Introduction

As a member of G protein-coupled receptor superfamily, calcium-sensing receptor (CaSR) has been detected in a variety of tissues and cells, and plays an essential role in cell differentiation, proliferation, apoptosis, hormone secretion, etc., in addition to maintaining Ca2+ homeostasis in the body [1–4]. In previous studies, we have found that CaSR is involved in rat cardiac ischemia/reperfusion injury, apoptosis and hypertrophy, and that CaSR expression of myocardium in atherosclerotic rat was elevated, resulting in hypersensitivity to acute myocardial infarction [5, 6].

Monocytes-macrophages, as scavenger cells in the body, participate in the non-specific immunity as well as in many diseases and pathological processes [7–9], especially, atherosclerosis and multiple organ failure. Yamaguchi et al. have previously detected the expression of CaSR in human and murine monocytes isolated from peripheral blood [10–12]. However, the effect of CaSR activation on cytokine secretion and the pathway by which intracellular calcium was elevated by CaSR activation were not clarified in these studies.

THP-1, a human acute monocytic leukemia cell line, has been extensively used as a model for studying the differentiation of monocytes and macrophages in vitro [13]. When treated with phorbol-12-myristate-13-acetate (PMA), the THP-1 cells (monocyte-like) can differentiate into cells with macrophages-like phenotype [14–16]. Compared to other human myeloid cell lines, such as HL-60, U937, KG-1, or HEL cell lines, the differentiated THP-1 cells behave more like native monocyte-derived macrophages. For example, THP-1 cell line is the only cell line capable of producing lipoprotein lipase (LPL) in several human leukemia cell lines [15].

The imbalance of Ca2+ homeostasis is closely related with cardiovascular diseases such as hypertension, atherosclerosis, and coronary artery disease [17–19]. It is well known that monocytes-macrophages play important roles in onset and deterioration of atherosclerosis, shock, multiple organ failure, etc. [20–22]. Whether the monocyte/macrophage-mediated pathological processes are related with activation of CaSR remains unclear so far.

In this study, we determined whether CaSR is expressed in differentiated THP-1 cells stimulated with PMA, by which signal transduction pathway the CaSR activation induced increase of intracellular calcium concentration, and what effect CaSR activation had on cytokine release.

Materials and methods

Chemicals

LPS, GdCl3, NPS2390, PMA, NiCl2, CdCl2, U73122, U73343 and thapsigargin (TG) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluo-3/Am was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).

Cell line and cell cultures

The human acute monocytic leukemia cell line THP-1 was obtained from the American Type Culture Collection (Rockville, MD).THP-1 cells were maintained in RPMI 1640 (Thermo, Grand Island, NY), supplemented with 10% FBS (GIBCO, Grand Island, NY, USA) at pH 7.4, 37°C in a 5% CO2 atmosphere. THP-1 cells (5 × 105 cells per 35-mm dish) were differentiated by stimulation with PMA (160 nmol/ml, final concentration) for 3 days to obtain a macrophage-like phenotype, which closely resembles human monocyte-derived macrophages, as previously reported [12, 14].

Morphological examination

THP-1 cells were plated in 24-well plates. After 24, 48 and 72 h of treatment with PMA (160 nM), the cells were examined with an optical phase-contrast microscope (Olympus, Japan) after washing thrice with PBS.

Tartrate resistant acid phosphatase staining

Tartrate resistant acid phosphatase (TRAP) staining was performed according to the manufacturer’s instructions of the TRAP kit (Sigma, St. Louis, MO, USA). The THP-1 or differentiated THP-1 cells adhering to slides were fixed in Fixative Solution for 30 s and rinsed thoroughly in deionized water. These cells were divided into two groups. One group was incubated in solution A (Deionized water prewarmed 45 ml; Diazotized Fast Garnet GBC Solution 1.0 ml; Naphthol AS-Bl Phosphate Solution 0.5 ml; Acetate Solution 2.0 ml), the other group was incubated in solution B (Tartrate Solution 1.0 ml was added into solution A above mentioned) for 1 h in 37°C from light. The groups incubated in solution B was negative control. Then the cells were counterstained 2 min in hematoxylin solution, and rinsed several minutes in alkaline tap water to blue nuclei. The cells were then examined and photographed microscopically.

Western blotting for CaSR in THP-1 cells

Total proteins of the 293 cells, differentiated or undifferentiated THP-1 cells were prepared according to the manufacturer’s instructions as described [23]. Samples of 40 μg of protein from different experimental groups were separated by 10% SDS–PAGE and transferred to nitrocellulose membranes by electro-blotting. The membranes were blocked in TBS-T [137 mM NaCl, 20 mM Tris (pH 7.6), and 0.1% (v/v) Tween 20] containing 5% (w/v) skimmed milk at 37°C for 1 h. The membranes were then incubated overnight at 4°C with antibody against CaSR (Alomone Labs Ltd, Israel, 1:200). The membranes were incubated with secondary antibody AP-IgG (Promega) (diluted 1:5000 in TBS-T) for 1 h at room temperature. The lysate of 293 cells was the positive control. A peptide pre-absorption experiment was the negative control. β-actin was used as an internal control for a semi-quantitative assay.

Immunofluorescence staining of differentiated THP-1 cells for CaSR

The differentiated THP-1 cells plated at a density of 3.5–5 × 105 cells/ml onto cover slips, fixed with 4% paraformaldehyde, were treated with 3% BSA for 1 h and then incubated overnight at 4°C with CaSR primary antibody (1:50, Alomone). After washing the cells three times with 0.5% bovine serum albumin in PBS for 10 min, FITC-coupled, goat anti-rabbit IgG (1:100, Santa Cruz) was added and incubated for 1 h at room temperature. A peptide pre-absorption experiment was the negative control. The cells were then washed with PBS three times for 10 min. The cells were then examined and photographed using a fluorescence microscope (Olympus, Japan).

Measurement of intracellular free Ca2+ concentration

The differentiated THP-1 cells were loaded with 10 μM Fluo-3/AM for 30 min at 37°C, then rinsed with PBS for three times to remove the extracellular Fluo-3/AM. First, the cells were treated with various concentration of Ca2+ (0–30 mM) or Gd3+ (0–3 mM) to observe the effect of CaSR agonist on increase of intracellular free Ca2+ concentration ([Ca2+]i). According these results, Ca2+ (10 mM) or Gd3+ (1 mM) was used in the following experiments. These cells were randomly divided into CaCl2 challenge group and GdCl3 challenge group. Other intervention agents, for sample, NPS2390 (10 μM, a specific CaSR inhibitor), NiCl2 (100 μM, an inhibitor of Na+/Ca2+ exchanger), CdCl2 (20 μM, an inhibitor of L-type calcium channel), U73122 (10 μM, a PLC specific blocker), U73343 (10 μM, an inactivated analogue of U73122), thapsigargin (10 μM, TG, Ca2+-ATPase inhibitor), were added into media for 30 min before 10 mM Ca2+ or 1 mM GdCl3 challenge (see Fig. 4 in detail).

[Ca2+]i was detected at 488 nm using Laser Confocal Scanning Microscope (Leica Corporation, Germany). Following a 15 s baseline recording, cells were exposed to 10 mM Ca2+ or 1 mM Gd3+ and then recorded for 6 min. [Ca2+]i changes were showed with fluorescence intensity which represented FI and normalized to initial fluorescence intensity (FI0) [24].

Cytokine ELISA assays

Differentiated THP-1 cells were rinsed with PBS for three times before the experiments. Afterward, the cells were incubated in fresh serum-free medium containing 1 mg/L LPS, 10 μM NPS2390, GdCl3 (0–2 mM), or 1 mM GdCl3 together with 10 μM NPS2390, respectively, for 24 h.

Cytokine assays were performed with the following commercially available kits with a sensitivity (>10pg/ml): IL-1β and TNF-α human enzyme-linked immune absorbent assay (ELISA) system (Genetimes Technology Inc, Shanghai, China), according to the manufacturer’s instructions. Briefly, 100 μl of supernatant or recombinant protein standards were added to each well coated with primary antibodies against the specific cytokine, rinsed with buffer, incubated with the corresponding biotinylated secondary antibodies, and rinsed again before adding the streptavidin-horseradish peroxidase solution and developing the reaction with the tetramethylbenzidine (TMB) substrate solution. Absorbance of end products was determined at 450 nm within 10 min after developing the color.

Statistical analysis

All the data were obtained from at least three independent experiments that were replicated two or four times for each condition. All values are expressed as means ± SE. Comparisons among the groups were performed using one-way variance (ANOVA) followed by LSD test or Dunnett’s test. Values of P < 0.05 were considered statistically significant.

Results

Differentiation of THP-1 to macrophage-like cells

Morphological examination

After stimulated with PMA for 24 h (160 nmol/l), most of THP-1 cells adhered to dish surface, aggregated, and followed by elongation and pseudopodia formation in a time-dependent manner. More macrophage-like phenotypes appeared with time (Fig. 1).

Fig. 1
figure 1

Morphological examination. Morphological changes of the THP-1 cells treated with 160 nM of PMA for 0, 24, 48, and 72 h were photographed

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TRAP staining

The red staining, which indicated positive TRAP expression, was observed in the differentiated THP-1 cells induced by PMA for 72 h. There was no red staining in the undifferentiated THP-1 cells (Fig. 2).

Fig. 2
figure 2

Tartrate resistant acid phosphatase staining. TRAP staining was used for identification of macrophages differentiation derived from THP-1 cells. In the negative groups, the cells were incubated in the solution containing the tartrate (Fig. 2)

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CaSR expression in differentiated THP-1 cells

The protein expression of CaSR with two bands at 110 and 130 kD was detected in differentiated THP-1 cells and 293 cells (positive control) using Western blotting. In the negative control group, no bands could be observed (Fig. 3a).

Fig. 3
figure 3

The expression of calcium-sensing receptor (CaSR) in THP-1 cells. a Protein expression of CaSR in 293 cells (lanes 1, 6), undifferentiated (lanes 3, 4) or differentiated THP-1 cells (lane2, 5) was detected by western blot. A peptide pre-absorption experiment was the negative control (lanes 1, 2, 3). b The nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). A peptide pre-absorption experiment was the negative control (a).The immunofluorescence staining in differentiated THP-1 cells showed that CaSR was mainly expressed on the membrane and in the cytoplasm (b)

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Subcellular localization of CaSR in differentiated THP-1 cells was determined by an immunoflurescence staining method. Excited FITC emits green fluorescence. Nuclei stained with 4′-6-diamidino-2-phenylindole (DAPI) display blue fluorescence. Strong green fluorescence was observed on the differentiated THP-1 cell surface and cytosol, indicating the expression of CaSR protein across the plasma membrane and inside the cells. In the negative control group, no green fluorescence could be observed (Fig. 3b).

Increase of [Ca2+]i induced by CaSR activation via G-PLC pathway

We found that extracellular Ca2+ and Gd3+ could induce increase of [Ca2+]i in differentiated THP-1 cells in concentration dependent manner at [Ca2+]o ranging from 0 to 30 mM and [Gd3+]o ranging from 0 to 3 mM (Fig. 4a, b).

Fig. 4
figure 4

The effects of different agents on increase of intracellular calcium ([Ca2+]i) induced by extracellular Ca2+ or Gd3+ in differentiated THP-1 cells. a The cells in the group were exposed to various concentration of CaCl2. b The cells in the group were exposed to various concentration of GdCl3. c The cells in the experimental group were pretreated with NiCl2 (100 μM) and CdCl2 (20 μM) in the absence or presence of NPS2390 (10 μM), U73122 (10 μM), U73343 (10 μM) or TG (10 μM) for 30 min, then they were exposed consecutively to CaCl2 (10 mM). d The cells in experimental groups were exposed to GdCl3 (1 mM) with or without pre-exposure to NPS2390 (10 μM), U73122 (10 μM), U73343 (10 μM) or TG (10 μM) for 30 min. The cells in control group were incubated in Ca2+-free Krebs buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.2 mM NaHCO3, 10 mM glucose, 10 mM Hepes, pH 7.4) (c, d). The intracellular calcium record was performed for 6 min (c, d)

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The pre-treatment of 100 μM NiCl2(an inhibitor of Na+/Ca2+ exchanger)and 20 μM CdCl2 (an inhibitor of L-type calcium channel) for 30 min significantly diminished the increase of [Ca2+]i induced by 10 mM Ca2+, but could not be restored [Ca2+]i to the basal level. Meanwhile, NPS2390 (an antagonist of CaSR) completely abolished the increase of [Ca2+]i induced by 10 mM Ca2+ or 1 mM Gd3+(Fig. 4c, d).

Under the normal condition, the intracellular Ca2+ is from extracellular Ca2+ entry and release of intracellular Ca2+ store. To verify whether the change of [Ca2+]i induced by CaSR activation is dependent on intracellular Ca2+ store, cells were pretreated with 10 μM thapsigargin (TG, Ca2+-ATPase inhibitor) or 10 μM U73122 (a PLC specific blocker) for 30 min. The results showed that both TG and U73122 eliminated the increase of [Ca2+]i caused by extracellular 10 mM Ca2+ or 1 mM Gd3+. On the other hand, pretreatment with 10 μM U73334 (an inactivated analogue of U73122) did not prevent this increase of [Ca2+]i (Fig. 4c, d).

Treatment with NiCl2 + CdCl2, NPS2390, TG, and U73122 alone had no effect on [Ca2+]i of differentiated THP-1 Cells (The data were not supplied).

The increase of TNF-α and IL-1β release in differentiated THP-1 macrophages induced by GdCl3

ELISA assays showed that GdCl3 increased IL-1β and TNF-α in the centrifuged supernatant depending on the GdCl3 concentration, and 1 mM GdCl3 induced the secretion of IL-1β and TNF-α significantly, as well as LPS (1 mg/l). The increase of IL-1β and TNF-α induced by GdCl3 was attenuated by 10 μM NPS2390. Treatment with NPS2390 alone did not affect secretion of IL-1β and TNF-α (Fig. 5).

Fig. 5
figure 5

Effect of GdCl3 loading on IL-1β and TNF-α release in differentiated THP-1 cells. Differentiated THP-1 cells were incubated in serum-free medium for 4 h at 37°C. Then cells were incubated with LPS (1 mg/l), or various concentration of GdCl3 (0–2 mM) for 24 h, or pre-treated with 10 μM NPS2390 for 1 h before adding GdCl3. The concentration of IL-1β (a, c) and TNF-α (b, d) in the culture media was detected with ELISA kit. Results are expressed as mean ± SE of four independent experiments. * P < 0.05 versus control group, ** P < 0.01 versus control group, # P < 0.05 versus 1 mM GdCl3 group, $ P > 0.05 versus control group

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Discussion

Systemic inflammatory response is involved in many diseases and pathological processes, for example, atherosclerosis, shock, diabetes, organ failure. The cytokines released by monocyte/macrophage play important roles in systemic inflammatory response. Whether the more like native macrophages derived from THP-1 express CaSR functionally remains unclear so far.

To confirm the existence of functional CaSR expression in some tissues, the following evidence must be provided: (1) the existence of CaSR mRNA and protein in these tissues or cells; (2) the increase of [Ca2+]i is owing to extracellular calcium ([Ca2+]o) increment; (3) the [Ca2+]o-induced increase of [Ca2+]i is dependent on the release of Ca2+ from intracellular stores, but not on the calcium influx via voltage-dependent calcium channel; (4) the CaSR agonist- Gd3+ can cause similar response induced by an elevation of [Ca2+]o [25–28].

In this study, the morphological observation and TRAP staining indicated that THP-1 cells treated with PMA could differentiate into cells with the phenotype of macrophages. Western blotting analysis showed that the two specific protein bands at 110 and 130 kD, which represented as the precursor and mature form of CaSR, were displayed in differentiated THP-1. It is generally agreed that bands of 130–170 kDa are fully glycosylated mature form of the CaSR [28, 29]. The size of CaSR protein detected by western blotting varies considerably depending on the tissue and cell type, cellular fraction analyzed (membrane or cytoplasm), and the degree of posttranslational modification (glycosylation) of the CaSR protein. In our experiment, the same bands were detected in 293 cell expressing CaSR cDNA and did not appear in the peptide pre-absorption group. These results demonstrated that the 110- and 130-kDa bands were specific to CaSR. Meanwhile, the immunofluorescence staining indicated that CaSR proteins were located in the membrane and cytoplasm of differentiated THP-1 cells. Based on these data, we defined the expression of CaSR in differentiated THP-1 cells. Yamaguchi et al. reported that the CaSR expression exists in peripheral blood monocytes and several mononuclear cell strains, such as, human leukemia cell line HL-60 [30].

