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
Abstract
:1. Introduction
2. Results
2.1. Hypoxia Upregulates AK4 in Primary Human Pulmonary Arterial Smooth Muscle Cells (PASMCs)
2.2. AK4 Is Upregulated in Hypoxic PASMCs in a HIF-1α-Dependent Manner
2.3. AK4 Regulates the Viability and Proliferation of PASMCs under Both Normoxia and Hypoxia
2.4. Downregulation of AK4 Augments the Mitochondrial Respiratory Function and Reduces the Glycolytic Metabolism in PASMCs
2.5. AK4 Interacts with HIF-1α and Akt Signaling Pathways in PASMCs
2.6. AK4 Is Increased in the Lungs of IPAH Patients and Regulates the Glycolytic Metabolism of IPAH-PASMCs
3. Discussion
4. Materials and Methods
4.1. Human Lung Samples
4.2. Cell Culture and Hypoxia Exposure of PASMCs
4.3. RNA Interference by Synthetic siRNA
4.4. Cell Viability Assay
4.5. Apoptosis Assay
4.6. Cell Proliferation Assay
4.7. Lactate Production Assay
4.8. Measurement of PASMC Mitochondrial Respiration Using High-Resolution Respirometry
4.9. Immunocytochemistry
4.10. Immunohistochemistry
4.11. RNA Isolation and RT-qPCR
4.12. Western Blot
4.13. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AK4 | adenylate kinase 4 |
Akt | protein kinase B |
B2M | beta-2-microglobulin |
BrdU | bromodeoxyuridine |
DFO | deferoxamine |
EPAS1 | endothelial PAS domain-containing protein 1 (HIF-2α) |
FCCP | carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone |
HIF | hypoxia inducible factor |
HK2 | hexokinase 2 |
HOX | hypoxia |
IPAH | idiopathic pulmonary arterial hypertension |
LDHA | lactate dehydrogenase A |
Ki-67 | marker of proliferation Ki-67 |
NOX | normoxia |
OCR | oxygen consumption rate |
OXPHOS | oxidative phosphorylation |
PASMCs | pulmonary arterial smooth muscle cells |
PBS | phosphate-buffered saline |
PDHK1 | pyruvate dehydrogenase kinase 1 |
PH | pulmonary hypertension |
RT | room temperature |
RT-qPCR | reverse transcription quantitative polymerase chain reaction |
scramble | scrambled siRNA (control siRNA) |
TCA | tricarboxylic acid |
VSMCs | vascular smooth muscle cells |
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Wujak, M.; Veith, C.; Wu, C.-Y.; Wilke, T.; Kanbagli, Z.I.; Novoyatleva, T.; Guenther, A.; Seeger, W.; Grimminger, F.; Sommer, N.; et al. 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. Int. J. Mol. Sci. 2021, 22, 10371. https://doi.org/10.3390/ijms221910371
Wujak M, Veith C, Wu C-Y, Wilke T, Kanbagli ZI, Novoyatleva T, Guenther A, Seeger W, Grimminger F, Sommer N, et al. 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. International Journal of Molecular Sciences. 2021; 22(19):10371. https://doi.org/10.3390/ijms221910371
Chicago/Turabian StyleWujak, Magdalena, Christine Veith, Cheng-Yu Wu, Tessa Wilke, Zeki Ilker Kanbagli, Tatyana Novoyatleva, Andreas Guenther, Werner Seeger, Friedrich Grimminger, Natascha Sommer, and et al. 2021. "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" International Journal of Molecular Sciences 22, no. 19: 10371. https://doi.org/10.3390/ijms221910371