Doctor of Philosophy (PhD), Wright State University, 2014, Environmental Sciences PhD
Transcriptional regulation of the murine immunoglobulin heavy chain gene involves several regulatory elements including the 3'Igh regulatory region (3'IghRR) composed of at least four enhancers (hs3A; hs1,2; hs3B; hs4). Enhancers hs1,2 and hs4 contain binding sites for several transcription factors including NF-κB/Rel proteins and the AhR. Interestingly, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) profoundly inhibits 3'IghRR and hs1,2 activation induced by the B-cell activator lipopolysaccharide (LPS), but enhances the activation of the hs4. Within the hs4, the AhR binding site overlaps an NF-κB/Rel binding site suggesting that both the AhR and the NF-κB together may modulate of the 3'IghRR. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR following LPS stimulation and TCDD treatment on 3'IghRR, hs1,2, and hs4. In our studies we utilized the CH12.LX B cell line; the CH12.IκBαAA cell line, which expresses an inducible IκBα super repressor (IκBαAA); the CH12.γ2b-3'IghRR cell line that stably expresses a γ2b-3'IghRR-regulated γ2b transgene reporter; and splenocytes derived from B6C3F1 mice. The stimulation of the CH12.γ2b-3'IghRR cell line with Toll-like receptor (TLR) agonists LPS, Resiquimod (R848), or Cytosine-phosphate-Guanine (CpG)-oligodeoxynucleotides combined with a co-treatment of TCDD significantly inhibited the TLR-induced activation of the 3'IghRR. Utilizing transiently expressed luciferase reporters, we found induction of IκBαAA expression partially attenuated LPS-induced activation of the 3'IghRR and hs4, partially reversed the effects of a TCDD and LPS co-treatment on the activity of the 3'IghRR and hs4, and the addition of an AhR antagonist, CH223191, markedly reversed the LPS and TCDD induced inhibition of the 3'IghRR and inhibited the synergistic activation of the hs4. Chromatin immunoprecipitation analysis of CH12.LX and murine splenocytes demonstrated a LPS and a LPS co-treatment with TCDD-dependent increase in Rel (open full item for complete abstract)
Committee: Courtney Sulentic Ph.D. (Advisor); Michael Leffak Ph.D. (Committee Member); Mill Miller Ph.D. (Committee Member); David Cool Ph.D. (Committee Member); Rodney DeKoter Ph.D. (Committee Member); Don Cipollini Ph.D. (Other)
Subjects: Environmental Science; Immunology; Molecular Biology; Toxicology