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Updated: Jun 28, 2026
Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin
Published on: July 14, 2017
uPAR-deficient mouse keratinocytes fail to produce EGFR-dependent laminin-5, affecting migration in vivo and in vitro
Silvia D'Alessio1, Laura Gerasi, Francesco Blasi
1Università Vita Salute San Raffaele and Istituto Scientifico H San Raffaele, via Olgettina 60, 20132 Milano, Italy.
Abstract:
The urokinase receptor (uPAR) is involved in a series of pathological processes, from inflammation to cancer. We have analyzed in detail the role of uPAR and the mechanisms involved in keratinocyte behavior during wound healing by exploiting uPAR-knockout (KO) mice. In vivo, uPAR-KO mice showed delayed wound healing, with abnormal keratinocyte migration and proliferation. In vitro, unlike wild-type cells, primary uPAR-KO keratinocytes did not proliferate in response to epidermal growth factor (EGF), their growth and migration were not inhibited by EGF-receptor (EGFR) inhibitors, and they did not adhere to uncoated surfaces. Whereas EGFR levels in uPAR-KO keratinocytes were normal, there was no tyrosine phosphorylation upon addition of EGF, and its downstream targets, extracellular-signal-regulated kinases 1 and 2 (ERK1/2), were not activated. Re-introduction of mouse uPAR rescued all phenotypes. In vitro adhesion and migration defects were associated with the failure of uPAR-KO keratinocytes to normally produce and secrete laminin-5 (LN5), an event that requires EGFR signaling. These results were confirmed in vivo, with LN5 being upregulated during wound healing in wild-type but not in uPAR-KO epidermis.
Insights
Urokinase receptor (uPAR) is crucial for skin wound healing. Lacking uPAR impairs keratinocyte migration and proliferation, delaying wound closure by disrupting epidermal growth factor signaling and laminin-5 production.
Area of Science:
- Cell biology
- Dermatology
- Molecular biology
Background:
- The urokinase receptor (uPAR) plays a role in various pathological processes, including inflammation and cancer.
- Understanding uPAR's function in keratinocyte behavior is vital for wound healing research.
Purpose of the Study:
- To investigate the specific role of uPAR in keratinocyte behavior during wound healing.
- To elucidate the molecular mechanisms underlying uPAR's function in epidermal repair.
Main Methods:
- Utilized uPAR-knockout (KO) mice and primary uPAR-KO keratinocytes for in vivo and in vitro experiments.
- Assessed keratinocyte migration, proliferation, adhesion, and response to epidermal growth factor (EGF) and its receptor (EGFR) signaling.
- Analyzed EGFR levels, tyrosine phosphorylation, ERK1/2 activation, and laminin-5 (LN5) production.
Main Results:
- uPAR-KO mice exhibited delayed wound healing with impaired keratinocyte migration and proliferation.
- uPAR-KO keratinocytes failed to proliferate in response to EGF, showed resistance to EGFR inhibitors, and lacked adhesion to uncoated surfaces.
- EGFR signaling was defective in uPAR-KO keratinocytes, evidenced by absent EGF-induced tyrosine phosphorylation and ERK1/2 activation, and impaired LN5 production and secretion.
Conclusions:
- uPAR is essential for normal keratinocyte migration, proliferation, and adhesion during wound healing.
- uPAR signaling is required for EGF-induced EGFR activation, downstream signaling, and laminin-5 production, which are critical for epidermal repair.
