Expression of the calcium sensing receptor in human peripheral blood T lymphocyte and its contribution to cytokine secretion through MAPKs or NF-κB pathways

Abstract

The calcium-sensing receptor (CaSR) has been reported to play an important role in many tissues and organs. However, studies about the expression and function of CaSR in T lymphocytes are still not very lucid. In this study, we investigated the above-mentioned issues using RT-PCR, immunofluorescence staining, Western blotting, and the ELISA techniques. We found that the CaSR protein was expressed, and mainly located in the membrane in the normal human peripheral blood T lymphocytes. GdCl₃ (an agonist of CaSR) increased the dose-dependency of the CaSR expression, which was abolished by NPS2390 (an inhibitor of CaSR). GdCl₃ and Ca²⁺ increased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 (one subgroup of MAPKs) and P65 (subunit of NF-κB),but, they had no significant effects on the JNK and P38 subgroups of MAPKs. Meantime, GdCl₃ and Ca²⁺ stimulated both the IL-6 and TNF-β releases and their mRNA expressions. However, these effects of GdCl₃ and Ca²⁺ were inhibited by NPS2390, U0126 (MAPKs pathway inhibitor) or Bay-11-7082 (NF-κB pathway inhibitor). These results suggested that CaSR was functionally expressed in the T cells, and the activated CaSR contributed to the cytokine secretion through the partial MAPK and NF-κB pathways.

Introduction

Calcium-sensing receptor (CaSR) is a member of the G protein-coupled receptor superfamily, which has been detected in many tissues and cells. CaSR maintains the Ca²⁺ homeostasis in the body. In addition, CaSR also plays an important role in the cell differentiation, apoptosis, hormone secretion, etc. (Tu et al., 2008, Molostvov et al., 2008, Kantham et al., 2009). In previous studies, Sun et al. had found that CaSR was involved in the rat cardiac ischemia/reperfusion injury and CaSR activation induced rat cardiomyocyte apoptosis combined with transient receptor potential channels (Sun et al., 2006, Sun et al., 2010, Feng et al., 2011).

Cytokines participate in the specific immunity as well as many diseases (Woollard and Geissmann, 2010, Hashizume et al., 2011, Nakagomi et al., 2010). Lymphocytes include the T lymphocytes, B lymphocytes, and NK cells. The T lymphocytes are mainly involved in the immune response by secreting cytokines. At present, many studies are available concerning CaSR and cytokines, but, few studies have established the relation between CaSR and cytokines.

Previously, the expression of CaSR in the human and murine monocytes isolated from the peripheral blood was detected (Olszak et al., 2000, Yamaguchi et al., 1998a, Yamaguchi et al., 1998b). Rothe et al. (2005) extracted DNA from the peripheral lymphocytes and platelets and investigated the CaSR gene polymorphism Arg990Gly. Platelets have been identified with the CaSR expression. However, the B cells do not express CaSR and the Ca²⁺-induced responses in the B cells cannot be blocked by specific CaSR inhibitors (Hammond et al., 2007). Subsequently, we need to clarify whether T lymphocytes exit CaSR.

Activation of immuno-responsive cells via a variety of membrane receptors are frequently coupled to the activation of various sequential kinase cascades. In general, it was involved in three pathways: mitogen-activated protein kinases (MAPKs), Ik-B kinases (IKKs) and Janus kinases (JAKs). In mammalian cells, MAPKs have been classified into at least three subfamilies (Kyriakis and Avruch, 2001): the ERK1/2 group; the N-terminal kinase (JNK) group; and p38 MAPK. MAPKs can be activated by many GPCRs. The IKKs mediate the activation of nuclear factor kB (NF-κB) via a series of phosphorylation events. NF-κB is a dimmer of members of the Rel family of proteins, which includes p50, p65, and cRel (Canaff and Hendy, 2005). Activities of these intracellular signaling proteins often result in the activation of immuno-responsive cells, and the subsequent enhanced production of cytokines. Hence, detection of stimulatory phosphorylation of the signaling proteins and the production of cytokines can serve as useful indicators of whether receptor activation is linked to immuno-modulatory effects (Chan et al., 2005).

Calcium deposition may be both a consequence as well as a cause of chronic inflammatory changes at the sites of injury, infection, atherosclerotic plaques etc. It is well established that lymphocytes play important roles in the onset and deterioration of atherosclerosis, rheumatoid arthritis etc. (Xi et al., 2010). Free ionized calcium is the most effective agonist of CaSR, but it is not special. Thus, we also selected the Gd³⁺ as the activator. However, whether the lymphocyte-mediated pathological processes are related with the activation of CaSR remains ambiguous so far. In this study, we aim to detect CaSR expression in T lymphocytes and intend to prove the hypothesis that CaSR on the T lymphocytes participates in modulating the production of cytokines and meantime to identify the relevant cellular signaling pathways.