Ca2+ ionophore A23187-dependent stabilization of granulocyte-macrophage colony-stimulating factor messenger RNA in murine thymoma EL-4 cells is mediated through two distinct regions in the 3'-untranslated region.
We analyze the role of the Ca2+ ionophore A23187 in the induction of GM-CSF mRNA expression in EL-4 thymoma cells. Northern analysis shows that A23187 increases the half-life of GM-CSF mRNA. To identify potential Ca2+ response elements in the GM-CSF mRNA, we produced stable transfectants containing pRSV-CAT (EL-4cat) or hybrid constructs in which most of the GM-CSF 3'-untranslated region (EL-4gm) or the adenosine-uridine boxes alone (EL-4au) were placed in a downstream position from the CAT coding region. A23187 induces a 4.4-fold increase in CAT activity in EL-4cat cells and a 210-fold and 48-fold increase in CAT activity in EL-4gm and EL-4au cells, respectively. Actinomycin D chase experiments in transfected cells demonstrate that A23187 increases the half-life of CAT mRNA from 15 min to 3 h in EL-4au cells and more than 3 h in EL-4gm cells, suggesting that the effect of Ca2+ is mediated predominantly by the adenosine-uridine boxes with a smaller contribution from upstream regions. To map these upstream regions, we transfected cells with constructs containing mutations of the 3'-untranslated region. With two of these mutations, corresponding to a region located about 160 bases upstream of the adenosine-uridine boxes, CAT activity was induced only 50-fold compared to 200-fold in EL-4gm cells. These data indicate that two regions within the GM-CSF 3'-untranslated region interact to modulate Ca2+ effects on GM-CSF mRNA half-life.
Iwai
Y
Y
Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.
Akahane
K
K
Pluznik
D H
DH
Cohen
R B
RB
D000818:Animals; D001483:Base Sequence; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D013601:T-Lymphocytes / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D000001:Calcimycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D002118:Calcium