Identification of functional aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in murine splenocytes.
The objective of the present studies was to determine whether the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein are present and functional in B6C3F1 (C57BL/6 x C3H) mouse splenocytes. Northern analysis of poly(A) RNA isolated from splenocytes revealed transcripts of approximately 6.6 kb which hybridized to the AhR complementary DNA (cDNA) probe. Anti-AhR antibodies identified two major cytosolic forms of the AhR in splenocytes, approximately 95 and 104 kDa, corresponding to the codominately expressed Ahrb alleles in the B6C3F1 mice. Northern analysis utilizing an oligomer probe for ARNT identified three messenger RNA (mRNA) transcripts, approximately 5.6, 2.0, and 1.1 kb, in spleen which was consistent with the banding pattern observed in the B6C3F1 mouse liver. Western blotting confirmed the presence of the approximately 87 kDa ARNT protein in splenocytes. Protein quantitation by slot blot analysis demonstrated approximately 2.0-fold more AhR in liver than in splenocytes. Interestingly, ARNT was approximately 2.4-fold more abundant in splenocytes than in liver. Consistent with these results, comparison by quantitative reverse transcriptase-polymerase chain reaction analysis of AhR and ARNT transcripts in liver and splenocytes demonstrated approximately 2.3-fold more AhR transcripts in liver than in splenocytes and approximately 3.2-fold more ARNT transcripts in splenocytes than in liver. In addition, comparisons between AhR and ARNT transcripts isolated from the liver and splenocytes indicated a greater number of ARNT transcripts as compared with AhR in both preparations. TCDD treatment of splenocytes induced binding of the AhR nuclear complex to the dioxin-responsive enhancer (DRE) as detected by the electrophoretic mobility shift assay. These findings confirm that the AhR and ARNT are present in mouse splenocytes and are capable of binding to the DRE.
Williams
C E
CE
Department of Pharmacology & Toxicology, Michigan State University, East Lansing 48824, USA.
Crawford
R B
RB
Holsapple
M P
MP
Kaminski
N E
NE
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
C497397:Arnt protein, mouse; D017931:DNA Primers; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator