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.
Highlights
Human epidermis is composed of layers of self-renewing keratinocytes that demonstrate remarkable versatility in responses to external environmental stimuli via complex programs of gene expression
FK1012 is capable of cross-linking intracellular proteins engineered to contain the FK506 binding protein (FKBP); an FKBP-linked Fas cytoplasmic domain served as a confirmation of antibody effects as well as allowed initiation of features of Fas signal transduction in different subcellular locations
While apoptosis has been implicated in a variety of processes in the skin, the role of Fas in triggering this process is as yet not fully characterized
Summary
Human epidermis is composed of layers of self-renewing keratinocytes that demonstrate remarkable versatility in responses to external environmental stimuli via complex programs of gene expression Such complex genetic programs are largely triggered by cell surface receptor signal transduction cascades in the skin and mediate adaptive tissue functions, such as cellular proliferation in wound healing, expression of cytokines in inflammation, and apoptosis in response to a range of injurious stimuli. Cell surface receptors initiating such complex programs of gene expression are activated, in many cases, by a cross-linking of their cytoplasmic domains induced by extracellular binding of cognate ligand [1]. Such ligandinduced receptor oligomerization appears to be a general mechanism of activation for a variety of receptor gene families,. Features of such a cell death signal transduction pathway in the epidermis may reveal a mechanism to eliminate injured keratinocytes in avoiding potential subsequent infectious and neoplastic complications in the skin
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