Transcriptional regulation of apolipoprotein A-I expression in Hep G2 cells by phorbol ester

AbstractThe regulation of apolipoprotein A-I (apo A-I) gene expression by 12-O-tetradecanoylphorbol 13-acetate (TPA) was investigated in the human hepatoma cell line Hep G2. TPA treatment decreased apo A-I mRNA levels in a time-dependent manner, by up to 50% versus control cells within 24 h. Nuclear run-on transcription assays demonstrated a transcriptional effect of TPA. Using transfection analysis with a plasmid construct containing the −1378/+11 apo A-I promoter fused to the secreted placental alkaline phosphatase (SPAP) reporter gene, we showed that the SPAP activity was decreased to 50% when Hep G2 cells were incubated in the presence of TPA. The inhibitory effect of TPA was still maintained when fragment −253 to −4 of apo A-I promoter was linked to the CAT reporter gene. These data indicate that transcriptional modulation of apolipoprotein A-I gene expression following phorbol ester treatment is transduced by gene elements located between −253 and −4 of the apo A-I promoter

ACTIVATION PROTEOLYTIQUE DU RECEPTEUR B 2 HUMAIN DE LA BRADYKININE PAR LA KALLICREINE ET D'AUTRES PROTEASES A SERINE

L'OBJECTIF DE LA PRESENTE RECHERCHE ETAIT DE DEMONTRER L'ACTIVATION DIRECTE DU RECEPTEUR B 2 HUMAIN DE LA BRADYKININE PAR LA KALLICREINE ET D'AUTRES PROTEASES A SERINE. LE SYSTEME KALLICREINE-KININE JOUE D'IMPORTANTS ROLES DANS LES FONCTIONS CARDIOVASCULAIRES ET RENALES. TOUTEFOIS LES ACTIONS PHYSIOLOGIQUES ET PATHOLOGIQUES DE LA KALLICREINE ONT ETE ATTRIBUEES A LA LIBERATION DES PEPTIDES VASOACTIFS, LES KININES, A PARTIR DES PRECURSEURS KININOGENES (BHOOLA ET AL., 1992). LA VALIDITE DE CETTE HYPOTHESE A ETE TESTEE ET LES RESULTATS ONT PROUVE QUE LES KALLICREINES NON SEULEMENT LIBERENT LES KININES A PARTIR DU SUBSTRAT KININOGENE MAIS AUSSI AGISSENT DIRECTEMENT SUR LE RECEPTEUR B 2 HUMAIN DE LA BRADYKININE. LES ACTIONS DES PROTEASES A SERINE ONT ETE EXAMINEES SUR DES CULTURES DE CELLULES PRIMAIRES CHO (CHINESE HAMSTER OVARY CELLS), HEK293 (HUMAN EMBRYONIC KIDNEY CELLS) OU MDCK (MADIN DARBY CANINE KIDNEY CELLS) STABLEMENT TRANSFECTEES AVEC LE RECEPTEUR B 2 DE LA BRADYKININE ET SUR LES CELLULES BPAE (BOVINE PULMONARY ARTERY ENDOTHELIAL CELLS) EXPRIMANT CONSTITUTIVEMENT LE RECEPTEUR. AFIN DE DETERMINER LE(S) SITE(S) POTENTIEL(S) DE CLIVAGE, L'EFFET DE LA KALLICREINE A ETE TESTE SUR UNE CONSTRUCTION DU RECEPTEUR B 2 DE LA BRADYKININE DELETE DE SES 19 ACIDES AMINES N-TERMINAUX, LE LYS 1 9 INCLUS, ET SUR DES CONSTRUCTIONS DU RECEPTEUR B 2 PONCTUELLEMENT MUTES AUX K172T, R270G ET R281G. L'AFFINITE DE LA KALLICREINE POUR LE RECEPTEUR B 2 DE LA BRADYKININE A ETE DAVANTAGE INVESTIGUEE PAR L'UTILISATION DE RADIOLIGANDS. IL EST TRES PROBABLE QUE LE RECEPTEUR B 2 DE LA BRADYKININE APPARTIENNE A UN NOUVEAU GROUPE DE RECEPTEURS ACTIVES PAR LES PROTEASES A SERINE. LE ROLE DIRECT DE LA KALLICREINE DANS L'ACTIVATION DU RECEPTEUR B 2 POURRAIT ETRE D'UNE IMPORTANCE CLINIQUE QUI PUISSE MENER AU DEVELOPPEMENT DE NOUVEAUX MEDICAMENTS CIBLES.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Effets des acides gras polyinsaturés n-3 et n-6 dans les cellules musculaires lisses d'aorte de rat (implication dans l'athérosclérose)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Oxysterol and 9-cis-retinoic acid stimulate the group IIA secretory phospholipase A2 gene in rat smooth-muscle cells.

