Hypoxia-Induced Epithelial-To-Mesenchymal Transition Mediates Fibroblast Abnormalities via ERK Activation in Cutaneous Wound Healing
Abstract
:1. Introduction
2. Results
2.1. p-ERK and HIF-1α Levels Are Elevated in Keloid Tissue and Fibroblasts
2.2. Hypoxia Activates TGF-β Signaling and Induces EMT in HDFs
2.3. The ERK/MAPK Pathway Is Involved in Hypoxia-Induced EMT
2.4. Phenotypic EMT Markers Are Expressed in HDFs under Hypoxia
2.5. Hypoxia Induces a Phenotypic Switch of Fibroblasts to Myofibroblasts
2.6. ERK Inhibition Reduces Hypoxia-Induced ECM Deposition
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Keloid-Derived Fibroblasts
4.2. Real-Time PCR Analysis
4.3. Western Blotting Analysis
4.4. Cell Viability Analysis
4.5. Immunohistochemistry
4.6. Antibodies and Reagents
4.7. Confocal Imaging
4.8. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AFAP | Actin filament associated protein 1 |
CTGF | Connective tissue growth factor |
ECM | Extracellular matrix |
EMT | Epithelial-mesenchymal transition |
ERK | Extracellular signal-regulated kinase |
HDF | Human dermal fibroblast |
HIF-1α | Hypoxia-inducible factor-1α |
KF | Keloid fibroblast |
MAPK | Mitogen-activated protein kinase |
MMP | Matrix metalloproteinase |
PBS | Phosphate-buffered saline |
PI3K | Phosphatidylinositol-4,5-bisphosphate 3-kinase |
TGF-β | Transforming growth factor-β |
TIMP | Tissue inhibitor of metalloproteinases |
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Target Gene | Primer Sequences (5′–3′) or Assay ID | Reference |
---|---|---|
E-cadherin | Forward: CACCACGGGCTTGGATTTTG Reverse: TGGGGGCTTCATTCACATCC | [47] |
N-cadherin | Forward: TCAGGCGTCTGTAGAGGCTT Reverse: ATGCACATCCTTCGATAAGACTG | [48] |
Vimentin | Forward: GACGCCATCAACACCGAGTT Reverse: CTTTGTCGTTGGTTAGCTGGT | [49] |
TGF-beta | Forward: ACCCACAACGAAATCTATGACA Reverse: GCTGAGGTATCGCCAGGAAT | [50] |
HIF1A | Forward: ACTCATCCATGTGACCACG Reverse: TAGTTCTCCCCCGGCTAG | [51] |
COL1A1 | Forward: AAGGTGTTGTGCGATGACG Reverse: TGGTCGGTGGGTGACTCTG | [50] |
Beta-actin | Forward: CTACCTCATGAAGATCCTCACCGA Reverse: TTCTCCTTAATGTCACGCACGATT | [52] |
CTGF | Hs00170014_m1 | |
MMP9 | Hs00957562_m1 | |
MMP2 | Hs01548727_m1 | |
TIMP2 | HS00234278_m1 | |
TIMP1 | Hs01092512_g1 | |
COL1A1 | Hs00164004_m1 | |
Beta-actin | Hs01060665_g1 |
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Kim, J.; Kim, B.; Kim, S.M.; Yang, C.E.; Song, S.Y.; Lee, W.J.; Lee, J.H. Hypoxia-Induced Epithelial-To-Mesenchymal Transition Mediates Fibroblast Abnormalities via ERK Activation in Cutaneous Wound Healing. Int. J. Mol. Sci. 2019, 20, 2546. https://doi.org/10.3390/ijms20102546
Kim J, Kim B, Kim SM, Yang CE, Song SY, Lee WJ, Lee JH. Hypoxia-Induced Epithelial-To-Mesenchymal Transition Mediates Fibroblast Abnormalities via ERK Activation in Cutaneous Wound Healing. International Journal of Molecular Sciences. 2019; 20(10):2546. https://doi.org/10.3390/ijms20102546
Chicago/Turabian StyleKim, Jihee, Bomi Kim, Soo Min Kim, Chae Eun Yang, Seung Yong Song, Won Jai Lee, and Ju Hee Lee. 2019. "Hypoxia-Induced Epithelial-To-Mesenchymal Transition Mediates Fibroblast Abnormalities via ERK Activation in Cutaneous Wound Healing" International Journal of Molecular Sciences 20, no. 10: 2546. https://doi.org/10.3390/ijms20102546