The BCL2 family of proteins are well known for their ability to both positively and negatively regulate mitochondrial mechanisms of apoptosis. The anti-apoptotic members of this family can decrease mitochondrial outer membrane permeability and cytochrome c release. By stabilizing the cell against apoptosis, these proteins allow cell survival even in states of low energy. However, despite this intimate link between the BCL2 family proteins and mitochondria, their direct effect on metabolism is less clearly understood. It is generally expected that metabolic changes induced by the BCL2 family of proteins will further impact cell survival as murine hepatoma and cancer cells overexpressing Bcl-xL are sensitive to Bcl-xL inhibition1 but BCL-xL is also known to be essential for erythroid differentiation and more recently was linked specifically to heme synthesis2 . We therefore set out to investigate whether there was a connection between BCL-xL induced changes in cellular respiration and erythroid differentiation.
Murine erythroleukemia (MEL) cells were differentiated by exposure to 2% DMSO for 5 days and then real time oxygen consumption was measured on the Seahorse extracellular flux analyzer (XFA). DMSO induced differentiation yielded a 4-fold decrease in oxygen consumption. Western blot analysis revealed that BCL-xL was induced during differentiation. We then generated cell lines in which BCL-xL was knocked down with small hairpin RNA (shRNA). As differentiation has previously been reported to be fatal in MEL cells without BCL-xL activity, both parental cells and BCL-xL knockdowns were infected with a vector over expressing BCL2. Differentiation over 5 days with 2% DMSO was performed on these new cell lines. Erythroid differentiation was confirmed using Benzidine staining. While the control cell line showed high rates of Benzidine staining after exposure to DMSO, the BCL-xL knockdown cell line consistently showed <5% benzidine positivity. Western blot analysis confirmed the absence of BCL-xL induction by DMSO exposure in the knockdown cell line. Using the Seahorse XFA the control cell line was shown to have significant decrease in oxygen consumption when exposed to DMSO, while DMSO exposed BCL-xL knockdown cells showed less than half this drop in oxygen consumption. However, both control and BCL-xL knockdowns have limited respiratory reserve as the response to CCCP, an uncoupler of electron transport, is diminished after DMSO exposure as compared to their undifferentiated counterparts.
Our results suggest that erythroid differentiation is associated with a significant decrease in cellular respiration. Although, not the only contributor to the decreased dependence on oxidative phosphorylation of cells undergoing erythroid differentiation, BCL-xL expression is clearly a necessary factor. Our data is able to connect BCL-xL expression to both erythroid differentiation and this distinct metabolic phenotype. As BCL-xL's role in erythroid differentiation has previously been reported to be associated with heme synthesis, future work will focus on identifying oxidative metabolic pathways associated with BCL-xL expression and heme synthesis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.