Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Figures

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  Figure 1. AK4 is upregulated in primary human pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia. (A) Relative mRNA expression of AK1–AK9 genes comprising the adenylate kinase family in PASMCs exposed to 21% O2 (normoxia, NOX) or 1% O2 (hypoxia, HOX) for 72 h; n = 3. (B) Relative mRNA expression of AK4 in PASMCs exposed to normoxia (NOX) or hypoxia (HOX, 1% O2) for the indicated time points, compared to NOX; n = 3. (C) Representative Western blot for AK4 followed by densitometric quantification of relative AK4 expression in PASMCs exposed to normoxia (NOX) or hypoxia (HOX, 1% O2) for the indicated time points, compared to NOX; n = 5. (D) Immunocytochemical localization of AK4 (in green) in PASMCs cultured under normoxia (NOX) or hypoxia (HOX, 1% O2) for 48 h. Nuclear staining with DAPI (in blue). Scale bar: 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, (A) unpaired Student’s t-test or (B,C) one-way ANOVA followed by Dunnett multiple comparisons test.

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  Figure 2. Hypoxia upregulates AK4 in an HIF-1α-dependent manner. (A) Representative Western blot for HIF-1α and AK4 followed by densitometric quantification of relative expression in PASMCs treated with 50 µM DFO for 24 h, compared to untreated control; n = 4. (B) Representative Western blot for HIF-1α and AK4 and densitometric quantification of relative expression in PASMCs transfected with HIF-1α siRNA (siHIF-1α), followed by hypoxic (HOX, 1% O2) exposure for 24 h, compared to non-targeting siRNA as a control (scrambled siRNA = scramble) under NOX or HOX; n = 3. (C,E) Relative mRNA expression of AK4 in PASMCs transfected with siHIF-1α (C) or siEPAS1 (E), followed by hypoxic (HOX, 1% O2) exposure for 24 h, compared to control (scramble); n = 3–5. (D) Representative Western blot for EPAS1 (HIF-2α) and AK4 and densitometric quantification of relative expression in PASMCs transfected with EPAS1 siRNA (siEPAS1), followed by hypoxic (HOX, 1% O2) exposure for 24 h, compared to control (scramble) under NOX or HOX; n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001, (A,C,E) unpaired Student’s t-test or (B,D) one-way ANOVA followed by Tukey multiple comparisons test.

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  Figure 3. AK4 regulates the viability and proliferation of PASMCs both under normoxia and hypoxia. For all experiments, PASMCs were transfected with siRNA-targeting AK4 (siAK4) or non-targeting control (scrambled siRNA = scramble) followed by exposure to normoxia (NOX) or hypoxia (HOX, 1% O2) for the indicated time points. (A) Relative mRNA expression of AK4 in PASMCs after AK4 knockdown (siAK4), compared to corresponding control (scramble) under NOX or HOX for 24, 48, and 72 h; n = 3. (B) Representative Western blot for AK4 followed by densitometric quantification of relative expression in PASMCs after AK4 knockdown (siAK4), compared to corresponding control (scramble) under NOX or HOX for 24 h; n = 5. (C) PASMC apoptosis measured by pSIVA-IANBD probe after AK4 knockdown (siAK4), compared to corresponding control (scramble) under NOX or 24 h HOX; n = 6. (D) PASMC viability measured by alamarBlue assay after AK4 knockdown (siAK4), compared to corresponding control (scramble) under NOX or HOX for 76 h; n = 6. (E) PASMC proliferation assessed by BrdU incorporation assay after AK4 knockdown (siAK4), compared to corresponding control (scramble) under NOX or HOX for 72 h; n = 5. (F) Relative…

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  Figure 4. AK4 regulates mitochondrial respiratory function of PASMCs. (A) Representative graphs of high-resolution respirometry of intact PASMCs after transfection with control (left) scramble and siAK4 (right) treated with 4.5 µM oligomycin, sequential injections of FCCP (0.45 µM each), and 4.5 µM antimycin A. Measurements were performed with an Oroboros Oxygraph-2k instrument. The blue line indicates the oxygen concentration and the red line the oxygen flux per 106 cells (O2 consumption). Gray vertical lines labeled 01, 02, 03, and 04 indicate the time points selected for quantification of respiration. (B) Schematic graph of O2 consumption rates after sequential addition of oligomycin, FCCP and antimycin A with depicted parameters of coupled and uncoupled respiration. (C) Mitochondrial respiration parameters of intact PASMCs after AK4 knockdown under normoxia, determined from the high-resolution respirometry measurements; n = 5. (D) Mitochondrial respiration parameters of intact PASMCs after AK4 knockdown followed by 50 µM DFO treatment for 24 h, determined from the high-resolution respirometry measurements; n = 3. * p < 0.05, ** p < 0.01, unpaired Student’s t-test.