The inflammation that occurs during rheumatoid arthritis or atherosclerosis is characterized by the release of large amounts of sPLA(2) (group IIA secretory phospholipase A(2)). We have shown previously that the sPLA(2) promoter in SMC (smooth-muscle cells) is activated by interleukin-1beta and cAMP-signalling pathways, through the interplay of multiple transcription factors [Antonio, Brouillet, Janvier, Monne, Bereziat, Andreani, and Raymondjean (2002) Biochem. J. 368, 415-424]. In the present study, we have investigated the regulation of sPLA(2) gene expression in rat aortic SMCs by oxysterols. We found that oxysterol ligands that bind to the LXR (liver X receptor), including 25-HC (25-hydroxycholesterol) and 22( R )-HC, cause the accumulation of sPLA(2) mRNA and an increased enzyme activity. Transient transfection experiments demonstrated that the sPLA(2) promoter is synergistically activated by 22( R )-HC in combination with 9- cis -retinoic acid, a ligand for the LXR heterodimeric partner RXR (retinoid X receptor). Promoter activity was also increased in a sterol-responsive fashion when cells were co-transfected with LXRalpha/RXRalpha or LXRbeta/RXRalpha. Mutagenesis studies and gel mobility-shift assays revealed that LXR/RXR heterodimers regulate sPLA(2) transcription directly, by interacting with a degenerated LXRE (LXR response element) at position [-421/-406] of the sPLA(2) promoter. Chromatin immunoprecipitation revealed the in vivo occupancy of LXR on the sPLA(2) promoter. In addition, the orphan nuclear receptor LRH-1 (liver receptor homologue-1) potentiated the sterol-dependent regulation of the sPLA(2) promoter by binding to an identified promoter element (TCAAGGCTG). Finally, we have demonstrated that oxysterols act independent of interleukin-1beta and cAMP pathways to activate the sPLA(2) promoter. In the present study, we have identified a new pathway activating sPLA(2) gene expression in SMCs

Thyroid Hormone Modulates Apolipoprotein-AI Gene Expression at the Post-Transcriptional Level in Hep G2 Cells

International audienc

The Modulation of Apolipoprotein E Gene Expression by 3,3'-5-triiodothyronine in HepG2 Cells Occurs at Transcriptional and Post-transcriptional Levels

International audienc

Semicarbazide-Sensitive Amine Oxidase in Vascular Smooth Muscle Cells

International audienceCultured vascular smooth muscle cells (VSMCs) derived from rat aortic media were used to examine semicarbazide-sensitive amine oxidase (SSAO) expression during their differentiation process. In a defined serum-free medium permissive for in vitro VSMC differentiation, there was a large increase in SSAO mRNA and protein levels and in the related enzyme activity during the course of cell culture. This pattern of expression was concomitant with that of some smooth muscle–specific mRNA markers of differentiation. mRNAs in differentiated cultured VSMCs were comparable to those detected in total aorta and media. Pharmacological properties of SSAO present in VSMCs were similar to enzyme activities previously described in the aortic wall. In this model, we also demonstrated that methylamine, a physiological substrate of SSAO, activated 2-deoxyglucose transport in a time- and dose-dependent manner. This methylamine effect was reproduced by other SSAO substrates and was prevented by the SSAO inhibitor semicarbazide. It was antagonized in the presence of catalase, suggesting that SSAO-activated glucose transport was mediated through H 2 O 2 production. In addition, methylamine promoted glucose transporter 1 accumulation at the cell surface. Thus, we demonstrate for the first time the differentiation-dependent expression of SSAO in VSMCs and its role in the regulation of VSMC glucose uptake

Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1beta in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-kappaB and Ets transcription factors.

The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter