Expression of the calcium sensing receptor in human peripheral blood T lymphocyte and its contribution to cytokine secretion through MAPKs or NF-κB pathways
Highlights
► We found that the calcium sensing receptor (CaSR) was expressed in the normal human peripheral blood lymphocytes. ► CaSR activation elicited an elevation in the expression and secretion of IL6, TNF-β. ► CaSR activation increased the phosphorylation of xtracellular signal-regulated kinase (ERK)1/2 and P65. ► These effects were reverted by MAPKs and NF-κB pathway inhibitors.
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.
Section snippets
Chemicals
GdCl3, NPS2390, PHA were purchased from Promega Corporation. (Madison, WI, USA). Anti-CaSR-Ab was from Alpha Diagnostic International (San Antonio, USA). Anti-P-ERK1/2-Ab, anti-P-JNK-Ab, anti-P-P65-Ab and anti-P-P38-Ab were from Cell Signaling Technology. Inc (Boston, MA, USA).
Blood samples and T cells purification
Heparinized blood (30 ml) was collected from the healthy volunteers with approval. The T lymphocytes were purified by negative selection using the RosetteSep™ human T cell enrichment cocktail (15021, StemCell Technologies,
Morphological examination
Most of the T cells enlarged with a clear nuclei and irregular outline after stimulated with PHA for 72 h. Cells aggregated and the cell colonies could be observed (Fig. 1).
CaSR expression in human peripheral blood T cells
The CaSR mRNA and CaSR protein with relative molecular mass of 120–170 kDa and 90–110 kDa were detected using RT-PCR and Western blotting (Fig. 2A and B). There were three bands of CaSR protein. We analyzed the WB results with the total protein amount. Subcellular localization of the CaSR in the T lymphocytes was determined by
Discussion
The cytokines released by T lymphocytes play important roles in the immune defense as well as systemic inflammatory response. Whether T lymphocytes expresses the CaSR functionally remains ambiguous so far. In the current study, Western blotting analysis showed that the specific protein bands at 120–170 kDa and 90–110 kDa were displayed in the naive and activated T cells. Moreover, the CaSR expression in activated T lymphocytes was increased in comparison to the naive T cells. In general, it is
Acknowledgments
This research is supported by National Natural Science Foundation of China (81101315) and Heilongjiang Province Technology Research Foundation of the Education Department (12511187).
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Both authors contributed equally to this work.