Biomedical subjects

Sébastien Jeay

Publications and source records attributed to Sébastien Jeay.

6 recordsLinked to original sources

PubMed2004

The propeptide domain of lysyl oxidase induces phenotypic reversion of ras-transformed cells.

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. beta-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-kappaB activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.

Aminopropionitrile↗

PubMed2004

Reciprocal control of Forkhead box O 3a and c-Myc via the phosphatidylinositol 3-kinase pathway coordinately regulates p27Kip1 levels.

B cell receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in NF-kappa B and c-Myc, and induction of the p27(Kip1) cyclin-dependent kinase inhibitor, which promotes growth arrest and apoptosis. BCR engagement was recently shown to induce a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the increase in p27. As induction of p27 is due to an increase in gene transcription, we investigated the role of the Forkhead box O (FOXO) transcription factor family, which has been shown to potently induce p27 promoter activity. We demonstrate that pharmacologic inhibitors of PI3K or BCR engagement lead to decreased inactive cytoplasmic levels and increased active functional nuclear FOXO3a. In contrast, inhibition of PI3K/Akt signaling decreased the levels of NF-kappa B and c-Myc, which has been shown to repress p27 promoter activity. To test the effects of ectopic c-Myc on endogenous p27 levels, WEHI 231 cells stably expressing c-Myc or empty vector DNA were prepared. Ectopic c-Myc blocked the induction of p27 expression upon either inhibition of PI3K or BCR engagement. Thus, p27(Kip1) is coordinately regulated via two arms of a signaling pathway that are inversely controlled upon inhibition of PI3K: induction of the activator FOXO3a and down-regulation of the repressor c-Myc.

Animals↗

PubMed2003

B-Myb repressor function is regulated by cyclin A phosphorylation and sequences within the C-terminal domain.

B-Myb is a widely expressed member of the myb oncogene family that has been shown to act as either an activator or repressor of gene transcription in a cell-type-specific fashion. For example, in aortic smooth muscle cells B-Myb represses transcription of the alpha2(V) collagen gene. Recently, phosphorylation of B-Myb by cyclin A was shown to enhance greatly its ability to transactivate. Here, we have tested the effects of cyclin A on the ability of B-Myb to repress. We report that coexpression of cyclin A abolished repression of the alpha2(V) collagen promoter, whereas a dominant-negative cyclin-dependent kinase 2 (cdk2) enhanced repression by ectopic and endogenous B-Myb protein. Mutation of 10 of 22 putative cyclin A sites, which greatly reduces the effects of cyclin A on transactivation by B-Myb, had no effect on the ability of cyclin A to alleviate B-Myb-mediated repression of alpha2(V) collagen promoter activity. Furthermore, the stability of the mutant B-Myb protein was largely unaffected by cyclin A, although ectopic expression of cyclin A enhanced the rate of decay of wild-type B-Myb protein. Thus, the mechanisms of repression and activation appear distinct, for example, mediated by different critical phosphorylation sites or protein-protein interactions. B-Myb mutants with either deletion of aa 374-581 (B-Myb-Mut3) or C-terminal truncation beyond aa 491 (B-Myb-491) positively regulated alpha2(V) collagen promoter activity, and were not affected by cyclin A. Thus, our findings indicate that the ability of B-Myb to function as a repressor of matrix promoter activity is abolished by cyclin A, and maps the sites mediating negative regulation by B-Myb to the region between aa 491 and 582.

Animals↗

PubMed2003

Lysyl oxidase inhibits ras-mediated transformation by preventing activation of NF-kappa B.

