Figure 1.
Expression of mitochondrial kinases in 7-week-old ICR mouse tissues.
(A) Tissue-specific expression of Ak2, Ckmt1, Ckmt2, and Ndpk-d mRNA in adult mouse tissues. Br, brain; He, Heart; Lu, lung; St, stomach; LI, large intestine; Li, liver; Ki, kidney; Th, thymus; Sp, spleen; BM, bone marrow. Mu, skeletal muscle; Te, testis; 18S rRNA is presented as a loading control. Sizes of PCR products are presented on the right side of the panel. (B) Tissue-specific expression of Ak2, Ckmt1, Ckmt2, and Ndpk-d proteins in adult mouse tissues. β-Actin and Gapdh are presented as loading controls. Molecular weight is shown on the right side of the panel. (C) Relative levels of mRNA and protein expression of each enzyme in adult mouse tissues. The relative level is shown compared to the value of heart as 1. Sample numbers are shown as follows; N = 4 for Ak2 and Ndpk-d, N = 5 for Ckmt1, Ckmt2, Ak2, Ckmt1, Ckmt2 and Ndpk-d.
Figure 2.
Expression of mitochondrial kinases in mouse ES cells and embryos.
(A) RT-PCR analyses were performed on mouse ES cells and E8 embryos. ES, mouse ES cells; E8, mouse E8 embryos. Br, brain; Ki, kidney; and He, heart tissues were used as PCR controls. 18S rRNA is presented as a loading control. Sizes of PCR products are presented on the right side of the panel. (B) Western blot analysis was performed on mouse ES cells and E8 embryos. Pan-Actin antibody is used as a control. Molecular weight is shown on the right side of the panel. (C) Relative mRNA and protein expression values of each enzyme in mouse ES cells and E8 embryos. ES mRNA and all protein data, N = 3; E8 mRNA, N = 5.
Figure 3.
AK activity in mouse ES cells, E8 embryos, and 7-week-old adult mouse tissues.
(A) AK1 and AK2 activities in adult mouse tissues; (B) AK1 and AK2 activities in mouse ES cells and E8 embryos. The activity of each AK was normalized to total protein contents. Open and closed bars indicate AK1 and AK2 activity, respectively. Br, brain; He, Heart; Lu, lung; St, stomach; LI, large intestine; Li, liver; Ki, kidney; Th, thymus; Sp, spleen; BM, bone marrow; Mu, skeletal muscle; and Te, testis. N = 3. *, p<0.05.
Figure 4.
Regulation of mitochondrial kinase expression during HL-60 cell differentiation into macrophages or neutrophils.
(A) Morphological confirmation of macrophage- and neutrophil differentiation of HL-60 cells. HL-60 cells were treated with 20 nM PMA for macrophage differentiation or 10 µM ATRA for neutrophil differentiation. PMA-treated HL-60 cells were stained with Wright-Giemsa, and ATRA-treated HL-60 cells were stained with Giemsa. Scale bar, 100 µm. (B, C) Analysis of enzyme expression during macrophage- (B) and neutrophil differentiation (C) of HL-60 cells. CD11b was used as a marker of myeloid differentiation. Cont indicates a positive control used as follows; skeletal muscle was used for AK2, CKMT1 and CKMT2 in B and for CKMT1 and CKMT2 in C, and kidney was used for AK2 in C.
Figure 5.
Effects of AK2 knockdown on macrophage- and neutrophil differentiation of HL-60 cells.
(A) Effects of AK2 knockdown on macrophage- and neutrophil differentiation; N, control siRNA treatment; CD11b, differentiation marker; cont, mouse heart (AK2, CKMT1 and 2), human leukocyte (CD11b). (B) Rates of myeloid differentiation were assessed using NBT assays. Differentiated macrophage- and neutrophil HL-60 cells are NBT positive. N, control siRNA treatment; ** p<0.01. Experiments were performed in triplicate. (C) ROS measurement in HL-60 cells during macrophage- and neutrophil differentiations. Relative ratio of fluorescent intensity correlated to ROS production during myeloid differentiations was assessed at the each time point of the upper time course. Negative control siRNA treated samples, N = 3; AK2 siRNA treated samples, N = 4. * p<0.05.
Figure 6.
Working hypothesis for the role of AK2 during hematopoietic differentiation of bipotent HL-60 progenitor cells.
Human AK2-deficient hematopoietic progenitor cells can differentiate into macrophages but not into neutrophils (upper panel). During differentiation into macrophages (left panel), CKMT1 may interact with mitochondrial ATP synthase, ANT, and voltage-dependent anion channel (VDAC). This interaction recycles ATP-ADP in IMS without AK2. ATP from CKMT1-mediated ADP recycling could be used for cellular function including UPR to decrease ER stress induced by de novo neosynthesized proteins and to support macrophage differentiation. During neutrophil differentiation (right panel), AK2-deficient hematopoietic progenitor cells could not fully maintain mitochondrial adenine nucleotide homeostasis. The subsequent ER and oxidative stresses may impair differentiation and cellular functions. Disturbed adenine nucleotide metabolism in IMS may lead to ER stress and abnormal ROS production as shown in patients with RD and our AK2- deficient experimental models resulting in either neutropenia or impairment of neutrophil differentiation. IM and OM indicate mitochondrial inner and outer membranes, respectively. Open arrows and dotted-lines indicate the possible regulations, filled allows are the confirmed findings in this study.