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Specific triggering of the Fas signal transduction pathway in normal human keratinocytes
R A Freiberg1, D M Spencer, K A Choate
1Veterans Administration Palo Alto Health Care System, Palo Alto, California 94304, USA. khavari@cmgm.stanford.edu
Abstract:
The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.
Insights
Human skin cells (keratinocytes) eliminate damaged cells via Fas-mediated apoptosis. This study demonstrates that triggering Fas signaling, even without growth factors, effectively induces programmed cell death in keratinocytes.
Area of Science:
- Cell Biology
- Dermatology
- Immunology
Background:
- The epidermis faces constant genotoxic stress, necessitating mechanisms for eliminating damaged cells.
- The Fas receptor initiates apoptosis in various cell types, including keratinocytes, crucial for tissue homeostasis.
Purpose of the Study:
- To investigate the functionality of the Fas signal transduction pathway in human keratinocytes.
- To determine if Fas-mediated apoptosis can be triggered independently of growth stimuli in keratinocytes.
Main Methods:
- Engineered human keratinocytes expressing Fas cytoplasmic domains linked to FK506 binding protein (FKBP).
- Utilized FK1012 to induce oligomerization and cross-linking of engineered Fas receptors.
- Assessed keratinocyte viability and apoptosis markers following Fas receptor activation.
Main Results:
- FK1012-induced oligomerization of membrane-targeted Fas.FKBP decreased keratinocyte viability by up to 89%.
- Fas receptor cross-linking, either chemically or via antibodies, induced apoptosis-like cellular changes.
- A specific Fas point mutation (lpr) abolished the apoptotic response; growth factors did not confer protection.
Conclusions:
- The Fas signal transduction pathway is constitutively active and functional in human keratinocytes.
- Fas-mediated apoptosis in keratinocytes can be triggered without prior induction and overrides growth signals.
- This pathway represents a potent mechanism for eliminating genetically altered keratinocytes.