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A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue
Published on: August 25, 2018
BPAG1-e restricts keratinocyte migration through control of adhesion stability
Magdalene Michael1, Rumena Begum2, Kenneth Fong3
1Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London, UK; St Johns Institute of Dermatology, King's College London, Guys Campus, London, UK; Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.
The Journal of Investigative Dermatology
|September 13, 2013
Summary
Bullous pemphigoid antigen 1 (BPAG1-e) mutations cause skin fragility by impairing keratinocyte adhesion and migration. This highlights BPAG1-e's crucial role in regulating cell behavior and integrin function in epithelial cells.
Area of Science:
- Cell Biology
- Dermatology
- Biochemistry
Background:
- Bullous pemphigoid antigen 1 (BPAG1-e), a cytoskeletal linker protein, is crucial for hemidesmosome integrity.
- Mutations in the dystonin (DST) gene encoding BPAG1-e cause inherited skin fragility disorders.
- Previous studies suggested BPAG1-e's role in skin structure, but its specific cellular functions were unclear.
Purpose of the Study:
- To investigate the cellular and molecular consequences of BPAG1-e loss-of-function mutations in keratinocytes.
- To elucidate the role of BPAG1-e in regulating keratinocyte adhesion, spreading, migration, and integrin expression.
- To compare the effects of naturally occurring mutations versus gene silencing on keratinocyte behavior.
Main Methods:
- Isolation and analysis of keratinocytes from patients with homozygous nonsense mutations in the DST isoform encoding BPAG1-e.
- Assessment of keratinocyte adhesion, cell spreading, and migration.
- Quantitative analysis of cell surface and total protein levels of key adhesion molecules, including integrins and keratins.
- Comparison with keratinocytes where BPAG1-e was silenced using short hairpin RNA (shRNA).
Main Results:
- Keratinocytes from patients exhibited significant defects in adhesion, increased cell spreading, and enhanced migration.
- Mutant keratinocytes showed reduced cell surface β4 integrin levels and increased total keratin-14 and β1 integrin levels.
- Short hairpin RNA-mediated knockdown of BPAG1-e in control keratinocytes did not replicate the observed cellular defects.
- Naturally occurring mutations revealed a critical role for BPAG1-e in keratinocyte adhesion, migration, and integrin switching.
Conclusions:
- BPAG1-e is essential for normal keratinocyte adhesion and migration, independent of its structural role in hemidesmosomes.
- Loss of BPAG1-e function leads to altered integrin expression and functional switching in epithelial cells.
- Naturally occurring mutations provide unique insights into BPAG1-e function that cannot be fully recapitulated by gene silencing techniques.
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