Cyrille Lejczak's research while affiliated with French Institute of Health and Medical Research and other places

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Publications (2)

Fig. 1. Effect of ACTH on Expression of VEGF, Tis11b, and HuR mRNAs A, Representative ethidium bromide staining of VEGF, Tis11b, and HuR mRNAs amplified by RT-PCR. Primary cultures of BAC cells were treated with 10 n M ACTH for the 
Fig. 2. Effect of ACTH on Tis11b and HuR Protein Levels in Total Cell Extracts and Subcellular Fractions A, BAC cells were treated with 10 n M ACTH for the indicated periods of time. Tis11b and HuR protein levels of whole-cell extracts (10 ␮ g) were analyzed by Western blot as outlined in Materials and Methods . The Western blot was subsequently probed with an anti- ␣ -tubulin monoclonal antibody to assess equal loading of samples. B, Quantitation of HuR and Tis11b protein levels of total cell extracts in three independent experiments. Protein level values were normalized to ␣ -tubulin protein levels. C, BAC cells were treated with 10 n M ACTH as indicated in panel A. Nuclear (5 ␮ g) and cytoplasmic (20 ␮ g) fractions were prepared as described in Materials and Methods and subjected to Western blot analysis to monitor Tis11b and HuR expression. The same membranes were sequentially probed with antibodies recognizing cytoplasm- and nucleus-specific proteins ( ␣ -tubulin and lamin A/C, respectively) to assess the quality of the fractionation process and to check for equal protein loading. D, Cytoplasmic and nuclear HuR protein levels were normalized to ␣ -tubulin and lamin protein levels, respectively, and are expressed as a fraction of total cytoplasmic or total nuclear protein content (n ϭ 2). 
Fig. 3. Coimmunoprecipitation of HuR from BAC Cytoplasmic and Nuclear Extracts Using Anti-pp32 Antibodies A, BAC cells were treated with 10 n M ACTH for the indi- 
Fig. 4. Effect of LMB on ACTH-Induced Increase in VEGF mRNA Levels A, BAC cells were treated with 10 n M ACTH for the indicated periods of time, in the presence or in the absence of LMB (5 ng/ml). When used, LMB was added 15 min before ACTH treatment. Cytoplasmic (30 ␮ g) and nuclear fractions (5 ␮ g) were subjected to Western blot analysis of HuR. B, Representative ethidium bromide staining of VEGF mRNA levels amplified by RT-PCR in BAC cells stimulated with ACTH in the presence or in the absence of LMB (5 ng/ml). C, Quantitation of VEGF mRNA levels in BAC cells stimulated with ACTH in the presence or in the absence of LMB, expressed as fold induction of mRNA levels at time 0 (unstimulated cells). Each point is the mean value from two separate experiments. 
Fig. 5. Effect of HuR Repression by RNAi on ACTH-Induced Increase in VEGF mRNA Levels A, BAC cells were transfected either with HuR-specific siRNA or a negative control siRNA as described in Materials and Methods . Culture medium was changed 48 h later, and cells were treated for the indicated periods of time with or without 10 n M ACTH. At each time point of stimulation, total RNA was isolated and RT-PCR analysis was performed to determine HuR, VEGF, or HPRT mRNA expression levels. B, Western blot analysis of HuR protein levels in whole-cell extracts (10 ␮ g), showing that HuR siRNA was effective in knocking down HuR protein levels. In this particular experiment, blots for HuR were exposed for 2 min. Despite prolonged exposure of the membrane (15 min), HuR was barely detectable in protein extracts derived from HuR siRNA-treated cells. C, Quantitation of the RT-PCR experiment represented in panel A, in which HuR repression was evaluated to 85%. Results obtained from three independent experiments revealed that ACTH-induced increase in VEGF mRNA levels was altered to a lesser extent when HuR repression was about 70% (data not shown). 

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Antagonistic Functions of Tetradecanoyl Phorbol Acetate-Inducible-Sequence 11b and HuR in the Hormonal Regulation of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Stability by Adrenocorticotropin
  • Article
  • Full-text available

May 2006

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206 Reads

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42 Citations

Molecular Endocrinology

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Cyrille Lejczak

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Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3'-untranslated region (3'-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3'-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3'-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

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Citations (2)

... Interestingly, the level of expression of angiogenic factors, such as vascular endothelial growth factor-A (VEGF-A), is high in these organs despite the absence of active angiogenesis, suggesting that these factors play an important role in the homeostasis and the hormonal control of the microvasculature (Shweiki et al. 1993). Our team has previously established that VEGF expression is under the hormonal control of adrenocorticotropin (ACTH) both in primary cultures of bovine steroidogenic adrenocortical cells in vitro and in mouse adrenal gland in vivo (Gaillard et al. 2000, Thomas et al. 2004, Cherradi et al. 2006. However, besides VEGF, a number of other angiogenic factors have been described, which may also be expressed in the adrenal cortex and participate in the regulation of its vascularization. ...

Reference:

Mitogenic functions of endocrine gland-derived vascular endothelial growth factor and Bombina variegata 8 on steroidogenic adrenocortical cells
P192 - Antagonistic functions of tis11B and HUR in the hormonal regulation of vascular endothelial growth factor MRNA stability by adrenocorticotropin
  • Citing Article

  • October 2005

Annales d Endocrinologie

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C. Lejczak

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... In contrast, much less is known concerning the role of TIS11b in cancer progression. We have previously shown that fusion of TIS11b to cell-penetrating peptides (CPPs) allows delivery of TIS11b into cells and decreased vascular endothelial growth factor (VEGF) expression, lung tumor growth, and vascularization [14][15][16]. More recently, TIS11b was reported to promote cell quiescence [17] and to belong to a tumor-suppressor network in T-cell leukemia [18]. ...

Reference:

Targeting AU-rich element-mediated mRNA decay with a truncated active form of the zinc-finger protein TIS11b/BRF1 impairs major hallmarks of mammary tumorigenesis
Antagonistic Functions of Tetradecanoyl Phorbol Acetate-Inducible-Sequence 11b and HuR in the Hormonal Regulation of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Stability by Adrenocorticotropin

Molecular Endocrinology

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Cyrille Lejczak

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