Étude du rôle d'une isoforme du récepteur-alpha des œstrogènes (ERa46) dans le processus de prolifération des cellules épithéliales mammaires humaines (propriétés fonctionnelles et gènes cibles)

Deux protéines ERa sont exprimées dans les cellules cancéreuses mammaires humaines MCF7: ERa 66, forme native et ERa 46, délétée de la région A/B. ERa 46 possède une seule (AF-2) des deux fonctions de transactivation de ERa 66 en raison de cette délétion et se situe au niveau du noyau des MCF7. Ces deux ERa semblent avoir des effets opposés sur la croissance cellulaire. Notre étude montre qu'en présence d'E2, si ERa 66 semble potentialiser la prolifération, ERa 46 l'inhibe. Cette protéine possède aussi des propriétés transcriptionnelles spécifiques: elle inhibe l'activité AF-1 de ERa 66. L'étude de la régulation transcriptionnelle du gène pS2 via ERa 66 et ERa 46 montre que les deux formes se lient cycliquement à ce promoteur. En présence d'E2, les deux ERa se conduisent de manière similaire et recrutent des séquences protéiques permettant d'activer la transcription de pS2. En absence d'E2, seule ERa 46 interagit avec des co-répresseurs et réprime la transcription basale de ce gène.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Rapid nitration of adipocyte phosphoenolpyruvate carboxykinase by leptin reduces glyceroneogenesis and induces fatty acid release.

Fatty acid (FA) release from white adipose tissue (WAT) is the result of the balance between triglyceride breakdown and FA re-esterification. The latter relies on the induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme for glyceroneogenesis. We previously demonstrated that long-term (18 h) leptin treatment of rat epididymal WAT explants reduced glyceroneogenesis through nitric oxide (NO)-induced decrease in PEPCK-C expression. We investigated the effect of a short-term leptin treatment (2 h) on PEPCK-C expression and glyceroneogenesis in relation to NO production. We demonstrate that in WAT explants, leptin-induced NO synthase III (NOS III) phosphorylation was associated with reduced PEPCK-C level and glyceroneogenesis, leading to FA release, while PEPCK-C gene expression remained unaffected. These effects were absent in WAT explants from leptin receptor-deficient Zucker rat. Immunoprecipitation and western blot experiments showed that the leptin-induced decrease in PEPCK-C level was correlated with an increase in PEPCK-C nitration. All these effects were abolished by the NOS inhibitor Nω-nitro-L-arginine methyl ester and mimicked by the NO donor S-nitroso-N-acetyl-DL penicillamine. We propose a mechanism in which leptin activates NOS III and induces NO that nitrates PEPCK-C to reduce its level and glyceroneogenesis, therefore limiting FA re-esterification in WAT

NO-dependent effects of leptin on glycerol (A) and FA (B) release from rat WAT explants.

<p>Explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or IFN-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA (lipolysis medium). Results are expressed as the percent of glycerol or FA relative to the corresponding untreated control. Crude values for control were 4.57±0.13 nmol.mg⁻¹ tissue.2 h⁻¹ for glycerol and 4.60±0.47 nmol.mg⁻¹ tissue. 2 h⁻¹ for FA. Each value represents the mean ± SEM, (n = 4) *, <i>P</i><0.01 <i>vs</i>. control; ^a<i>P</i><0.01 <i>vs.</i> leptin-treated explants.</p

NO-dependent effects of leptin on PEPCK-C protein in WAT explants from rats.

<p>WAT explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or INF-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA. PEPCK-C and β-actin proteins were revealed by western blotting performed on cytosolic proteins. (A) Densitometry scanning in ImageJ software. Each value represents the mean ± SEM (n = 4) *, <i>P</i><0.01 <i>vs</i>. control;^a<i>P</i><0.01 <i>vs.</i> leptin-treated explants. (B) Representative autoradiograms.</p

NO-dependent effect of leptin on the nitration of PEPCK-C protein in WAT explants from rats.

<p>WAT explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or IFN-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA. We first immunoprecipited cytosolic proteins using the anti-PEPCK-C antibody then proceeded to western blotting with the anti-nitroprotein antibody. (A) Densitometry scanning in ImageJ software. Each value represents the mean ± SEM, (n = 4) *, <i>P</i><0.01 <i>vs</i>. control;^a<i>P</i><0.01 <i>vs.</i> leptin-treated explants. (B) Representative autoradiogram.</p

Rat probes used in real time PCR.

<p>Rat probes used in real time PCR.</p

NO-dependent effects of leptin on glyceroneogenesis and NOS III phosphorylation in WAT explants from rats.

<p>Explants from SD (A) or Zucker rats (B, C) were pre-treated or not with L-NAME (1 mmol/L) or AG490 (10 µmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or IFN-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA and [1-¹⁴C]-pyruvate. Glyceroneogenic flux was measured by the [1-¹⁴C]-pyruvate incorporation into neutral lipids. Each value represents the mean ± SEM, (n = 4) *, <i>P</i><0.01 <i>vs</i>. control; ^a<i>P</i><0.01 <i>vs.</i> leptin-treated explants. (C) Representative autoradiogram of a western blot performed on WAT cytosolic proteins from Zucker rats reveals total NOS III and its Ser¹¹⁷⁹ phosphorylated form.</p

The relative contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor alpha transcriptional activity depends upon the differentiation stage of the cell.

International audienceThe activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor alpha (ERalpha) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ERalpha AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ERalpha activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ERalpha transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ERalpha signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ERalpha in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ERalpha-positive cell lines tested strongly suggest that the transcriptional activity of ERalpha relies on its AF-1 in most estrogen target cell types

Effects of leptin and IFN-γ on PEPCK-C, NOS II and NOS III mRNA in rat WAT explants.

<p>Explants were treated or not with leptin (10 µg/L) or IFN-γ (50 µg/L) for 2 h. PEPCK-C, NOS II and NOS III mRNA levels were analysed by RT-qPCR. Results are normalized using 18S rRNA. Each value represents the mean ± SEM, (n = 4).</p

The human estrogen receptor-alpha isoform hERalpha46 antagonizes the proliferative influence of hERalpha66 in MCF7 breast cancer cells

11 pagesInternational audienceThe expression of two human estrogen receptor-alpha (hERalpha) isoforms has been characterized within estrogen receptor-alpha-positive breast cancer cell lines such as MCF7: the full-length hERalpha66 and the N terminally deleted hERalpha46, which is devoid of activation function (AF)-1. Although hERalpha66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERalpha46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERalpha46 is mainly expressed in the nucleus at relatively low levels, whereas hERalpha66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERalpha46 accumulating within the nucleus. Although hERalpha46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G(0)/G(1) phases. To gain further details on the influence of hERalpha46 on cell growth, we used PC12 estrogen receptor-alpha-negative cell line, in which stable transfection of hERalpha66 but not hERalpha46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERalpha46 inhibits the hERalpha66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERalpha46 antagonizes the proliferative action of hERalpha66 in MCF7 cells in part by inhibiting hERalpha66 AF-1 activity