Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway

Porcine bone marrow mesenchymal stem cells (pBMSCs) have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium ([Ca2+]o) on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM [Ca2+]o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, [Ca2+]o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, [Ca2+]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca2+]o. Moreover, [Ca2+]o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca2+]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair.


Introduction
Bone marrow mesenchymal stem cells (BMSCs) not only regulate hematopoietic and other stem cells niches but also have multipotential capacities to differentiate toward osteocyte, chondrocyte, adipocyte, and myocyte [1], performing an important role in regenerative medicine, wound healing, and disease therapy [2]. Pigs exhibit similar structure and function to those of humans and have been widely used as a valuable model in biomedical research such as tissue engineering and cell therapy [3]. In addition to the studies on the isolation and differentiation capacities of porcine BMSCs (pBMSCs) [4,5], the investigation on pBMSCs proliferation, which ensures that a large amount of pBMSCs is obtained, is very important. Thus, controlling the proliferation of pBMSCs is an attractive approach to determine the size of the pBMSCs pool and subsequently its possible influence on the maintenance of stem cell niches and multilineage differentiation potential.
Calcium ion, one of the most widely occurring second messengers, is an important cellular signaling component, which has been shown to play a pivotal role in controlling cell proliferation [6,7]. Extracellular calcium ([Ca 2+ ] o ) modulates cell proliferation in various cells, such as myeloma cells 2 Stem Cells International [8], rat bone marrow-derived progenitor cells [9], osteoblasts [10], preadipocytes [11], and synovium-derived mesenchymal stromal cells [12]. [Ca 2+ ] o exerts its role in regulating cell proliferation either via calcium influx through calcium channels or by activating the calcium-sensitive receptor (CaSR) [13]. CaSR is a G protein-coupled receptor on plasma membrane which senses [Ca 2+ ] o [14]. Several studies have shown that CaSR is expressed in mesangial cells [15], osteoblasts [10], and preadipocytes [11] and plays a vital role in the regulation of cell proliferation [16].
[Ca 2+ ] o always leads to the increase of intracellular calcium ([Ca 2+ ] i ) and subsequent activation of the intracellular signaling pathway, which regulates cell proliferation. The MAPK signaling pathway, which consists of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun kinase (JNK), and p38 MAPK, plays an important role in controlling cell proliferation in mammalian cells [17]. In particular, ERK1/2 is involved in the proliferation of various cells, such as kidney epithelial cells [18], smooth muscle cell [19], melanoma cell [20], and preadipocytes [11]. ERK elicits its role in cell proliferation possibly through its effect on the cell cycle transition from the G0/G1 phase to the S phase [21] and/or on the expression of cyclin D [22,23].
Although many studies described that [Ca 2+ ] o is involved in the regulation of cell proliferation, the role of [Ca 2+ ] o in pBMSCs proliferation and the possible mechanisms underlying this process remain unclear. Thus, the present study was designed to investigate the effects of [Ca 2+ ] o on pBMSCs proliferation by determining the cell numbers, cell cycle progression, and expression levels of proliferative marker genes. In addition, we sought to explore the underlying mechanisms involved in this process, including the contribution of CaSR and the relevant intracellular signaling pathway. Our results revealed that the enhanced pBMSCs proliferation in response to [Ca 2+ ] o was associated with the activation of the CaSR and ERK1/2 signaling pathway.

Cell
Culture and Treatment. pBMSCs were isolated and purified from the bone marrow of postnatal Landrace pigs aged between 5 and 7 days as we previously described [4]. The purified pBMSCs were seeded in a 96-well plate with density of 5000 cells/well and cultured in DMEM/F12 medium (containing 1 mM calcium) supplemented with 10% FBS, 100 U/mL of penicillin sodium, and 100 g/L of streptomycin sulfate in a humidified cell incubator with atmosphere of 5% CO 2 at 37 ∘ C. The pBMSCs were treated with various concentrations (1,2,4

Cell Cycle
Analysis. pBMSCs were seeded at 1 × 10 6 cells/per 25 cm 2 flask and cultured in presence of 4 mM [Ca 2+ ] o and/or CaSR antagonist NPS2143 supplementation for 5 days. Cell cycle status was determined by measuring cellular DNA content following staining with propidium iodide using flow cytometry as previously described [24]. Briefly, the cells were centrifuged and washed twice with ice-cold phosphate-buffered saline (PBS) and then fixed overnight in 70% ethanol at 4 ∘ C. Fixed cells were centrifuged at 2500 rpm for 5 min and the supernatant was discarded. Pellets were washed twice and incubated with propidium iodide/RNase Staining Buffer (BD Pharmingen, USA) for 30 min at room temperature in the dark.  2.6. Western Blot Analysis. At the end of incubation, the pBMSCs were harvested and washed twice with PBS. Then Western blot was conducted as previously described [26]. Cells were lysed by lysis buffer, and the cell lysates were centrifuged to remove insoluble materials and the protein concentration of each sample was measured. Equal protein amounts of each sample were separated by SDS-PAGE and electroblotted to PVDF membranes (Millipore, Billerica,

