Study of the effects of a sublethal and transient endoplasmic reticulum stress on mitochondrial population of HepG2 cells

PhD thesis defended by Kayleen VANNUVEL (Prof. Thierry ARNOULD) - 09/10/2015

Prof. Thierry ARNOULD, UNamur, Laboratory of Cellular and Molecular Biology (URBC), Organelle dysfunction (DYSO) group


Thierry Arnould (Supervisor), Martine Raes (Co-Supervisor), Michel Jadot (Jury), Jean-Jacques Letesson (President), Jean-Christophe Jonas (Jury), Jennifer Rieusset (Jury) & Jean-Paul Thissen (Jury)


Over the past years, growing knowledge and evidence about the existence of crosstalks between cellular organelles and their potential effects on cell survival or cell death have emerged. Especially, evidence accumulated showing an intimate relationship/interactions between the endoplasmic reticulum (ER) and mitochondria. These close contacts not only establish physical and functional interactions allowing exchange of calcium and lipids but are also involved in several biological processes including mitochondrial bioenergetics and regulation of apoptotic cell death pathways. It is now well established that severe ER stress/dysfunction and activation of the unfolded protein response (UPR) can trigger the impairments of mitochondrial functions leading to the subsequent apoptosis having an impact on several pathologies. However, it becomes more and more evident that cells might also regularly deal with transient and sublethal ER stresses which do not necessarily lead to cell death but might still affect the function and/or the morphology of other organelles such as mitochondria.

The aim of this work was to study the putative effects of a mild and transient ER stress on the mitochondrial population bioenergetics and morphology in HepG2 (human hepatocellular carcinoma) cells. The first part of this work was to characterize the induction of a non-lethal and transient ER stress that triggers UPR (Unfolded protein response) by using two well-known ER stress inducers : thapsigargin (TG), an inhibitor of the SERCA pumps, inhibiting the entry of calcium in the ER lumen and leading to an ER stress due to a depletion of calcium necessary for ER folding chaperones, and brefeldin A (BFA), an inhibitor of the transport of proteins from the ER lumen to the Golgi apparatus causing an accumulation of proteins within the ER. We provide evidence that both molecules trigger a mild (defined as a non-lethal stress) and transient ER stress and activate the unfolded protein response without inducing apoptosis.
In the second part, we focused on the putative effects of the induction of an ER stress on the cell mitochondrial population. We first observed that a non-lethal stress provokes fragmentation of the mitochondrial network which is accompanied with a decrease in the mitochondrial membrane potential, mitochondrial ROS production and respiration. Moreover, we observed that these mitochondrial impairments are reversible as not sustained during a recovery period during which cells are no longer exposed to the ER stress inducers. These results suggest that cells respond and are able to adapt to the applied stress. Mechanistically, we found that these mitochondrial dysfunctions are, at least partly, dependent on the activation of JNK. Indeed, we showed that in the presence of a JNK inhibitor, ER stress induced changes in the mitochondrial morphology are no longer observed. Moreover, the reduction of mtROS production induced by the ER stress was less severe in both BFA- and TG-treated cells in the presence of the JNK inhibitor even if this molecule does not have any effect on the reduction of the mitochondrial membrane potential triggered by the ER stress.

Our study brings new evidence on the close relationship existing between the ER and mitochondria under non-lethal and transient ER stress showing that cells are able to respond and to adapt to the stress without inducing cell death. However, the cytoprotective mechanisms underlying these observations remain to be determined.