Cancer du sein et micro-environnement tumoral: rôle de la protéase ...

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LRP1, Adipogenesis, ObesityFigure 5. LRP1 expression is required for adipogenesis. (A)Staining of neutral lipids. Plates of 3T3F442A transfected with LucsiRNA (panel a) or LRP1 siRNA1 (panel b) at day 7 are presented. Twodayspost Luc or LRP1 siRNA transfection, confluent 3T3F442A cellswere grown in the presence of the adipogenic differentiation medium.After 7 days of differentiation, the extent of cellular lipid accumulationwas revealed by oil Red O staining. A representative experiment out of3 is shown. (B) Micrographs of 3T3F442A preadipocytes. Cellswere transfected with Luc siRNA (panels a and c) or LRP1 siRNA1 (panelsb and d) and micrographs were performed after 3 (panels a and b) and7 (panels c and d) days of differentiation. A representative experimentout of 3 is shown. (C) Quantification of neutral lipids andtriglycerides. In panel a, lipid content was quantified at 3, 5 and 7days of differentiation in 3T3F442A transfected with Luc or LRP1 siRNAs.Mean 6 SD of 3 independent experiments is shown. *P,0.0025 whencompared with Luc siRNA transfected cells. In panel b, triglycerideswere quantified at day 5 and 7 of differentiation in 3T3F442Atransfected with Luc or LRP1 siRNAs. Results were normalised per mgof DNA. Data are presented as mean 6 SD of an experiment performedin triplicate. *P,0.0025 when compared with Luc siRNA transfectedcells.doi:10.1371/journal.pone.0007422.g005Figure 6. Silencing of LRP1 in pre-adipocytes inhibits lipolysis.(A) Basal lipolysis. Glycerol release in the medium after 18 h wasquantified in 3T3F442A transfected with Luc or LRP1 siRNA1 after 7 daysof differentiation. Results were normalised with mg of DNA. Data arepresented as mean 6 SD of one experiment performed in quadruplicate.P,0.025 when compared with Luc siRNA transfected cells. (B)Induced lipolysis. Glycerol and non esterified fatty acid (NAFA)releases were quantified after 7 days of differentiation in 3T3F442A cellstransfected with Luc or LRP1 siRNA1 and treated with increasingconcentrations of isoproterenol. Results were normalised per mg ofDNA. Mean 6 SD of an experiment performed in quadruplicate ispresented. P,0.025 when compared with Luc siRNA transfected cells.doi:10.1371/journal.pone.0007422.g006adipogenic process. Our experiments using 3 LRP1 siRNAs showthat LRP1 extinction abrogates adipocyte differentiation in aLRP1 dose-dependent manner. Our results also pinpoint thatextinction of LRP1 expression in pre-adipocytes prevents theirlipolytic response both at a basal level and after isoproterenolinduction. Therefore the inhibition of lipid accumulation andtriglyceride levels observed in LRP1-silenced preadipocytes is notthe consequence of a stimulation of lipolysis, indicating a directeffect of LRP1 on the control of the adipogenic process. PPARc isknown to be crucial for the initiation of adipocyte differention [2].Interestingly, PPARc has been described to induce LRP1transcription [4] and our results indicate that LRP1 silencinginhibits PPARc expression. This suggests the existence of aregulatory loop between these two factors. Our findings reveal thatLRP1 silencing inhibits the expression of PPARc by 80% at day 3of differentiation, indicating an early effect of LRP1 at thecommencement of adipogenesis. It is therefore important todetermine how LRP1 lost down-regulates PPARc expression. Arecent study demonstrates that LRP1 controls gene transcriptionvia Regulated Intramembrane Proteolysis through the cytoplasmicPLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7422
LRP1, Adipogenesis, ObesityFigure 7. LRP1 expression is up-regulated in human and mouseobese adipose tissues. (A) LRP1 expression in adipose tissuefrom lean and obese human. LRP1 mRNA level was quantified inhuman intra-abdominal visceral adipose tissue samples (VAT) obtainedfrom 27 morbide grade III obese patients (44.5+/21.8 year old, BMI: 47.6+/2 1.3 kg/m 2 ) before a bariatric surgery and from 10 control patientsundergoing abdominal lipectomy for plastic surgery (42.7 +/2 4.5 yearold, BMI: 23.1 +/2 3.3 kg/m 2 ). Results are mean values +/2 SEM,*P,0.01 when compared with controls (normal VAT). (B) LRP1expression in adipocytes from obese mouse. LRP1 mRNA levelwas quantified in adipocytes isolated from intra-abdominal adiposetissues from 30-week-old overweight C57Bl6/J mice fed in high-fat diet(HFD) and from C57Bl6/J control mice fed in normal diet (ND). Resultsare mean value +/2 SEM from 5 mice for ND group and 4 mice for HFDgroup. *P,0.05 when compared with ND controls.doi:10.1371/journal.pone.0007422.g007release of LRP1 intracellular domain, LRP1-ICD [18]. Studies inthe future will investigate whether LRP1-ICD regulates PPARcexpression in preadipocytes.Our results also indicate, for the first time, that LRP1 expressionis up-regulated in human obese tissue. This up-regulation of LRP1expression in the VAT of overweight/obese