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Abstract:
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The aryl hydrocarbon receptor (AhR) is a ligand- activated transcription factor which plays a role in the inhibition of immunoglobulin (Ig) expression by the potent environmental toxicant
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Some studies have postulated that the TCDD- inhibited Ig expression may be mediated through a direct binding of AhR to dioxin response elements (DRE). Our previous studies have identified functional DRE binding sites in the 3`Igh regulatory region (3`IghRR) and hypothesized that activated AhR inhibits Ig expression by inhibiting 3`IghRR activity. We have developed a new cell line model that expresses a ?2b transgene under the regulation of the 3’IghRR and through lentiviral transduction expresses shRNA targeted to Ahr message. This shAhR cell line has a heterozygous population of cells that express a reduction of AhR as seen with western blot analysis. In our current study we have utilized limiting dilution to select the greatest AhR-knockdown clonal populations. Our western blot analysis demonstrated a marked reduction in AhR expression in comparison to both the well-characterized parental cell line (CH12.LX) and the CH12.LX variant expressing the 3’IghRR-regulated ?2b transgene (CH12.LX ?2b-3’IghRR). Further studies include evaluating the ability of these cells to induce Cyp1A1 expression (hallmark endpoint of AhR activation), and the effect of TCDD on 3’IghRR-regulated ?2b expression and endogenous Ig expression. (Supported by NIH RO1ES014676 and ARRA supplement 04S1 and NIH PREP 5R25GM086257-02. |
