-6R. This is the first demonstration that IL-6, secreted by macrophages, can induce a STAT3-mediated IL-10 production by colon tumor cells.

GPL, a novel cytokine receptor related to GP130 and leukemia inhibitory factor receptor.

We describe a novel cytokine receptor named GP130 Like receptor, or GPL, that displays similarities with the interleukin-6 and interleukin-12 family of signaling receptors. Four different isoforms diverging in their carboxyl terminus were isolated, corresponding to proteins encompassing 560, 610, 626, and 745 amino acids. Sequences included a signal peptide of 32 amino acids, followed by a cytokine binding domain containing four conserved cysteines, a WSDWS motif, and a region consisting of three fibronectin type III domain repeats. No immunoglobulin-like module was identified in the GPL sequences. The intracellular part of longer isoforms contained a proline-rich region defining a box1 motif for interaction with the Janus kinases. The Gpl gene is organized in 15 exons and is located on 5q11.2 in tandem with the gp130 gene. Both genes were only separated by 24 kilobases, with opposite transcriptional orientations. The GPL receptor displayed a 28% identity with gp130. Specific GPL transcripts were observed in tissues involved in reproduction. Transcripts were also found in blood cells and in bone marrow, revealing expression of GPL in all of the myelomonocytic lineage, from hematopoietic stem cells to activated dendritic cells. In monocytes and dendritic cells, expression of GPL was strongly up-regulated by interferon-gamma, indicating a possible involvement of GPL in Th1-type immune responses. The molecular basis of cell signaling mediated by GPL was studied using chimeric receptors where external portions of alpha or beta interleukin-5 receptor subunits were fused to the internal portion of GPL or of related receptors. Results indicated that association of GPL to the intracellular portions of gp130, or LIF receptor, allowed the signaling cascade.

Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling.

The extracellular protein Spätzle is required for activation of the Toll signaling pathway in the embryonic development and innate immune defense of Drosophila. Spätzle is synthesized as a pro-protein and is processed to a functional form by a serine protease. We show here that the mature form of Spätzle triggers a Toll-dependent immune response after injection into the hemolymph of flies. Spätzle specifically bound to Drosophila cells and to Cos-7 cells expressing Toll. Furthermore, in vitro experiments showed that the mature form of Spätzle bound to the Toll ectodomain with high affinity and with a stoichiometry of one Spätzle dimer to two receptors. The Spätzle pro-protein was inactive in all these assays, indicating that the pro-domain sequence, which is natively unstructured, acts to prevent interaction of the cytokine and its receptor Toll. These results show that, in contrast to the human Toll-like receptors, Drosophila Toll requires only an endogenous protein ligand for activation and signaling.

Leukemia inhibitory factor (LIF), cardiotrophin-1, and oncostatin M share structural binding determinants in the immunoglobulin-like domain of LIF receptor.

Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and oncostatin M (OSM) are four helix bundle cytokines acting through a common heterodimeric receptor composed of gp130 and LIF receptor (LIFR). Binding to LIFR occurs through a binding site characterized by an FXXK motif located at the N terminus of helix D (site III). The immunoglobulin (Ig)-like domain of LIFR was modeled, and the physico-chemical properties of its Connolly surface were analyzed. This analysis revealed an area displaying properties complementary to those of the LIF site III. Two residues of the Ig-like domain of LIFR, Asp214 and Phe284, formed a mirror image of the FXXK motif. Engineered LIFR mutants in which either or both of these two residues were mutated to alanine were transfected in Ba/F3 cells already containing gp130. The F284A mutation impaired the biological response induced by LIF and CT-1, whereas the response to OSM remained unchanged. The Asp214 mutation did not alter the functional responses. The D214A/F284A double mutation, however, totally impaired cellular proliferation to LIF and CT-1 and partially impaired OSM-induced proliferation with a 20-fold increase in EC50. These results were corroborated by the analysis of STAT3 phosphorylation and Scatchard analysis of cytokine binding to Ba/F3 cells. Molecular modeling of the complex of LIF with the Ig-like domain of LIFR provides a clue for the superadditivity of the D214A/F284A double mutation. Our results indicate that LIF, CT-1, and OSM share an overlapping binding site located in the Ig-like domain of LIFR. The different behaviors of LIF and CT-1, on one side, and of OSM, on the other side, can be related to the different affinity of their site III for LIFR.

Functionally active fusion protein of the novel composite cytokine CLC/soluble CNTF receptor.

The heterodimeric cytokine composed of the soluble ciliary neurotrophic factor receptor (sCNTFR) and the IL-6 family member cardiotrophin-like cytokine (CLC) was recently identified as a new ligand for gp130-leukemia inhibitory factor receptor (LIFR) complex [Plun-Favreau, H., Elson, G., Chabbert, M., Froger, J., deLapeyriere, O., Lelievre, E., Guillet, C., Hermann, J., Gauchat, J. F., Gascan, H. & Chevalier, S. (2001) EMBO J. 20, 1692-1703]. This heterodimer shows overlapping biological properties with LIF. Although CLC contains a putative signal peptide and therefore should enter into the classical secretory pathway, the protein has been shown to be retained within transfected mammalian cells, unless coexpressed with either sCNTFR or cytokine like factor (CLF) [Elson, G. C., Lelievre, E., Guillet, C., Chevalier, S., Plun-Favreau, H., Froger, J., Suard, I., de Coignac, A. B., Delneste, Y., Bonnefoy, J. Y., Gauchat, J. F. & Gascan, H. (2000) Nat. Neurosci. 3, 867-872]. In the present study, we demonstrate that a fusion protein comprising CLC covalently coupled through a glycine/serine linker to sCNTFR (CC-FP) is efficiently secreted from transfected mammalian cells. CC-FP shows enhanced activities in respect to the CLC/sCNTFR native complex, on a number of cells expressing gp130 and LIFR on their surface. In addition, CC-FP is able to compete with CNTF for cell binding, indicating that both cytokines share binding epitope(s) expressed by their receptor complex. Analysis of the downstream signaling events revealed the recruitment by CC-FP of the signal transducer and activator of transcription (STAT)-3, Akt and mitogen-activated protein (MAP) kinase pathways. The monomeric bioactive CLC/sCNTFR fusion protein is therefore a powerful tool to study the biological role of the recently described cytokine CLC.