TGF-beta1 activates mitochondrial biogenesis and energetic remodeling of NIH/3T3 fibroblasts

Aurora Affiliations

Center for Integrative Research on Cardiovascular Aging, Immunotherapy Research

Presentation Notes

Presented at 2013 Aurora Scientific Day, Milwaukee, WI


Background/significance: Differentiation of fibroblasts (FB) into myofibroblasts (myoFB) is the key event in the wound healing process of damaged tissue. Following tissue injury and initial migration and intensive proliferation, FB differentiate into myoFB, cells managing remodeling of extracellular matrix and depositing collagen. Phenotypic switch from proto-myoFB into myoFB is accompanied by remodeling cell metabolism and conversion of non-excitable precursors into the contracting cells capable of synthesis and deposition of structural proteins which are orchestrated by metalloproteinase(s) into extracellular matrix. In addition to remodeling of extracellular matrix myoFB also became excitable cells, capable of mechanical contraction.

Purpose: We hypothesized that TGF-beta1 mediated differentiation of naive NIH/3T3 FB into myoFB will be associated with energetic remodeling and increased presence of mitochondria as more efficient source of intracellular ATP.

Methods: NIH/3T3 cell line obtained from ATCC. Current study used confocal microscopy, Seahorse Extracellular Flux Respirometry, multiwell plate reader and standard biochemical and biophysical protocols. Vimentin, alpha-SMA and mitochondrial proteins were determined using Western blotting mt-DNA was quantified with Mitochondrial DNA kit; matrix metalloproteinase(s) were determined using Zymogel.

Results: TGF-beta1 exposure induced differentiation of naïve NIH 3T3 cells into differentiated myofibroblasts as verified by increased expression of characteristic alpha-SMA from 0.87±0.2 to 1.44±0.27 RU (normalized to housekeeping GAPDH). At the same time, TGF-beta1 exposure increased the number of mitochondria per cell, doubled baseline respiration of differentiated cells, increased average mitochondrial DNA/cell as well as expression of mitochondria specific proteins such as ANT. TGF-beta1 exposure increased baseline respiration of NIH/3T3 cells from 1.13±0.1 nmol O2/min/106 cells in naïve culture to 2.25±0.03 nmol O2/min/106 cells in TGF-beta1 treated cells. Mitochondrial DNA more than doubled from 307±9 μg DNA/106 cells in control to 530±12 μg DNA/106 cells in TGF-beta1 exposed cells and was paralleled by increased expression of mitochondrial specific proteins: VDAC 58 % and ANT by 57 %.

Conclusion: TGF-beta1 dependent differentiation of NIH/3T3 fibroblasts results in energetic remodeling and activation of mitochondrial biogenesis due to increased energy demand, which is compensated and fulfilled by increased mitochondrial biogenesis as the source of more efficient source of ATP.

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