Faculty Collaboration Database

Calcium ionophore upregulation of AUUUA-specific binding protein activity is contemporaneous with granulocyte macrophage colony-stimulating factor messenger RNA stabilization in AML14.3D10 cells. Am J Respir Cell Mol Biol 1999 Nov;21(5):621-8

Date

10/28/1999

Pubmed ID

10536121

DOI

10.1165/ajrcmb.21.5.3694

Scopus ID

2-s2.0-0033220421 (requires institutional sign-in at Scopus site)   18 Citations

Abstract

Eosinophils produce granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances their survival and function. In T cells and fibroblasts, GM-CSF production is controlled predominantly by variable messenger RNA (mRNA) stability involving 3' untranslated region (3' UTR) adenosine-uridine-rich elements (AREs) and sequence-specific mRNA binding proteins. However, the mode of regulation of this critical cytokine remains unknown in eosinophils. Therefore, we measured GM-CSF mRNA decay in an eosinophil-like cell line (AML14.3D10) and, with a radiolabeled GM-CSF RNA probe, asked whether ARE-specific, mRNA binding proteins were present in cytoplasmic lysates of these cells. Human GM-CSF mRNA transfected into unstimulated AML14.3D10 cells decayed with a half-life of 6 min, which increased to 14 min after 1 h, and to 22 min after 2 h, of ionophore-mediated activation. GM-CSF RNA mobility shift assays using cytoplasmic extracts from resting or ionophore-stimulated AML14.3D10 cells revealed multiple RNA-protein complexes of 55, 60, 85, 100, and 125 kD. A 47-kD complex was also detected with an 80-base RNA probe containing four consecutive AUUUA motifs. On the basis of competition studies, all of the observed binding protein activities interacted with the 3' UTR AREs. In addition, binding activity increased 2.5-fold in cytoplasmic lysates from cells stimulated with calcium ionophore for 2 h, contemporaneous with GM-CSF mRNA stabilization. These data provide direct evidence that ionophore stabilizes GM-CSF mRNA in AML14.3D10 cells and simultaneously increases the activity of a series of AUUUA-specific mRNA binding proteins. We conclude that the interaction of AU-specific binding proteins may stabilize GM-CSF mRNA in activated eosinophil-like cell lines.

Author List

Ruth JH, Esnault S, Jarzembowski JA, Malter JS

Author

Jason A. Jarzembowski MD, PhD Sr Associate Dean, CEO CSG, Professor in the Pathology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

3' Untranslated Regions
Binding, Competitive
Calcium
Carrier Proteins
Cell Line
Eosinophils
Granulocyte-Macrophage Colony-Stimulating Factor
Humans
Ionomycin
Ionophores
RNA Stability
RNA, Messenger
RNA-Binding Proteins
Time Factors
Transfection
Up-Regulation