Lysyl oxidase (LO), which catalyzes the oxidation of lysine residues, was previously shown to have anti-oncogenic activity on ras-transformed cells. Since oncogenic Ras mediates transformation, in part, through the activation of the transcription factor nuclear factor-kappa B (NF-kappa B), we tested here the effects of LO on NF-kappa B activity. Expression of LO in ras-transformed NIH 3T3 cells led to decreased NF-kappa B binding and activity, as well as the expression of the NF-kappa B target gene c-myc. Importantly, ectopic expression of LO led to a dramatic decrease in colony formation by ras-transformed NIH 3T3 cells, a finding comparable to the expression of the I kappa B alpha dominant-negative mutant, which could be rescued by p65/p50 NF-kappa B subunit expression. LO was unable to directly inhibit the activity of ectopically expressed p65 and c-Rel NF-kappa B subunits, suggesting that LO affected an upstream signaling pathway(s) induced by Ras. Consistent with this hypothesis, LO expression decreased both the rate of I kappa B alpha turnover and the activities of IKK alpha and IKK beta. Moreover, the ectopic expression of a constitutively active version of either kinase reversed the negative effects of LO. Ras can induce NF-kappa B via both the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK pathways. LO potently downregulated the PI3K and Akt kinases, while partially inhibiting MEK kinase activity. Expression of a constitutively activated, myristylated Akt or PDK1 was able to counteract the effect of LO on NF-kappa B, whereas constitutively activated Raf was only partially effective. Importantly, LO blocked membrane localization of Akt and PDK1 in Ras-transformed cells. Overall, these results strongly argue that the anti-oncogenic effects of LO on ras-mediated transformation are due to its ability to inhibit signaling pathways that lead to activation of NF-kappa B.

3T3 Cells↗

PubMed2002

Growth hormone can act as a cytokine controlling survival and proliferation of immune cells: new insights into signaling pathways.

While growth hormone (GH) is classically defined as a peptide hormone, recent evidence supports a role for GH acting as a cytokine in the immune system under conditions of stress, counteracting immunosuppression by glucocorticoids. Lymphoid cells express the GH receptor, which belongs to the cytokine receptor superfamily, and GH can be produced by immune tissues, suggesting an autocrine/paracrine mode of action of GH. GH can act as a cytokine, promoting cell cycle progression of lymphoid cells and preventing apoptosis. These effects of GH were shown to be mainly mediated by the PI-3 kinase/Akt pathway and the transcription factor NF-kappaB. Expression of several cell cycle mediators, as well as Bcl-2, c-Myc and cyclin proteins were found to be regulated by GH. Survival of immune cells under conditions of stress was promoted by NF-kappaB. Thus, GH acts not only as a hormone but also as a cytokine, playing a potentially important role in immune system cells. Lastly, in this mini-review, we will discuss whether the discovery of these molecules in GH signaling pathways offers new insights into additional mechanisms of action whereby GH regulates apoptosis, proliferation and neoplastic transformation of cells of the immune system.

Animals↗

PubMedDate not supplied

New homologous bioassays for human lactogens show that agonism or antagonism of various analogs is a function of assay sensitivity.

The reference bioassay for lactogens is the Nb2 cell proliferation assay, whose extreme sensitivity allows the detection of very low amounts of lactogenic activity in biologic fluids. The use of rat Nb2 cells raises the problem of species specificity when analyzing lactogens of other origin, including human lactogenic hormones for which no reference bioassay currently exists. In this article, we describe two new homologous bioassays for human lactogens. One is a transcriptional bioassay generated by stably transfecting 293 human embryonic kidney fibroblasts using two plasmids, encoding the human prolactin receptor (hPRLR) and the PRL-responsive lactogenic hormone response element luciferase reporter gene. The second is a proliferation assay obtained by stably transfecting Ba/F3 cells with a plasmid encoding the hPRLR. We provide characterization of the various clones or cell populations that were isolated, and we describe experiments that were performed to achieve optimized protocols for both bioassays. These new assays were compared with other cells types exhibiting well-recognized PRL-mediated responses (proliferation of Nb2 or of human breast tumor cell lines), using various lactogen analogs. This comparative analysis provides strong evidence that the intrinsic characteristics of each bioassay dramatically affect the biologic properties attributed to the lactogen of interest. Depending on the assay, a given analog can exhibit agonistic or antagonistic properties. We hypothesize that in addition to species specificity, assay sensitivity is the key parameter in directing the apparent bioactivity of lactogens. Of course, in the end, it will be necessary to confirm the agonistic or antagonistic properties of the tested analogs, in vivo.

Animals↗