Le médicament épigénétique 5-Azacytidine stabilise l’ARN messager du récepteur des lipoprotéines de basse densité (LDLR) via une voie IRE1α/EGFR/ERK1/2- dépendante

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in Canada and around the world. Several clinical trials have shown that reducing lowdensity lipoprotein (LDL-c) cholesterol is an effective strategy for preventing atherosclerosis and coronary heart disease to reduce mortality. The low density lipoprotein receptor (LDLR) is a transmembrane glycoprotein that serves for the binding and endocytosis of LDL particles. In humans, the LDLR is strongly expressed in the liver, and is responsible for the elimination of 70% to 80% of LDL-c from plasma. Genetic mutations can lead to dysfunctional LDLR and cause familial hypercholesterolemia (FH), a disease characterized by a very high level of blood LDL-c and the formation of premature atherosclerotic plaques.  The expression of LDLR is strictly controlled by transcription factors called sterol regulatory element-binding proteins (SREBPs) in response to excessive accumulation or depletion of intracellular cholesterol. Post-translational regulation of LDLR is mediated by its intracellular degradation via the proprotein convertase subtilisin / kexin type 9 (PCSK9) or by the ubiquitin ligase E3, the inducible degrader of LDLR (IDOL). On the other hand, our knowledge about the post-transcriptional regulation of LDLR mRNA is still very little advanced.  The post-transcriptional regulation of LDLR mRNA stability has been shown to be mediated through the mRNA 3’ untranslated region (3’UTR). Human LDLR mRNA contains a 2.5-kb-long stretch of 3’UTR in which 3 AU-rich elements (AREs) were previously identified as a mediators of rapid turnover rate of LDLR mRNA in liver cells.   In this thesis, we sought answers to the following questions: (1) At which level does 5-Azacytidine (5-AzaC), an antimetabolite used for the treatment of leukemia and myelodysplastic syndrome, induce regulation of the LDLR? (2) Which cell signaling pathways are involved in 5-AzaC LDLR stabilization? (3) Is the 5-AzaC LDLR mRNAstabilization - ER stress- dependent?  1) We first demonstrated that 5-AzaC increased the expression of LDLR in human hepatic cells (HepG2 and Huh7) by a new mechanism independent of the transcription factors SREBP. In order to elucidate the effects of 5-AzaC on lipoprotein metabolism, we investigated its effect on LDLR mRNA expression. We observed a significant stabilization of its mRNA as well as an increase in the level of the membrane protein. Our results also show that 5-AzaC induces the stabilization of the LDLR mRNA by a post-transcriptional mechanism requiring the presence of its 3'UTR sequence. Systematic mutations of the three AREs (ARE1-3) motifs located at the 3'UTR sequence of the LDLR mRNA and their luciferase-like expression in Huh-7 cells showed that the ARE1 motif is both the primary determinant of LDLR mRNA lability and the target of 5-AzaCinduced mRNA stabilization.  2) We pinpointed a new signaling pathway required in LDLR post-transcriptional regulation involving the inositol-requiring enzyme 1α (IRE1α), the c-Jun N-terminal kinase (JNK), the epidermal growth factor receptor (EGFR) and the extracellular signaling-regulated kinase1/2 (ERK1/2). In hepatocytes, 5-AzaC induced the activation of the EGFR receptor after 6 h of treatment. This effect could be explained by the increase in receptor-specific ligands such as amphiregulin (AREG) (40-fold) and epiregulin (EREG) (60-fold). The phosphorylation of ERK1/2 was increased 2 h after the JNK pathway activation by IRE1α.  3) As opposed to the results obtained with thapsigargin (Th), a potent ER stress inducer, our results showed that 5-AzaC activates the IRE1α pathway independently of the ER stress. Blocking of the IRE1α kinase domain with the specific inhibitor (KIRA 6) blunted the increase of LDLR mRNA and protein levels in 5-AzaC-treated cells. Contrarily, the inhibition of the endonuclease domain of IRE1α, using the specific inhibitor STF 083010 or by its mutation, did not affect the 5-AzaC LDLR up-regulation neither IRE1α and p-ERK1/2 activation.  In summary, this work allowed us to identify 5-AzaC as an important stabilizer of the LDLR mRNA in hepatocytes via the IRE1α/JNK-EGFR-ERK1/2 signaling cascade. This mechanism started by the specific activation of the IRE1α kinase domain independently to the ER stress and finished by the phosphorylation of ERK1/2 and the binding of the RNA-binding proteins (RBPs) ensuring LDLR mRNA stability. This study revealed that the 3'UTR sequence is necessary for the stabilization of the LDLR mRNA  and that the first ARE1 motif closest to the stop codon is the main cause of stabilization via 5-AzaC.  The identification of this new regulatory pathway will reveal potential targets to enhance plasma LDL-c clearance in the liver as well as new therapeutic strategies to reduce cholesterol levels.

Keyphrases: 5-AzaC, LDLR, 3'UTR, ARE1, IRE1α, C-JNK, EGFR, ERK1/2