Enolase 1 and calreticulin regulate the differentiation and function of mouse mast cells

Abstract

It has become widely accepted that the role of mast cells is not restricted to allergic processes. Thus, mast cells play an important role in innate and adaptive immune responses, but study of proteins related to differentiation of mast cells has not been done yet. Enolase 1 is a glycolytic enzyme expressed in most tissues and calreticulin, known as endoplasmic reticulum (ER) resident chaperon, has multifunctional responses. This study aimed to investigate the effects of these proteins on the differentiation and functions of mouse bone marrow-derived mast cells (BMMCs). To identify the target proteins related to the differentiation of BMMCs, we examined the protein expression pattern of BMMCs using 2-dimensional electrophoresis (2-DE) and MALDI-TOF analysis. Expressions of Fc epsilon RI alpha, surface molecules (c-kit, CD40, CD40L, VCAM-1). tryptase, and cytokines were examined in BMMCs using FACS analysis, Western blot, and RT-PCR respectively. Enolase 1 and calreticulin were transfected into BMMCs, and [Ca2+]i levels were determined by confocal microscope, while amounts of TNF-alpha and LTs were measured by ELISA. Eight proteins were identified by proteomic analysis. Enolase and calreticulin siRNA transfection inhibited the expressions of Fc epsilon RI alpha, surface molecules, tryptase, and cytokine mRNA, which are gradually enhanced during culture periods of BMMCs. Enolase 1 and calreticulin siRNA reduced the [Ca2+]i levels, amounts of total TNF-alpha, and the release of TNF-alpha and leukotrienes, all of which are increased in the BMMCs activated with antigen/antibody reaction. The data suggest that enolase 1 and calreticulin are important proteins in regulating the differentiation and functions of BMMCs. (C) 2011 Elsevier Inc. All rights reserved.