Vasoactive intestinal peptide receptor-1 (VPAC-1) is a novel gene target of the hemolymphopoietic transcription factor Ikaros.
Vasoactive intestinal peptide and its G-protein-coupled receptors, VPAC-1 and VPAC-2, are highly expressed in the immune system and modulate diverse T cell functions. The human VPAC-1 5'-flanking region (1.4 kb) contains four high affinity Ikaros (IK) consensus sequences. Ikaros native protein from T cell nuclear extracts and IK-1 and IK-2 recombinant proteins recognized an IK high affinity binding motif in the VPAC-1 promoter in electrophoretic mobility shift assays by a sequence-specific mechanism, and anti-IK antibodies supershifted this complex. Stable NIH-3T3 clones overexpressing IK-1 or IK-2 isoforms were generated to investigate Ikaros regulation of endogenous VPAC-1 expression as assessed by quantifying VPAC-1 mRNA and protein. By traditional and fluorometric-based kinetic reverse transcription-PCR and (125)I-labeled vasoactive intestinal peptide binding, both IK-1 and IK-2 suppressed endogenous VPAC-1 expression in NIH-3T3 clones by a range of 50-93%. When a series of nested deletions of the VPAC-1 luciferase reporter construct were transiently transfected into IK-2 clones there was up to a 41% decrease in transcriptional activity compared with vector control. Two major IK-2 binding domains also were identified at -1076 to -623 bp and at -222 to -35 bp, respectively. As both Ikaros and its novel target VPAC-1 are highly expressed in T cells, this system may be a dominant determinant of the VPAC-1 expression in immune responses.
Dorsam
Glenn
G
Department of Medicine, University of California Medical Center, San Francisco, California 94143-0711, USA. Glenn-Dorsam@excite.com
Goetzl
Edward J
EJ
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D051740:Ikaros Transcription Factor; D019169:Jurkat Cells; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497239:IKZF1 protein, human; D010455:Peptides; D020033:Protein Isoforms; D012333:RNA, Messenger; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011994:Recombinant Proteins; D014157:Transcription Factors; C495508:Vipr1 protein, rat; C497238:Zfpn1a1 protein, mouse; D051740:Ikaros Transcription Factor; D008156:Luciferases