patients is inconsensus with our results in obese mice. Obesity is characterizedby the increase of intracellular lipid accumulation which shows asignificant correlation with adipocyte differentiation. Terminallydifferentiated adipocytes cannot divide. Hence, alterations in thenumber of fat cells within the body must be accomplished by thedifferentiation of preadipocytes, which act as a renewable source ofadipocytes. Interestingly, our in vitro study in 3T3F442A cellsindicates that LRP1 expression is required for adipocytedifferentiation and since LRP1 is abundantly expressed inadipocytes, we propose that LRP1 may participate in the onsetFigure 8. LRP1 silencing in fully-differentiated adipocytes leadsto lipid-depleted cells. (A) Silencing of LRP1 in fully-differentiatedadipocyte. 3T3F442A pre-adipocytes were maintained for 10 daysin adipogenic differentiation medium (panel a) and were transfected withLuc siRNA or LRP1 siRNA1. LRP1b protein expression was monitored byimmunoblotting 2 days post-transfection (panel b). ERK2 was used as aloading control. (B) Staining of neutral lipids. Plates of differentiated3T3F442A transfected with Luc siRNA (panel a) or LRP1 siRNA1 (panel b)are shown. After siRNA transfection, 3T3F442A mature adipocytes weregrown in DMEM + 10% FCS. The extent of cellular lipid accumulation wasdetermined at day 2 and 7 of culture by oil Red O staining. Arepresentative experiment out of 3 is shown. (C) Micrographs of3T3F442A adipocytes. Micrographs of differentiated adipocytestransfected with Luc siRNA (panel a) or LRP1 siRNA1 (panel b) wereperformed 7 days post-transfection. A representative experiment out of 3is shown. (D) Quantification of lipid content. Lipid content wasquantified at days 2 and 7 post-tranfection of differentiated adipocytestransfected with Luc or LRP1 siRNA1. Mean 6 SD of 3 independentexperiments is shown. *P,0.005 when compared with Luc siRNAtransfected cells.doi:10.1371/journal.pone.0007422.g008of obesity. The crucial role of LRP1 in obesity is also supported bya recent study showing that adipocyte LRP12/2 mice have anoverall decrease in fat mass and are protected from high-fat dietinducedobesity [12]. Our experiments further show that silencingPLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7422
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Université Montpellier I UFR Méd
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Un grand merci à tous mes collabor
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TitleBreast cancer and tumoral micr
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Cancer du sein et micro-environneme
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C. PRESENTATION DU TRAVAIL DE THESE
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But de la thèseLa cathepsine-D (ca
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I. Le tissu adipeux1) Généralité
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(Adenosine triphosphate). Cette pro
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Cette synthèse de novo a lieu dans
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ABFigure 6 : Schéma de la oxydati
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d. Ladipocyte : une cellule sécré
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3) Ladipogenèsea. Les différentes
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. Les facteurs de transcriptionLa d
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Lexpression de C/EBP est quant à e
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adipeux. Les analyses histologiques
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adipocyteFigure 10 : Schéma repré
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II. La cathepsine-D1) Synthèse, ma
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les cath-B et L, en une forme matur
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Il existe deux RM6P : le RM6P/IGFII
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2) Fonctions de la cath-Da. Dans la
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. Dans les pathologiesEn plus de se
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c. Rôles et mécanismes daction da
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carcinome de prostate serait respon
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La cath-D joue donc un rôle import
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Une fois le marquage des peptides e
Page 52 and 53: Afin de réaliser ce projet, nous a
Page 54 and 55: III. Le récepteur LRP11) Organisat
Page 56 and 57: 2) Trafic intra-cellulaireComme le
Page 58 and 59: LRP1αLRP1Membrane associatedprotea
Page 60 and 61: Tableau 3 : Ligands connus du LRP1(
Page 62 and 63: I. Etude du rôle de la cathepsine
Page 64 and 65: Cathepsin-D, a key protease in brea
Page 66 and 67: IntroductionConsumption of meals ri
Page 68 and 69: (Fig. 1A, panel a). This differenti
Page 70 and 71: differentiated adipocyte (Fig. 4B).