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  Figure 5. AK4 downregulation decreases the glycolytic metabolism in PASMCs. (A) Representative Western blots for HK2, LDHA, and PDHK1 followed by densitometric quantification of relative expression in PASMCs transfected with siAK4 followed by exposure to normoxia (NOX) or hypoxia (HOX, 1% O2) for 72 h (HK2 and LDHA detection) or 24 h (PDHK1 detection), compared to corresponding control (scrambled siRNA = scramble) under NOX or HOX; n = 4–6. (B) Lactate concentration in PASMC cultured media after AK4 knockdown followed by exposure to normoxia (NOX) or hypoxia (HOX, 1% O2) for 72 h, compared to corresponding control (scramble) under NOX or HOX; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni multiple comparisons test.

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  Figure 6. AK4 downregulation reduces HIF-1α levels and Akt signaling in PASMCs. (A,B) Representative Western blots for AK4 and HIF-1α and densitometric quantification of relative HIF-1α expression in PASMCs after AK4 downregulation followed by (A) treatment with 50 µM DFO for 6 h or (B) exposure to hypoxia (HOX, 1% O2) for 48 h; n = 3. (C) Relative mRNA expression of HIF-1α in PASMCs after AK4 knockdown (siAK4) under hypoxia (HOX, 1% O2) for 48 h, compared to control (scrambled siRNA = scramble); n = 3. (D) Representative Western blots for phospho-Akt (p-Akt) and total-Akt (t-Akt) followed by densitometric quantification of p-Akt levels in PASMCs transfected with siAK4 followed by exposure to normoxia (NOX) or hypoxia (HOX, 1% O2) for 72 h, compared to corresponding control (scramble) under NOX or HOX; n = 3–5. * p < 0.05, *** p < 0.001, **** p < 0.0001, (A–C) unpaired Student’s t-test or (D) two-way ANOVA followed by a Bonferroni multiple comparisons test.

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  Figure 7. AK4 is increased in the pulmonary vasculature of IPAH patients and regulates glycolytic metabolism of IPAH-PASMCs. (A) Top: Representative images of immunohistochemical staining for AK4 (in pink) in donor and IPAH lungs; Bottom: Representative images of von Willebrand factor (vWF) (brown)-stained and α-smooth muscle actin (α-SMA) (violet)-stained lung tissue from donor and IPAH patients. Nuclear staining with hematoxylin (blue). Scale bar: 100 µm. (B) Quantification of AK4 immunohistochemical staining area and intensity in lung tissue from donor and IPAH patients; n = 15. (C) IPAH-PASMC proliferation assessed by BrdU incorporation assay after AK4 knockdown (siAK4), compared to control (scrambled siRNA = scramble); n = 3. (D) Representative Western blots for HK2, LDHA, PDHK1, and AK4 followed by densitometric quantification of their relative expression in IPAH-PASMCs transfected with siAK4 for 72 h, compared to control (scramble); n = 3. (E) Lactate concentration in IPAH-PASMC cultured media after AK4 knockdown for 72 h, compared to control (scramble); n = 3. * p < 0.05, ** p < 0.01, **** p < 0.0001, unpaired Student’s t-test.

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  Figure 8. Schematic diagram depicting a possible role of AK4 in promoting a glycolytic, pro-proliferative, and pro-survival phenotype of PASMCs. Hypoxia upregulates AK4 in an HIF-1α-dependent manner. AK4 upregulation leads to Akt activation and increases HIF-1α levels, possibly by increasing ROS, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. The AK4-HIF-1α axis gives rise to increased levels of glycolytic enzymes (HK2, LDHA, PDHK1), promotes a metabolic shift from OXPHOS towards glycolysis (glycolytic shift), and increases PASMC proliferation and viability. This abnormal phenotype of PASMCs is a hallmark of the pulmonary vascular remodeling in pulmonary hypertension. Thus, targeting AK4 may represent a new therapeutic strategy for PH.