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A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue
Published on: August 25, 2018
Ras-induced spreading and wound closure in human epidermal keratinocytes
Michael Tscharntke1, Ruth Pofahl, Thomas Krieg
1Department Dermatology, University of Cologne and Centre for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany.
Abstract:
Although it is known that growth factor signaling cascades are active during epithelial wound healing, signals that regulate reepithelialization after wounding are not very well characterized. The small GTP binding protein Ras is a molecular switch involved in the regulation of signals originating from different growth factor receptors. We have investigated consequences of its activation in primary human keratinocytes. We provide evidence that activation of Ras can lead to shape changes of keratinocytes caused by rearrangements of the actin cytoskeleton that result in membrane protrusion and ruffling. Similar shape changes were found in the migrating tip of newly formed epithelium in mouse wounds. These cytoskeletal changes occur independently of keratinocyte terminal differentiation, and they can determine the speed of wound epithelialization in vitro. Using various mutant constructs and specific pharmacological inhibitors, we found that the effects of activated Ras on the cytoskeleton of keratinocytes are mediated by a phosphatidylinositol 3 kinase-independent activation of Rac. Our results suggest that growth factor-induced, Ras-mediated changes of keratinocyte shape may be an important mechanism that determines the speed of wound epithelialization.
Insights
Activated Ras protein influences keratinocyte shape and cytoskeleton, impacting wound healing speed. This Ras-mediated pathway, independent of phosphatidylinositol 3 kinase, involves Rac activation and is crucial for reepithelialization.
Area of Science:
- Cell Biology
- Wound Healing Research
- Molecular Signaling
Background:
- Growth factor signaling is active in epithelial wound healing, but regulators of reepithelialization remain unclear.
- The small GTP binding protein Ras acts as a molecular switch for growth factor receptor signaling.
Purpose of the Study:
- Investigate the consequences of Ras protein activation in primary human keratinocytes.
- Elucidate the role of Ras-mediated signaling in regulating keratinocyte shape and wound epithelialization speed.
Main Methods:
- Activation of Ras in primary human keratinocytes.
- Analysis of keratinocyte shape changes and actin cytoskeleton rearrangements.
- In vitro studies on wound epithelialization speed.
- Utilized mutant constructs and pharmacological inhibitors to study signaling pathways.
Main Results:
- Ras activation induces keratinocyte shape changes, including membrane protrusion and ruffling, via actin cytoskeleton rearrangement.
- These cytoskeletal changes were observed in migrating epithelium of mouse wounds.
- Ras-mediated effects on keratinocyte cytoskeleton are independent of keratinocyte terminal differentiation.
- The observed shape changes correlate with and can determine the speed of wound epithelialization in vitro.
- Ras-induced effects are mediated by phosphatidylinositol 3 kinase-independent activation of Rac.
Conclusions:
- Ras-mediated changes in keratinocyte shape are a significant factor in determining wound epithelialization speed.
- This pathway, involving Rac activation, represents a key mechanism in growth factor-induced reepithelialization.
- Understanding Ras signaling offers potential therapeutic targets for enhancing wound healing.
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