Suppression of LFA-1 Expression by Spermine Is Associated with Enhanced Methylation of ITGAL, the LFA-1 Promoter Area

Yoshihiko Kano,Kuniyasu Soda,Fumio Konishi,Nupur Gangopadhyay

doi:10.1371/journal.pone.0056056

Abstract

Spermine and spermidine, natural polyamines, suppress lymphocyte function-associated antigen 1 (LFA-1) expression and its associated cellular functions through mechanisms that remain unknown. Inhibition of ornithine decarboxylase, which is required for polyamine synthesis, in Jurkat cells by 3 mM D,L-alpha-difluoromethylornithine hydrochloride (DFMO) significantly decreased spermine and spermidine concentrations and was associated with decreased DNA methyltransferase (Dnmt) activity, enhanced demethylation of the LFA-1 gene (ITGAL) promoter area, and increased CD11a expression. Supplementation with extracellular spermine (500 µM) of cells pretreated with DFMO significantly increased polyamine concentrations, increased Dnmt activity, enhanced methylation of the ITGAL promoter, and decreased CD11a expression. It has been shown that changes in intracellular polyamine concentrations affect activities of -adenosyl-L-methionine-decaroboxylase, and, as a result, affect concentrations of the methyl group donor, S-adenosylmethionine (SAM), and of the competitive Dnmt inhibitor, decarboxylated SAM. Additional treatments designed to increase the amount of SAM and decrease the amount of decarboxylated SAM–such as treatment with methylglyoxal bis-guanylhydrazone (an inhibitor of S-adenosyl-L-methionine-decaroboxylase) and SAM supplementation–successfully decreased CD11a expression. Western blot analyses revealed that neither DFMO nor spermine supplementation affected the amount of active Ras-proximate-1, a member of the Ras superfamily of small GTPases and a key protein for regulation of CD11a expression. The results of this study suggest that polyamine-induced suppression of LFA-1 expression occurs via enhanced methylation of ITGAL.

Highlights

Lymphocyte function-associated antigen 1 (LFA-1), which consists of an alpha-L chain (CD11a) and a beta-2 chain (CD18), is one of the adhesion molecules expressed on cell membranes of a wide variety of leukocytes
Flow cytometric analysis revealed that spermine treatment for 72 h decreased the Mean Fluorescent Intensities (MFIs) of CD11a and CD18 staining on peripheral blood mononuclear cells (PBMCs) gated in the lymphocyte and monocyte light-scattered regions, in a dose-dependent manner (Fig. 1)
difluoromethylornithine hydrochloride (DFMO), which inhibits ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis

Summary

Introduction

Lymphocyte function-associated antigen 1 (LFA-1), which consists of an alpha-L chain (CD11a) and a beta-2 chain (CD18), is one of the adhesion molecules expressed on cell membranes of a wide variety of leukocytes. LFA-1 binds intercellular adhesion molecules (ICAMs) expressed on endothelial cells. Because chronic inflammation is thought to be involved in the pathogenesis of many, if not all, age-associated chronic diseases, increased pro-inflammatory status is thought to be one of the major factors that accelerate such diseases and aging itself. Among several predisposing factors favoring susceptibility to age-dependent chronic inflammatory diseases [9,10,11], an age-dependent increase in the proportion of cells expressing high levels of LFA-1 is one frequently documented in previous reports [11,12]. The mechanisms of action of agingassociated changes in LFA-1 expression are not fully elucidated; mechanisms known to be involved in the regulation of LFA-1 expression are DNA methylation of the LFA-1 promoter area and intracellular signaling [13,14,15]

Methods

Results

Conclusion