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Updated: Dec 30, 2025
Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue
Published on: July 28, 2023
TSG-6 Inhibits the Growth of Keloid Fibroblasts Via Mediating the TGF-β1/Smad Signaling Pathway
Xin-Yi Li1, Xiao-Juan Weng1, Xiao-Jing Li1
1Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University, China.
Background:
The cytokine TNF-α-stimulated gene-6 (TSG-6) had been verified to have a certain inhibitory effect on the inflammation. During wound healing, fibroblasts increasingly proliferated and deposited collagen fibers, leading to the formation of pathological scars. We sought to elucidate the mechanism by which the TGF-β1/Smad pathway was mediated by TSG-6 in human keloid fibroblasts.
Materials And Methods:
Human keloid fibroblast cells were isolated from keloid tissue by enzyme digestion and identified by immunocytochemistry. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed which were then transfected into human keloid fibroblasts. The mRNA and protein levels of TSG-6 were detected respectively by RT-PCR and western blot assay. The intracellular localization of TGF-β1-induced proteins and phosphorylated (p)-Smad2/3 in keloid fibroblasts were investigated using an immunofluorescence assay. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was detected by RT-PCR.
Results:
TSG-6 could effectively interfere the TGF-β1/Smad signal transduction pathway in keloid fibroblasts rather than in normal fibroblasts. The phosphorylation levels of Smad2/3 were notably reduced by TSG-6 treatment. TSG-6 blocked the complex formation of Smad2/3/4, and their nuclear translocation. However, it upregulated Smad7 expression, presenting dose dependence. PAI-1 was also suppressed by TSG-6 treatment.
Conclusions:
TSG-6 inhibits proliferation by inducing apoptosis in keloid fibroblasts, which may be associated with TGF-β1/Smad pathway.
Insights
Tumor necrosis factor-alpha-stimulated gene-6 (TSG-6) inhibits keloid fibroblast proliferation by interfering with the TGF-β1/Smad pathway. This mechanism involves reduced Smad2/3 phosphorylation and nuclear translocation, potentially offering new therapeutic strategies for scar reduction.
Area of Science:
- Biochemistry
- Cell Biology
- Dermatology
Background:
- Tumor necrosis factor-alpha-stimulated gene-6 (TSG-6) exhibits anti-inflammatory properties.
- Fibroblast proliferation and collagen deposition contribute to pathological scar formation.
- Understanding TSG-6's role in keloid pathogenesis is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the mechanism of TSG-6 in human keloid fibroblasts.
- To determine TSG-6's effect on the transforming growth factor-beta 1 (TGF-β1)/Smad signaling pathway.
Main Methods:
- Isolation and culture of human keloid fibroblasts.
- Transfection with lentiviral vectors to modulate TSG-6 expression.
- Analysis of TSG-6, TGF-β1, and Smad protein/mRNA levels using RT-PCR and Western blot.
- Immunofluorescence assays to assess Smad2/3 phosphorylation and nuclear translocation.
- Assessment of plasminogen activator inhibitor-1 (PAI-1) transcriptional activity.
Main Results:
- TSG-6 significantly interfered with the TGF-β1/Smad pathway in keloid fibroblasts.
- TSG-6 reduced Smad2/3 phosphorylation, blocked Smad2/3/4 complex formation, and inhibited nuclear translocation.
- TSG-6 upregulated Smad7 expression in a dose-dependent manner and suppressed PAI-1 activity.
Conclusions:
- TSG-6 inhibits keloid fibroblast proliferation, potentially through inducing apoptosis.
- The observed effects are associated with the modulation of the TGF-β1/Smad signaling pathway.
- TSG-6 represents a potential therapeutic agent for managing keloid scarring.
