Séverine Planel
Protein transduction domain-mediated intracellular delivery of the TTP family member TIS11b/BRF1 downregulates VEGF expression in vitro and in vivo
Published on 17 November 2008
Body text 1
Thesis presented November 17, 2008
Abstract:
In 2002, our team showed that stimulation of primary bovine adrenocortical cells byadrenocorticotropic hormone ACTH leads to a transcription-independent increase in VEGF expression. ACTH-induced VEGF expression is post-transcriptionally regulated through mRNA stabilization/destabilization via AU-rich elements (ARE) located in 3’-untranslated region (3’UTR) of VEGF mRNA. We previously characterized TIS11b as a zinc finger protein that is induced by ACTH concomitantly with VEGF regulation. TIS11b is a member of a protein family known for destabilizing short-lived mRNAs via ARE sequences. TIS11b interaction with VEGF mRNA has been subsequently confirmed and the implicated site has been identified.
In this context, the
first aim of my thesis was to evaluate the possibility to develop a new anti-angiogenic and anti-tumoral strategy using the mRNA-destabilizing ability of TIS11b in order to decrease VEGF levels in living tumor cells. To this end, TIS11b cDNA has been fused with PTDs (protein transduction domain, Tat derived from HIV, or the polyarginine peptides R7 and R9). PTDs allowed TIS11b to enter living cells and to intracellularly target VEGF mRNA. We report here that 100 nM of Flag-R7- and Flag-R9-TIS11b fusion proteins decrease VEGF mRNA level as well as VEGF protein level, by 40% after 24h in cultured cells. Moreover, we showed that a single injection of Flag-R9-TIS11b fusion protein into mouse adrenal glands decreases VEGF expression levels to 50% of control. Preliminary encouraging results of tumor growth inhibition were obtained with fusion protein injection into pre-established LL2 tumors in nude mice.
ACTH is a pituitary hormone that activates cAMP synthesis and the protein kinase A (PKA) pathway. The
second aim of my thesis was to characterize PKA-induced TIS11b phosphorylation in response to ACTH and to study its effect on TIS11b-mediated VEGF mRNA decay. For the first time, our team demonstrated that PKA phosphorylates TIS11b on serine 54
in vitro. A second ACTH-induced phosphorylation site, probably PKA-independent, has been identified on TIS11b-serine 334. These observations open a new field of investigation about the regulation of TIS11b mRNA destabilizing activity by specific phosphorylations.
Keywords:
VEGF, TIS11b, BRF1, ZFP36L1, Protein transduction, mRNA stability, Adrenal cortex, phosphorylation
Download this thesis.
Top page