Binding of the ELAV-like protein in murine autoimmune T-cells to the nonameric AU-rich element in the 3′ untranslated region of CD154 mRNA

Abstract

The CD154 molecule is important for experimental allergic encephalomyelitis (EAE) which is mediated by autoimmune CD4⁺ T-cells. Post-transcriptional instabilization/stabilization of mRNAs, which contain an adenylate uridylate rich element (ARE) in their 3′ untranslated region (3′UTR), is regulated in part by binding of ARE-binding proteins to the element. We have investigated the protein which binds to the nonameric ARE in the 3′UTR of CD154 mRNA. A protein which binds to the CD154 ARE was found to exist in a extract prepared from murine autoimmune T-cells activated with myelin basic protein (MBP), and turned out to be mHuR which is a ubiquitous ELAV-like protein. It was found that mHuR was upregulated upon stimulation of the T-cells with a MBP antigen. The CD154 ARE and the ARE in the 3′UTR of tumor necrosis factor-α (TNF-α) mRNA were competed in binding to mHuR, indicating that both AREs bind to the same site on mHuR. The presence of the CD154 ARE downstream of the luciferase cDNA in a reporter plasmid decreased the translational efficiency, and co-expression of the mHuR slightly increased the translation. These results suggest the possibility that the ELAV-like protein participates in the regulation of the expression of CD154 on the autoimmune T-cells. Modification of the expression of CD154 on autoimmune T-cells by regulating the ELAV-like protein may provide effective therapy for EAE and human multiple sclerosis.

Introduction

The CD154 molecule, the ligand for CD40, is expressed primarily by activated CD4⁺ T-cells, and its binding to the CD40 molecules on antigen presenting cells mediates both humoral and cell-mediated immune responses. experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis, is an autoimmune disease mediated by the autoimmune CD4⁺ T-cells. The expression of the CD154 molecules on the EAE autoimmune T-cells is crucial for their encephalitogenicity (Ford et al., 1999a, Abromson-Leeman et al., 2001). The previous studies have revealed that the CD154 knockout mice fail to develop clinical EAE (Grewal et al., 1996), that interruption of CD40–CD154 signaling limits the clinical course of EAE (Gerritse et al., 1996), and that blockade of CD40–CD154 interactions directly interferes with the effector functions of effector cells (Howard and Miller, 2001).

The CD154 expression is regulated in part by post-transcriptional mechanisms (Ford et al., 1999a, Rigby et al., 1999, Suarez et al., 1997, Barnhart et al., 2000). For post-transcriptional regulation, the instabilizing/stabilizing elements in the 3′ untranslated region (3′UTR) of mRNA play crucial roles. These elements are bound by transacting proteins and thereby stabilize or destabilize the mRNA. The adenylate uridylate rich element (ARE) is one of the instability elements (Shyu et al., 1991, Winstall et al., 1995). CD154 mRNA contains scattered four copies of the pentameric ARE, AUUUA, and a single copy of the nonameric ARE, UUAUUUAUU, in the 3′UTR. Previous studies have indicated that there are no proteins that bind to the pentameric AREs of CD154 mRNA in T-cells (Rigby et al., 1999, Barnhart et al., 2000). However, it has not been determined whether the protein that binds to the nonameric ARE of CD154 mRNA exists or not in T-cells. Previous study has shown that the nonameric ARE but not the pentameric ARE is the minimal ARE that effectively destabilizes mRNA (Zubiaga et al., 1995). In this communication, we have examined the CD154 nonameric ARE-binding protein in the autoimmune T-cells established from the EAE mice.