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Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Combined screening of thymocytes using apoptosis-specific cDNA array and promoter analysis yields novel gene targets
Michael T Fisher1, Mitzi Nagarkatti, Prakash S Nagarkatti
1Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23113, USA.
Abstract:
We have used pathway-specific cDNA arrays coupled with analysis of gene promoter regions to identify novel genes that may mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the thymus. C57BL/6 mice were injected ip with 50 microg/kg TCDD, and 3, 6, or 24 h later, RNA was extracted from the thymus and subjected to microarray analysis. Several members of the TNF and TNFR family were induced following TCDD exposure, including receptor/ligand pairs Ltbeta-R/LIGHT, OX40/OX40L and TNF-alpha/TNFR1. In addition, Fas and CD30 were also upregulated. Pro-apoptotic bcl-2 gene family members Bax and Hrk, among others, were also induced, as were pro-survival bcl-2 family genes Bcl-x and Bcl-w. Cell-cycle regulator p21Cip1 was also induced. In addition, we analyzed the promoter regions of genes induced by TCDD for the presence of dioxin-responsive elements (DREs). The Fas and LIGHT gene promoters were found to contain DREs as analyzed by Matinspector Web-based search algorithm. Furthermore, binding of the aryl hydrocarbon receptor (AhR) to the DREs present on these genes was confirmed by chromatin immunoprecipitation. Given that several of the genes, including Fas, LIGHT, and CD30 are involved in negative selection of T cells in the thymus, our studies suggest that TCDD-induced upregulation of these genes may enhance negative selection leading to thymic atrophy.
Insights
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure upregulates genes involved in T cell selection in the thymus. This suggests TCDD may cause thymic atrophy by enhancing T cell negative selection.
Area of Science:
- Immunology
- Toxicology
- Molecular Biology
Background:
- 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxicant.
- The thymus is a primary organ for T cell development and immune tolerance.
- Mechanisms underlying TCDD-induced thymic toxicity are not fully understood.
Purpose of the Study:
- Identify novel genes mediating TCDD toxicity in the thymus.
- Investigate the role of TCDD in regulating genes involved in T cell selection.
- Elucidate the molecular pathways affected by TCDD exposure in thymocytes.
Main Methods:
- Pathway-specific cDNA arrays for gene expression profiling.
- Analysis of gene promoter regions for dioxin-responsive elements (DREs).
- Chromatin immunoprecipitation to confirm aryl hydrocarbon receptor (AhR) binding.
Main Results:
- TCDD exposure upregulated several TNF and TNFR family members, including Fas, LIGHT, CD30, OX40, and TNF-alpha.
- Pro-apoptotic (Bax, Hrk) and pro-survival (Bcl-x, Bcl-w) bcl-2 family genes were induced.
- Fas and LIGHT gene promoters contained DREs, with confirmed AhR binding.
- Upregulation of Fas, LIGHT, and CD30 suggests enhanced T cell negative selection.
Conclusions:
- TCDD exposure induces genes critical for T cell negative selection in the thymus.
- AhR binding to DREs in Fas and LIGHT promoters mediates TCDD's effects.
- Enhanced negative selection likely contributes to TCDD-induced thymic atrophy.
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