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

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LRP1, Adipogenesis, ObesityFigure 3. Inhibition of LRP1 expression by silencing during adipocyte differentiation. (A) Silencing of LRP1 by siRNAs. 3T3F442Apreadipocytes were transiently transfected with Luc siRNA or LRP1 siRNAs. LRP1b protein expression was monitored by immunoblotting 2 days posttransfection.a tubulin was used as a loading control. A representative experiment out of 3 is shown. (B) Time-course of LRP1b proteinexpression after Luc siRNA or LRP1 siRNAs transfection during adipogenesis. 3T3F442A preadipocytes were transiently transfected withLuc siRNA or LRP1 siRNAs. Two-days post-transfection, LRP1b protein expression was monitored by immunoblotting 0, 3, 5 or 7 days afterdifferentiation induction. ERK2 was used as a loading control.doi:10.1371/journal.pone.0007422.g003expression in adipocytes isolated from C57BI6/J mice either fed aHFD or ND (Fig. 7B). As attempted HFD-fed C57BI6/J miceexhibited a significant increase in body mass (47.661.4 g) whencompared to their control littermates (31.161.2 g) (data not shown).LRP1 expression was significantly enhanced in adipocytes of HFDobese mice when compared to ND control mice (Fig. 7B).Altogether, our results indicate that LRP1 expression is upregulatedin human and mouse obese adipose tissues.Silencing of LRP1 expression inhibits the cellular lipidcontent of fully-differentiated adipocytesSince we observed that LRP1 expression was up-regulated inobese adipose tissues, we finally investigated whether extinction ofLRP1 expression in fully-differentiated adipocytes could diminishtheir lipid content (Fig. 8), as recently suggested in adipose-specificLRP12/2 mouse model [12]. 3T3F442A preadipocytes werecultured for 10 days in adipogenic differentiation medium (Fig. 8A,panel a) and fully-differentiated adipocytes were transientlytransfected with Luc siRNA or LRP1 siRNA1 (Fig. 8A, panel b).Oil red O staining revealed that, after 7 days of culture, the lipiddroplet size and number were decreased in LRP1 siRNA1transfected adipocytes (Fig. 8B–C). Quantification of lipids atdays 2 and 7 of culture revealed that, in Luc siRNA transfectedadipocytes, the level of cellular lipid remained unchanged whereas,in LRP1 siRNA1 transfected cells, the amount of lipid wassignificantly diminished by 27.8% (Fig. 8D). Interestingly, basallipolysis was significantly stimulated in LRP1 siRNA1 transfectedadipocytes (Fig. 9, panel a). We postulate that, since cells could notinternalized triglycerides in the absence of LRP1 as previouslyshown [12], they were metabolizing their intracellular lipid stock.However, induced lipolysis was not different in LRP1 and LucsiRNA transfected cells (Fig. 9, panel b). Altogether, these findingshighlight, for the first time, the crucial role of LRP1 in controllingadipogenesis and maintaining the lipid content in fully-differen-PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7422
LRP1, Adipogenesis, ObesityFigure 4. Silencing of LRP1 inhibits expression of adipocyte differentiation markers in a LRP1 dose-dependent manner. (A) mRNAexpression of LRP1, PPARc, HSL and aP2 in LRP1 siRNA1-silenced preadipocytes. RNA expression of LRP1, PPARc, HSL and aP2 was analysedin 3T3F442A preadipocytes transfected with Luc siRNA or LRP1 siRNA1. RNA was quantified by RT-PCR in 3T3F442A cells after 3 and 7 days in adipogenicdifferentiation medium. Mean 6 SD of 4 independent experiments is shown. *P,0.025 when compared with Luc siRNA transfected cells. (B) Inhibitionof PPARc, HSL and aP2 mRNA expression in LRP1-silenced preadipocytes in a LRP1 dose-dependent manner. RNA levels of LRP1, PPARc,HSL and aP2 in 3T3F442A preadipocytes transfected with Luc siRNA or the 3 LRP1 siRNA1-3 were performed as described in panel A.doi:10.1371/journal.pone.0007422.g004tiated adipocytes, and suggest that LRP1 may be an importanttherapeutic target in obesity.DiscussionHere, we show that LRP1 is highly expressed in 3T3F442Amurine preadipocyte cell line, as previously described for 3T3L1preadipocytes and embryonic fibroblasts [16,17]. Since LRP1 isexpressed at high levels in preadipocytes, it therefore can beinvolved in the early steps of adipocyte differentiation. Indeed, ourreport demonstrate that LRP1 silencing by siRNAs in 3T3F442Apreadipocytes significantly inhibits the expression of PPARc, HSLand aP2 adipocyte markers of differentiation and leads to lipiddepletedcells, indicating that LRP1 is a key regulator of thePLoS ONE | www.plosone.org 5 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
Page 50 and 51: 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 102 and 103: LRP1, Adipogenesis, ObesityFigure 5
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
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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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