Wang and Xu have reported that the increase of [Ca2+]o or other CaSR agonists (Gd3+, spermine, etc.) caused the release of calcium from sarcoplasmic reticulum and led to a quick multiplication of [Ca2+]i via the G protein-PLC-IP3 signaling transduction pathway [31].

In this study, we observed that extracellular Ca2+ and Gd3+ could induce increase of [Ca2+]i in differentiated THP-1 cells in concentration-dependent manner, and the increase of [Ca2+]i caused by 10 mM Ca2+ could be partially inhibited by NiCl2 (100 μM) + CdCl2 (20 μM) or abolished by NiCl2 + CdCl2 with NPS2390 (10 μM), U73122 (10 μM) or thapsigargin (10 μM). In GdCl3 challenge group, we obtained similar results. Besides activating CaSR, Gd3+ shows calcium-related functions, such as acting on stretch-activated and other calcium channels, so we chose NPS2390 (a specific inhibitor of CaSR) as an approach to evaluate the specificity of this activity on CaSR in this study.

As we all know, extracellular calcium entry is a triggering factor, and calcium release from endoplasmic reticulum is critical for increase of [Ca2+]i. Therefore, TG inhibiting the endoplasmic reticulum ATP-ase (that pumps Ca2+ into the ER) would elevate [Ca2+]i transiently, but a continuous inhibition of Ca2+ pump on ER could induce calcium exhaustion in ER, and resulte in decrease of [Ca2+]i. IP3 receptor-dependent calcium release from ER is also an important mechanism for increase of [Ca2+]i. The activated phospholipase C (PLC) could hydrolyze phosphatidylinositol 4,5-bisphosphate(PIP2) into IP3 and DAG, which can cause IP3 receptor-dependent calcium release from ER and extracellular calcium entry through L-type of calcium channel, respectively. U73122 is a special inhibitor of PLC, so pretreatment with U73122 could induce decline of [Ca2+]i. NiCl2 (an inhibitor of Na+/Ca2+ exchanger) and CdCl2 (an inhibitor of L-type calcium channel) could decrease extracellular calcium entry. These data suggest that the increase of intracellular calcium induced by activation of CaSR in differentiated THP-1 cells is through the G protein-PLC-IP3 signaling transduction pathway, although Na+/Ca2+ exchanger and L-type calcium channel are also involved in the increase of [Ca2+]i caused by increased [Ca2+]o.

The production and release of inflammatory factors and cytokines are an important function of monocyte-macrophages. Sugimoto reported that elevated [Ca2+]o contributes to the augmentation of peripheral blood mononuclear cells chemotaxis [32]. In this study, we found that GdCl3 treatment, as well as LPS, could induce an increase of IL-1β and TNFα level in the supernatant of THP-1 cells with concentration- dependent manner, and that NPS2390 could partially inhibit the Gd-induced IL-1β and TNF-α release in the cells, while this drug had pretty efficiency on blocking the Gd-induced increase of intracellular calcium. How to interpret such a discrepancy if cytokine secretion is mainly mediated by the CaSR? We think that Gd3+ is not only an agonist of CaSR, but also a kind of heavy metal which is harmful to body and can activate immune system. There might be other way, besides CaSR activation, by which Gd3+ induces IL-1β and TNFα release in THP-1 cells. Wermuth et al. reported that Gd3+ could induce expression and release of numerous cytokines [33]. Therefore, NPS2390, as an antagonist of CaSR, could only inhibit release of cytokines induced by Gd3+ via activating CaSR.

In conclusion, this study demonstrated that CaSR was functionally expressed in differentiated THP-1 cells with the phenotype of macrophages and that the activation of CaSR could promote the secretion of IL-1β and TNFα in differentiated THP-1 cells. These results suggested that CaSR was involved in a variety of mononuclear macrophage related diseases possibly, and that it should be considered as a promising and potential therapeutic target.