Page 72 and 73: DiscussionOur results demonstrate t
Page 74 and 75: from normal and peri-al breast adip
Page 76 and 77: mouse RS9 (sens 5CGGCCCGGGAGCTGTTGA
Page 78 and 79: agreement of local ethic committee.
Page 80 and 81: Statistical analysis. Results are e
Page 82 and 83: Loncarek J, Freiss G, Vignon F and
Page 84 and 85: Aa3000 *b4000*B8000**cath-D mRNA(ar
Page 86 and 87: ABcath-D mRNA(ratio RS9)PPARg mRNA(
Page 88 and 89: A D3 D7 D14D0BaD0 D3 D7 D14D0 D3 D7
Page 90 and 91: AshLucshcath-DF442A C34 C37 A4 D10c
Page 92 and 93: AF442-AC34C37A4D10BF442-AC34C37A4D1
Page 94 and 95: II. Etude du rôle du LRP1 dans les
Page 96 and 97: 2) Article 2:LRP1 receptor controls
Page 98 and 99: LRP1, Adipogenesis, Obesityrelative
Page 100 and 101: LRP1, Adipogenesis, ObesityFigure 3
Page 104 and 105: LRP1, Adipogenesis, ObesitySome ins
Page 106 and 107: LRP1, Adipogenesis, ObesityLipolysi
Page 108 and 109: CONCLUSIONLa cathepsine D (cath-D)
Page 110 and 111: la souris obèses. Enfin, linhibiti
Page 112 and 113: carcinogénique sont encore mal con
Page 114 and 115: Ce travail de thèse a étudié, po
Page 116 and 117: REFERENCESAhima, R. S. (2006). Adip
Page 118 and 119: implicates them as antigen presenti
Page 120 and 121: Cataldo, A. M., Barnett, J. L., Ber
Page 122 and 123: Folkman, J. (2003). Fundamental con
Page 124 and 125: Hofmann, S. M., Zhou, L., Perez-Til
Page 126 and 127: Langin, D. (2006a). Adipose tissue
Page 128 and 129: Ludwig, T., Ovitt, C. E., Bauer, U.
Page 130 and 131: Nirde, P., Derocq, D., Maynadier, M
Page 132 and 133: Rozanov, D. V., Hahn-Dantona, E., S
Page 134 and 135: Taleb, S., Cancello, R., Clement, K
Page 136 and 137: Xiao, Y., Junfeng, H., Tianhong, L.
Page 138 and 139: F. ANNEXE98
Page 140 and 141: Cathepsin D is a new ligand for ext
Page 142 and 143: INTRODUCTIONLysosomal aspartic prot
Page 144 and 145: pcDNA3.1(+)Myc-tagged LRP1b into pc
Page 146 and 147: (Protein refolding kit, Novagen) fo
Page 148 and 149: anti-mouse-gold (Aurion). Sections
Page 150 and 151: not secrete detectable levels of pr
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using cath-D-/- MEF transfected wit
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co-culture outgrowth assays with ca
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REFERENCES1. Vignon, F., Capony, F.
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steps in vivo: proliferation, angio
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28. Laurent-Matha, V., Lucas, A., H
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42. Zurhove, K., Nakajima, C., Herz
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Figure 1. Cath-D interacts with the
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Figure 2. Cath-D binds to residues
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Figure 3. Cath-D interacts with LRP
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Figure 5. Silencing LRP1 in cath-D
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Figure 1. Cath-D interacts with the
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Figure 6. LRP1 is the receptor medi
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Beaujouin, Figure Sup. 2cath-D-/-ME
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RésuméLaspartyl protéase catheps
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