Delineation of a novel pathway that regulates CD154 (CD40 ligand) expression.
The expression of CD154 (CD40 ligand) by activated T lymphocytes plays a central role in humoral and cellular immunity. The fundamental importance of this protein in mounting an immune response has made it an attractive target for immunomodulation. Several studies have demonstrated that CD154 expression is regulated at the level of mRNA turnover in a manner distinct from other cytokine genes. We have purified, sequenced, and characterized the two major proteins that bind the CD154 3' untranslated region (3'UTR) as members of the polypyrimidine tract binding protein (PTB) family. One of these proteins is a previously unreported alternatively spliced PTB isoform, which we call PTB-T. These proteins interact with a polypyrimidine-rich region within the CD154 3'UTR that lacks any known cis-acting instability elements. The polypyrimidine-rich region of the CD154 3'UTR was both necessary and sufficient to mediate changes in reporter gene expression and mRNA accumulation, indicating the presence of a novel cis-acting instability element. The presence of a cis-acting instability element in the polypyrimidine-rich region was confirmed using a tetracycline-responsive reporter gene approach. The function of this cis-acting element appears to be dependent on the relative cytoplasmic levels of PTB and PTB-T. Cotransfection of vectors encoding PTB-T consistently decreased the CD154 3'UTR-dependent luciferase expression. In contrast, transfection of plasmids encoding PTB tended to increase CD154 3'UTR-dependent luciferase expression. Thus, the CD154 3'UTR contains a novel cis-acting element whose function is determined by the binding of PTB and PTB-T. These data identify a specific pathway that regulates CD154 expression that can potentially be selectively targeted for the treatment of autoimmune disease and allograft rejection.
Hamilton
B JoNell
BJ
Departments of Medicine. Microbiology and Immunology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA.
Genin
Anna
A
Cron
Randy Q
RQ
Rigby
William F C
WF
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D023201:CD40 Ligand / Q000096:biosynthesis*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008213:Lymphocyte Activation; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D020033:Protein Isoforms; D012333:RNA, Messenger; D038941:Polypyrimidine Tract-Binding Protein; D023201:CD40 Ligand; D012313:RNA; D008156:Luciferases