Serge Champion

Role of Endoproteolytic Processing in the Adhesive and Signaling Functions of αvβ5 Integrin

Some integrin α subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the α subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing α1-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that α3, α6 and αvintegrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the αvβ5integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by αv integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of αv integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion.

Insulin-like growth factor-1 downregulates nuclear factor κB activation and upregulates interleukin-8 gene expression induced by tumor necrosis factor α

Pretreatment of HT29-D4 epithelial adenocarcinoma colic cells with des-IGF-1 upregulated TNF alpha-mediated activation of IL-8 expression at different levels (protein, mRNA, and hnRNA). RNA transcription but not RNA stabilization was found to be involved. In this cell line, cooperation of NF-kappa B with other factors appeared essential for IL-8 expression. Indeed, TNF alpha-induced NF-kappa B translocation was not sufficient to support enhancement of the transcription and des-IGF-1 did not promote but partly inhibited both the TNF alpha-induced NF-kappa B activation and I kappa B alpha degradation through a PI-3K-dependent pathway. A CCAAT/enhancer binding protein (C/EBP) site located on the IL-8 gene enhancer cooperated with a NF-kappa B binding site and led to the upregulation of IL-8 expression. Binding of C/EBP alpha to this sequence disappeared in IGF-1 treated cells. This event may be important for the cross-talk between IGF-1- and TNF alpha-mediated pathways leading to the control of inflammatory processes and the decision concerning apoptosis or cell survival.

Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag-Pol frameshifting in males of Drosophila melanogaster.

Extensive analyses of Drosophila melanogaster retrotransposon transcriptions in cultured cells or during development have been reported, but little is known about their translation during the development of the fly. Analysis of the translational products of the 1731 Drosophila melanogaster retrotransposon in Kc Drosophila cultured cells has been reported, showing the existence of primary products (Gag and Pol) and of processed polypeptides of various sizes. Study of 1731 retrotransposon expression at both levels of transcription and translation during the development of Drosophila melanogaster, is presented. 1731 transcripts were detected by in situ hybridization and 1731 proteins were detected by immunostaining and immunoblotting in embryos and in adult gonads. 1731 transcripts and proteins were detected in the mesoderm and central nervous system during embryonic development, in nurse cells and follicle cells in adult ovaries and in primary spermatocytes in adult testes. Moreover, Western blot analysis of the 1731 proteins with anti-Gag or anti-Pol antibodies in gonads revealed that the 1731 mRNA could be translated differentially according to the expressing tissue: essentially, ovarian translation and/or processing of 1731 products is different from that operating in testes, where the Gag-Pol fusion polyprotein is the most prominent product. Our results indicate that expression of the 1731 mobile element is regulated not only at the transcriptional level but also at the translational level, and that this regulation is different in the two sexes.

THE EFFECTS OF RIBAVIRIN ON THE GTP LEVEL AND THE VIP RECEPTOR DYNAMIC OF HUMAN IGR39 CELLS

GTP is one of the major cellular molecules involved in fundamental functions of cell life. Ribavirin, and antiviral and antitumoral agent, the primary site of action of which is the IMP deshydrogenase, was used in order to depress the intracellular GTP level. Consequential effects were tested on the property and dynamic of the VIP receptor on human melanoma IGR 39 cells. A concentration of 100 microM of Ribavirin reduced the intracelluar GTP level by more than 60% and induced a reversible growth arrest. Nevertheless this drug displayed no effect on: i) the VIP binding parameters (Kd and Bmax) of both high and low affinity receptors; ii) the cycling of the VIP receptor; iii) the based and VIP-stimulated cAMP production and iv) the subcellular GTP distribution. We show that Ribavirin, in the range of concentrations used, is very efficient to inhibit GTP synthesis in the human melanoma cell line IGR 39 and its growth, without affecting VIP receptor functions.

UVB irradiation-induced transcription from the long terminal repeat of intracisternal A particles and UVB-induced secretion of an extracellular factor that induces transcription of the intracisternal A particles in unirradiated cells

Intracisternal A particles (IAPs) are endogenous defective retroviral-like elements encoded by a family of proviral sequences present as a thousand copies in the mouse genome. In order to analyse the regulation of the long terminal repeat (LTR) directed transcription by UVB, D152 murine cells were transfected with a chimeric construct carrying the LTR of IAP linked to the bacterial chloramphenicol-acetyl-transferase reporter gene and then subjected by UVB irradiation in a dose- and time-dependent manner. Like the human immunodeficiency virus 1 type LTR and in spite of the lack of the nuclear factor kappa B consensus sequence, the IAP LTR could be activated by UVB. In addition, the D152 cells produced an extracellular factor are factors in response to UVB irradiation which activated the IAP LTR in unirradiated cells. This factor was detected both when responding cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures.