Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle
Abstract
:1. Introduction
2. Results
2.1. Dissociated Bronchiolar Cells Exhibited hASM Phenotype
2.2. TNFα Increased Mitochondrial Volume Density in hASM Cells
2.3. TNFα Increases mtDNA Copy Number and Mitochondrial Biogenesis in hASM Cells
2.4. TNFα-Induced pCREBS133 and pATF1S63 Phosphorylation
2.5. pCREBS133 Transcriptionally Activates PGC1α in TNFα-Treated hASM Cells
2.6. TNFα Increases the Expression of PGC1α in hASM Cells
2.7. PGC1α Transcriptionally Activates Expression of Downstream Gene Targets
3. Discussion
3.1. TNFα Increases Mitochondrial Volume Density in hASM Cells
3.2. TNFα Increases Mitochondrial Biogenesis in hASM Cells
3.3. TNFα Increases pCREBS133 and pATF1S63 Phosphorylation in hASM Cells
3.4. pCREBS133 Transcriptionally Activates PGC1α Expression in hASM Cells
3.5. PGC1α Activates Transcription of NRFs and TFAM in hASM Cells
3.6. TNFα Induces an Increase in Metabolic Demand in hASM Cells
3.7. Clinical Significance
3.8. Experimental Limitations and Future Studies
4. Materials and Methods
4.1. Dissociation of Cells from Bronchiolar Tissue
4.2. Phenotyping Dissociated hASM Cells
4.3. Experimental Design
4.4. Protein Extraction and Western Blot
4.5. Labeling and Confocal Imaging of Mitochondria in hASM Cells
4.6. Measurement of Mitochondrial Volume Density
4.7. Genomic DNA Extraction and Quantification of mtDNA
4.8. Bioinformatic Analysis for Transcription Factor Binding Site Prediction
4.9. Chromatin Immunoprecipitation Assay (ChIP)
4.10. RNA Extraction, cDNA Preparation and qPCR
4.11. Statistical Analysis
5. Summary and Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primary Ab | Manufacturer | Catalog Number | Application | Dilution |
---|---|---|---|---|
α-SMA | Abcam, Boston, MA, USA | ab5694 | ICC | 1:500 |
FSP1/S100a4 | Abcam, Boston, MA, USA | ab124805 | ICC | 1:500 |
α-SMA | Abcam, Boston, MA, USA | ab5694 | Western blot | 1:1000 |
PGC1α | Novus Biologicals, Centennial, CO, USA | NBP1-04676 | Western blot | 1:1000 |
pCREBS133/pATF1S63 | Cell Signaling Technology, Danvers, MA, USA | 9198S | Western blot | 1:1000 |
Total CREB | Cell Signaling Technology, Danvers, MA, USA | 9104S | Western blot | 1:1000 |
Total CREB | Cell Signaling Technology, Danvers, MA, USA | 4820S | ChIP | 1:1000 |
NRF1 | Invitrogen, Carlsbad, CA, USA | MA5-32782 | Western blot | 1:1000 |
Gene Name | Primer Name | Primer Sequence |
---|---|---|
NRF1 | NRF1-F | 5′-GCAACAGTAGCCACATTGGCT-3′ |
NRF1-R | 5′-GTCGTCTGGATGGTCATCTCAC-3′ | |
NRF2 | NRF2-F | 5′-CACATCCAGTCAGAAACCAGTGG3′ |
NRF2-R | 5′-GGAATGTCTGCGCCAAAAGCTG-3′ | |
PGC1α | PGC1α -ChIP-F | 5′-TGCTTGAAGCCTCCAAAAGT-3′ |
PGC1α -ChIP-R | 5′-AGTAGGCTGGGCTGTCACTC-3′ | |
RPS16 | RPS16-F | 5′-GTCTGTGCAGGTCTTCGGACGC-3′ |
RPS16-R | 5′-GACCATTGCCGCGTTTGCAGTG-3′ | |
TFAM | TFAM-F | 5′-GTGGTTTTCATCTGTCTTGGCAAG-3′ |
TFAM-R | 5′-TTCCCTCCAACGCTGGGCAATT-3′ |
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Dasgupta, D.; Mahadev Bhat, S.; Price, A.L.; Delmotte, P.; Sieck, G.C. Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle. Int. J. Mol. Sci. 2023, 24, 5788. https://doi.org/10.3390/ijms24065788
Dasgupta D, Mahadev Bhat S, Price AL, Delmotte P, Sieck GC. Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle. International Journal of Molecular Sciences. 2023; 24(6):5788. https://doi.org/10.3390/ijms24065788
Chicago/Turabian StyleDasgupta, Debanjali, Sanjana Mahadev Bhat, Alexis L. Price, Philippe Delmotte, and Gary C. Sieck. 2023. "Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle" International Journal of Molecular Sciences 24, no. 6: 5788. https://doi.org/10.3390/ijms24065788