A Dynamic Role for Bcl-xL in Cellular Metabolism and Apoptosis: Calcium Regulation at the ER-Mitochondria Interface

Abstract

Chinese hamster ovary (CHO) cells are used to produce nearly 70% of all recombinant protein products. High levels of apoptosis; however, are typical during production processes. As such, overexpression of Bcl-2 family proteins is commonly used in CHO cells to delay apoptosis and increase production. Bcl-2 family proteins are recognized for their apoptosis regulation at the mitochondria, yet new non-apoptotic roles for Bcl-2 family proteins have emerged including the mediation of intracellular calcium (Ca2+) homeostasis. Since the proper supply of Ca2+ in cells serves as an important mechanism for both cellular survival and bioenergetics, we explore the possibility of a dual purpose for anti-apoptotic Bcl-xL modulation of Ca2+ signaling. Firstly, we aim to clarify the multi-organelle localization and functions of Bcl-2 members in mediating Ca2+ signals and investigate the implications of Ca2+ as it relates to bioenergetics and apoptosis. Furthermore, to determine the mechanism by which anti-apoptotic proteins promote bioenergetics and cell survival, we investigated the localization of anti-apoptotic Bcl-xL in CHO cells. We found that Bcl-xL partially resides at the ER-mitochondria interface termed MAM (mitochondria-associated ER membrane). Since the MAM is known to house key Ca2+ signaling machinery, we were additionally interested in exploring the role of Bcl-xL in facilitating direct ER to mitochondrial Ca2+ signaling. We observed reduced cytosolic Ca2+ efflux and enhanced Ca2+ transfer directly from the ER to mitochondria following ER Ca2+ depletion. Bcl-2 expression additionally enhanced TCA cycle activity. Interestingly, prolonged thapsigargin treatment induced Bcl-xL translocation to the MAM and further promotes bioenergetics. Similar results were seen using CHO cells expressing a truncated form of Bcl-2 (Bcl-2∆) or a combination of anti-apoptotic proteins—E1B-19K, Aven and XIAP∆. Taken together, our data suggests that MAM-associated Bcl-xL may play a role in facilitating mitochondrial Ca2+ uptake to promote bioenergetics, which provides additional groundwork toward understanding CHO metabolism to further improve upon cell line development.