RIPK3 Expression in Fibroblasts in an in vivo and in vitro Skin Wound Model: A Controversial Result
I S Izumov1, M S Shitova1, M S Sabirov1
1Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, 119334 Russian Federation.
Abstract:
One of the major problems of regenerative medicine is the development of hypertrophic scars and keloids. The protein kinase RIPK3 is involved in necroptosis; however, recent evidence indicates that it also has non-canonical functions, including its involvement in the development of renal fibrosis. The aim of our work was to study the expression of RIPK3 in mouse and human skin models of fibrotic processes. A subpopulation of RIPK3+Vim+ cells was found in both human keloid and a mouse wound, with the cell number being significantly greater in the mouse wound bed compared to healthy skin. Real-time polymerase chain reaction (RT-PCR) detected expression of the Ripk3 and fibroblast biomarkers Acta2, Fap, Col1a1, and Fn1 in the cells isolated from the wound bed, indicating that RIPK3 can be expressed by wound bed fibroblasts. An analysis of the human fibroblasts stained with anti-RIPK3 antibodies demonstrated an increase in the fluorescence intensity in the presence of lipopolysaccharide (LPS) at concentrations of 5, 10, 25, 50, and 100 ng/ml and TGF-β at concentrations of 0.1, 1, 2, and 5 ng/ml compared to the control. At the same time, the expression levels of RIPK3 and fibroblast activation markers in the presence of TGF-β and LPS did not differ significantly from the control. It is possible that RIPK3 expression in wound fibroblasts is not directly associated with fibrotic processes, and that kinase plays a different, yet unknown role in wound healing. KEYWORDS scarring, keloid, skin, fibroblasts, cell culture, RIPK3.
Insights
Researchers investigated the role of RIPK3 in skin scarring and keloid development. They found RIPK3 is expressed in wound fibroblasts, but its direct association with fibrotic processes remains unclear, suggesting a novel role in wound healing.
Area of Science:
- Regenerative Medicine
- Dermatology
- Molecular Biology
Background:
- Hypertrophic scars and keloids are significant challenges in regenerative medicine.
- The protein kinase RIPK3, known for its role in necroptosis, also exhibits non-canonical functions, including involvement in renal fibrosis.
- Recent evidence suggests RIPK3 may play a role in fibrotic processes beyond its known functions.
Purpose of the Study:
- To investigate the expression of RIPK3 in mouse and human skin models of fibrotic processes.
- To determine if RIPK3 is expressed by fibroblasts in wound healing contexts.
- To explore the potential association between RIPK3 expression and fibrotic markers in skin.
Main Methods:
- Analysis of RIPK3 and Vimentin co-expression in human keloid and mouse wound skin.
- Isolation and gene expression analysis (RT-PCR) of wound bed fibroblasts for RIPK3 and fibroblast activation markers (Acta2, Fap, Col1a1, Fn1).
- In vitro stimulation of human fibroblasts with lipopolysaccharide (LPS) and TGF-β to assess RIPK3 and marker expression via antibody staining and RT-PCR.
Main Results:
- A subpopulation of RIPK3-positive Vimentin-positive cells was identified in human keloids and mouse wounds, with higher numbers in mouse wound beds.
- RIPK3 and key fibroblast activation markers were detected in isolated wound bed fibroblasts.
- While LPS and TGF-β increased RIPK3 protein levels in human fibroblasts, they did not significantly alter RIPK3 or fibroblast activation marker gene expression compared to controls.
Conclusions:
- RIPK3 is expressed in fibroblasts within fibrotic skin conditions and wound beds.
- The expression of RIPK3 in wound fibroblasts may not be directly linked to fibrotic processes.
- RIPK3 potentially plays an uncharacterized role in the complex mechanisms of wound healing.
