Rôle de l'apolipoprotéine D dans le métabolisme des lipides

L'apolipoprotéine D (apoD) est une glycoprotéine qui transporte plusieurs molécules hydrophobes, telles que l'acide arachidonique, le cholestérol et la progestérone. Afin d'étudier le rôle neuroprotecteur de l'apoD, des souris transgéniques (Tg) surexprimant l'apoD humaine (H-apoD) principalement au niveau du cerveau ont été générées. Bien que les souris Tg H-apoD résistent mieux à la neurodégénérescence, elles souffrent de stéatose hépatique et musculaire et sont résistantes à l'insuline. Le but de ce projet est de caractériser les mécanismes moléculaires associés à l'accumulation de lipides dans le foie des souris Tg H-apoD. L'expression de plusieurs gènes hépatiques impliqués dans le métabolisme lipidique ainsi que dans le stockage de ces lipides dans des gouttelettes lipidiques (GL) a été mesurée par RT-PCR semi-quantitative et par Western Blot. Notre étude a révélé une augmentation de plus de deux fois du facteur de transcription PPARyl dans le foie des souris Tg H -apoD par rapport aux souris sauvages. Plusieurs études suggèrent qu'une modulation de PPARyl peut mener à des troubles métaboliques, puisque PPARy induit la transcription de gènes impliqués dans l'accumulation de lipides et dans la formation des GL. Afin de mesurer son niveau d'activation, le niveau d'expression de ses gènes cibles a été mesuré. Le transporteur d'acides gras CD36, impliqué dans la captation des acides gras circulants par les hépatocytes, est surexprimé de 20 %. Cide a et Cide c, deux protéines impliquées dans la stabilisation des GL sont surexprimées de 50 %. Plin2, aussi localisée au niveau des GL et impliquée dans l'inhibition de la lipolyse, est aussi surexprimée (2 fois). Cela induit la formation de GL plus larges et donc la stéatose hépatique. Probablement en compensation, le récepteur nucléaire PPARα et son gène cible CPT1, enzyme limitant de la β-oxydation, sont surexprimés de 2,5 fois et de 20 % respectivement, augmentant ainsi la dégradation des lipides accumulés. L'AMPK est également activée, ce qui semble causer une diminution du niveau de lipogenèse (surtout par la phosphorylation de l'ACC) et de la néoglucogenèse. Ces résultats démontrent que la surexpression de l'apoD induit une surexpression et une activation de PPARyl. En induisant la transcription de ses gènes cibles, PPARy provoque l'accumulation de lipides, menant à une stéatose hépatique. Par contre, nos résultats démontrent qu'un mécanisme compensatoire est aussi induit (augmentation de la β-oxydation et diminution de la lipogenèse et néoglucogenèse) expliquant le phénotype peu sévère de la stéatose observée. Cette étude permettra de mieux comprendre le rôle de l'apoD dans le métabolisme des lipides.\ud ______________________________________________________________________________ \ud MOTS-CLÉS DE L’AUTEUR : ApoD, PPARy, stéatose hépatiqu

Expression and functions of galectin-7 in ovarian cancer.

International audienceThere is a critical need to develop effective new strategies for diagnosis and treatment of ovarian cancer. In the present work, we investigated the expression of galectin-7 (gal-7) in epithelial ovarian cancer (EOC) cells and studied its functional relevance. Immunohistochemical analysis of gal-7 expression in tissue microarrays showed that while gal-7 was not detected in normal ovarian tissues, positive cytoplasmic staining of gal-7 was detected in epithelial cells in all EOC histological subtypes but was more frequent in high grade tumors and metastatic samples. Gal-7 expression correlated with a significant difference in the overall survival of patients with ovarian serous cystadenocarcinoma. Furthermore, using human EOC cell lines, we found that gal-7 expression was induced by mutant p53. Mechanistically, Matrigel invasion assays and live cell imaging showed that gal-7 increased the invasive behavior of ovarian cancer cells by inducing MMP-9 and increasing cell motility. EOC cells can also secrete gal-7. Recombinant human gal-7 kills Jurkat T cells and human peripheral T cells, suggesting that gal-7 also has immunosuppressive properties. Taken together, our study validates the clinical significance of gal-7 overexpression in ovarian cancer and provides a rationale for targeting gal-7 to improve the outcome of patients with this disease

The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

<div><p><i>Galectin-7</i> is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing <i>galectin-7</i> expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ) in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. <i>In silico</i> analysis further revealed the presence of an established CEBP element in the <i>galectin-7</i> promoter. Mutation of this binding site abolished the transcriptional activity of the <i>galectin-7</i> promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous <i>galectin-7</i> promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of <i>galectin-7</i> gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.</p></div

<i>Galectin-7</i> expression induced by wild-type p53 required NF-κB.

<p>Inhibition of galectin-7 mRNA (<b>A</b>) and protein levels (<b>B</b>) by increasing doses of CAPE in MCF-7 and MDA-MB-231 cells transfected with an expression vector encoding wt p53. GAPDH was used as loading control.</p

Doxorubicin induces galectin-7 expression in MCF-7^p53wt.

<p>(<b>A</b>) Increased expression of <i>galectin-7</i> and <i>p21WAF1</i> in MCF-7 cells treated with increasing doses of doxorubicin. No such increase was observed in <i>galectin-3</i> mRNA levels. <i>GAPDH</i> was used as loading control. (<b>B</b>) Inhibition of <i>galectin-7</i> mRNA levels and p53 protein levels by p53 siRNA in MCF-7 cells treated with 0.5 µg/ml of doxorubicin. A siRNA CTRL vector was used as control and GAPDH or β-actin was used as loading control. (<b>C</b>) Confocal images showing galectin-7 expression in doxorubicin-treated MCF-7 as compared to untreated control cells. Fixed cells were labeled with a goat anti-human galectin-7 polyclonal antibody and an Alexa Fluor 488-conjugated donkey anti-goat IgG (green fluorescence). Nuclei were stained with DAPI (blue).</p

Galectin-7 expression is dependent of NF-κB signalling pathway.

<p>mRNA levels following treatment with increasing doses of TNFα (<b>A</b>) or at different time post-treatment with TNFα (<b>B</b>). (<b>C</b>) <i>In silico</i> analysis showing effect of parthenolide on TNFα-induced <i>galectin-7</i> expression in epidermal keratinocytes. Data were retrieved from a dataset (GDS : 1289) in the Gene Expression Omnibus (GEO) repository. (<b>D</b>) Effect of parthenolide on galectin-7 expression in HaCaT cells. mRNA levels of <i>galectin-7</i> was measured by semi-quantitative RT-PCR analysis 48h after treatment. No such effect was observed on C/EBPβ expression. GAPDH was used as loading control. (<b>E</b>) Dose-dependent inhibition of <i>galectin-7</i> mRNA levels in MDA-MB-468 cells treated with CAPE. GAPDH was used as loading control. (<b>F</b>) Dose-dependent inhibition of <i>galectin-7</i> mRNA levels in MDA-MB-468 cells treated with quercetin. GAPDH was used as loading control.</p