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TGF-β1-mediated differentiation of fibroblasts is associated with increased mitochondrial content and cellular respiration. PLoS One 2015;10(4):e0123046

Date

04/08/2015

Pubmed ID

25849590

Pubmed Central ID

PMC4388650

DOI

10.1371/journal.pone.0123046

Scopus ID

2-s2.0-84927534750 (requires institutional sign-in at Scopus site)   68 Citations

Abstract

OBJECTIVS: Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.

METHODS: Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.

RESULTS: Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).

CONCLUSIONS: TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.

Author List

Negmadjanov U, Godic Z, Rizvi F, Emelyanova L, Ross G, Richards J, Holmuhamedov EL, Jahangir A

Author

Gracious R. Ross Research Scientist II in the Cardiovascular Center department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Blotting, Western
Cell Differentiation
Cell Respiration
Cells, Cultured
Fibroblasts
Immunoenzyme Techniques
Mice
Mitochondria
Mitochondrial Proteins
NIH 3T3 Cells
RNA, Messenger
Transforming Growth Factor beta1