Functional analysis of Fas signaling in vivo using synthetic inducers of dimerization.
BACKGROUND
Genetic abnormalities in the Fas receptor or its trimeric ligand, FasL, result in massive T-cell proliferation and a lupus-like autoimmune syndrome, which was initially attributed to excessive lymphoproliferation but is now ascribed to the absence of Fas-mediated cell death. Although Fas is normally expressed on most thymocytes, negative selection seems to be unperturbed in Fas-deficient (lpr) mice. This suggests that Fas has an important function in peripheral, but not thymic, T cells.


RESULTS
To explore the Fas-mediated cell death pathway both in vitro and in vivo, we used conditional alleles of the Fas receptor that can be triggered by an intracellularly active chemical inducer of dimerization known as FK1012. We found that membrane attachment is important for Fas function and, unlike previous results with anti-Fas monoclonal antibodies, we show that dimerization is sufficient to trigger apoptosis. Finally, the administration of FK1012 in vivo to transgenic animals expressing the conditional FAS receptor in thymocytes demonstrates that sensitivity to FAS-mediated apoptosis is restricted to CD4+CD8+ thymocytes.


CONCLUSIONS
Here, we describe the first in vivo application of non-toxic, cell-permeable synthetic ligands to regulate signal transduction in transgenic mice expressing a conditional receptor. Using this system, we show that the Fas pathway is restricted to double-positive thymocytes in vivo, consistent with recent in vitro findings with thymocytes. This method promises to be more useful not only for developmental studies involving cell ablation, but also for studies involving the regulation of a wide variety of signaling molecules.
Spencer
D M
DM
Howard Hughes Medical Institute, Department of Developmental Biology, Stanford University Medical School, California 94305, USA. dspencer@bcm.tmc.edu
Belshaw
P J
PJ
Chen
L
L
Ho
S N
SN
Randazzo
F
F
Crabtree
G R
GR
Schreiber
S L
SL
D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D016923:Cell Death / Q000276:immunology; D002460:Cell Line; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology*; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology / Q000276:immunology*; D019014:fas Receptor / Q000235:genetics / Q000276:immunology*
C084260:FK 1012; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D016559:Tacrolimus