Differential Expression of Adenine Nucleotide Converting Enzymes in Mitochondrial Intermembrane Space: A Potential Role of Adenylate Kinase Isozyme 2 in Neutrophil Differentiation

Abstract

Adenine nucleotide dynamics in the mitochondrial intermembrane space (IMS) play a key role in oxidative phosphorylation. In a previous study, Drosophila adenylate kinase isozyme 2 (Dak2) knockout was reported to cause developmental lethality at the larval stage in Drosophila melanogaster. In addition, two other studies reported that AK2 is a responsible gene for reticular dysgenesis (RD), a human disease that is characterized by severe combined immunodeficiency and deafness. Therefore, mitochondrial AK2 may play an important role in hematopoietic differentiation and ontogenesis. Three additional adenine nucleotide metabolizing enzymes, including mitochondrial creatine kinases (CKMT1 and CKMT2) and nucleoside diphosphate kinase isoform D (NDPK-D), have been found in IMS. Although these kinases generate ADP for ATP synthesis, their involvement in RD remains unclear and still an open question. In this study, mRNA and protein expressions of these mitochondrial kinases were firstly examined in mouse ES cells, day 8 embryos, and 7-week-old adult mice. It was found that their expressions are spatiotemporally regulated, and Ak2 is exclusively expressed in bone marrow, which is a major hematopoietic tissue in adults. In subsequent experiments, we identified increased expression of both AK2 and CKMT1 during macrophage differentiation and exclusive production of AK2 during neutrophil differentiation using HL-60 cells as an in vitro model of hematopoietic differentiation. Furthermore, AK2 knockdown specifically inhibited neutrophil differentiation without affecting macrophage differentiation. These data suggest that AK2 is indispensable for neutrophil differentiation and indicate a possible causative link between AK2 deficiency and neutropenia in RD.

Mitochondrial Activity and Unfolded Protein Response are Required for Neutrophil Differentiation

“…During macrophage differentiation, we also observed reduction in BiP expression to that on day 1. We confirmed that neutrophil and macrophage differentiation is completed at days 4 and 1, respectively, based on morphological changes and marker expressions as shown previously [11]. Thus, we found the reduction in ER stress marker expression during myeloid differentiation in our system.…”

“…In our previous study, we demonstrated that HL-60 cells treated with ATRA in vitro for 4 days functionally differentiated and showed phagocytic activity and NBT-reducing capacity [11]. Therefore, we believe that XBP-1 may be essential at the late stage of neutrophil differentiation to become functionally matured neutrophils.…”

Recent advances in understanding the pathogenesis and management of reticular dysgenesis

“…These observations challenge the hypothesis that AK1 expression can compensate for an AK2 defect and for cell death in non‐leucocytes (Pannicke et al , ). Eventually, other mitochondrial inter‐membrane space kinases may substitute for a loss of AK2 function dependent on their respective expression patterns (Tanimura et al , ). In an additional model, neutrophil generation from the HL‐60 promyelocytic cell line with all‐ trans retinoic acid confirmed the AK2 dependence of neutrophil differentiation (Tanimura et al , ; Six et al , ).…”

Severe combined immunodeficiency—an update

“…The only available treatment for RD is allogeneic HSCT, which indicates that the inherited defect is cellular and not linked to the micro‐environment, as previously thought. Neutrophil differentiation abnormalities of RD patients are corrected by the restoration of AK2 expression in the bone marrow, thus confirming the specific role of AK2 in the development of the myeloid lineage . Moreover, AK2 is specifically expressed in the stria vascularis region of the inner ear, which explains the sensorineural deafness observed in these individuals .…”

“…Neutrophil differentiation abnormalities of RD patients are corrected by the restoration of AK2 expression in the bone marrow, thus confirming the specific role of AK2 in the development of the myeloid lineage. 12 Moreover, AK2 is specifically expressed in the stria vascularis region of the inner ear, which explains the sensorineural deafness observed in these individuals. 10 AK2 is localized in the mitochondrial intermembrane space where it regulates adenine nucleotide interconversion within the intermembrane space; 13 a very similar function is mediated by the cytoplasmatic enzyme AK1.…”