Abstract
Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways. Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells. Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions.
Footnotes
This work was supported by the National Institutes of Health National Center for Complementary and Alternative Medicine [Grants PO1-AT003961, R01AT006888]; the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01ES019313]; and the National Institutes of Health National Institute on Mental Health [Grant R01MH094755].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS:
- DES
- diethylstilbestrol
- AP-1
- activator protein-1
- COMP.
- competition
- DMSO
- dimethyl sulfoxide
- DRE
- dioxin responsive element
- EMSA
- electrophoretic mobility-shift assay
- ER
- estrogen receptor
- ERE
- ER element
- FasL
- Fas ligand
- GD
- gestational day
- Mut
- mutant
- NE
- nuclear extract
- NF-AT
- nuclear factor of activated T cells
- NF-κB
- nuclear factor κB
- PBS
- phosphate-buffered saline
- pCMV
- porcine cytomegalovirus
- PCR
- polymerase chain reaction
- PE
- phycoerythrin
- RLU
- relative light units
- RT
- reverse transcription
- SP1
- specificity protein 1
- TAM
- tamoxifen
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- VEH
- vehicle.
- Received May 4, 2012.
- Accepted August 9, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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