Real-Time Quantitative PCR.
The expressions of proliferative marker genes and CaSR were examined by real-time quantitative PCR as we previously described [27]. Briefly, total RNA was extracted from pBMSCs by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and cDNA was synthesized from 1 g of total RNA by the M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and random primers oligo(dT)18 according to the manufacturer's instructions. -actin was used as a candidate housekeeping gene. Real-time quantitative PCR was carried out in Mx3005p instrument (Stratagene, La Jolla, CA, USA) by using SYBR Green Real-Time PCR Master Mix reagents (Toyobo Co., Ltd., Osaka, Japan) and both sense and antisense primers (200 nM for each gene). Relative gene expression of each gene between experimental groups was analyzed using the 2 −ΔΔct method. Primer sequences (with their respective PCR fragment lengths) were shown in Table 1.

Statistical
Analysis. All data are presented as means ± standard error of the mean (SEM). Statistical analysis was performed using SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA). Differences between means were determined using Student's -test and a confidence level of < 0.05 was considered to be statistically significant.     (Figure 3(a)) or relative fluorescence (Figure 3(b) (Figure 5(a)). In agreement, the significant increase in the mRNA levels of cyclin A2, cyclin D3, cyclin E2, and PNCA and the significant decrease of p21 mRNA levels induced by 4 mM [Ca 2+ ] o were also abolished by U0126 ( Figure 5(b)). In addition, U0126 reversed the effects of [Ca 2+ ] o on the protein expression of cyclin D1 and p21 (Figures 5(c) and 5(d)). These results strongly suggested that [Ca 2+ ] o promoted pBMSCs proliferation through the activation of the ERK1/2 signaling pathway.

Discussion
In the present study, we determined that [Ca 2+ ] o promoted pBMSCs proliferation by regulating cell cycle progression and the expression levels of proliferative marker genes through the activation of plasma membrane receptor CaSR and intracellular ERK1/2 signaling pathway. The proproliferation effects of [Ca 2+ ] o have been reported in various cells such as rat bone marrow-derived progenitor cells [9], osteoblasts [10], preadipocytes [11], and porcine synoviumderived mesenchymal stromal cells [12]. In line with these results, we found that [Ca 2+ ] o promoted pBMSCs proliferation in a dose-dependent manner, with the similar promotive effects observed when [Ca 2+ ] o was greater than or equal to 4 mM. However, Liu et al. reported that the optimal [Ca 2+ ] o needed for rabbit BMSCs to proliferate was 1.8 mM and the higher level of [Ca 2+ ] o did not change cell proliferation [28]. In addition, it was demonstrated that the high level of [Ca 2+ ] o (7 or 10 mM) slowed the rate of porcine osteoblasts proliferation [29]. Furthermore, Lin et al. found that low calcium (0.09 mM) greatly enhanced the growth rate and extended the lifespan of human adipose-derived MSCs [30]. These discrepant effects of [Ca 2+ ] o on BMSCs proliferation might be attributed to the various species and/or culture conditions. In addition, the heterogeneous characteristic of BMSCs should also be considered for the different effects of [Ca 2+ ] o on BMSCs proliferation. The pBMSCs used in our study were positive for mesenchymal surface markers CD29 and CD44 and negative for hematopoietic marker CD45 and for the adhesion molecule CD31 and were able to differentiate into adipocytes and myocytes [4], which represented only a subpopulation of MSCs in porcine bone marrow. It should be noted that this study was performed in 20% oxygen tensions, which are generally used in standard culture. It has been reported that, in the presence of high oxygen tension (20% to 21% O 2 ) culture conditions, MSCs derived from human [31], mouse [32], and rat [33] show lower proliferative activity than that in the presence of low oxygen tension (2% to 5% O 2 ) culture conditions. Thus, the effects of [Ca 2+ ] o on pBMSCs proliferation in low oxygen tension should be further investigated in future study. The cell cycle progression and the expression levels of proliferative marker genes (cyclins, PCNA, and p21) were detected to elucidate the stimulatory effects of [Ca 2+ ] o on pBMSCs proliferation. In our study, we determined that [Ca 2+ ] o significantly decreased the ratio of G0/G1 phase and increased the percentage of the S phase and the PI in pBMSCs, compared with those of control group. These data indicated that [Ca 2+ ] o accelerated cell cycle progression from the G0/G1 phase to the S phase and promoted pBMSCs proliferation. It has been implicated that cyclin D and cyclin E are required for the transition from G1 to S phase of the cell cycle, whereas cyclin A is involved in the initiation and completion of DNA replication during the S phase [34,35]. In accord, we detected the elevated expressions of cyclin A2, cyclin D1/3, and cyclin E2 induced by [Ca 2+ ] o . In addition, the level of PCNA, an essential component for DNA replication machinery [36], was also improved by [Ca 2+ ] o . By contrast, p21, the inhibitor of cyclin-dependent kinase, was inhibited in the presence of [Ca 2+ ] o . Thus, these findings indicated that [Ca 2+ ] o stimulated pBMSCs proliferation by influencing the cell cycle progression and the expression levels of proliferative marker genes.
[Ca 2+ ] o elicits its effects on the regulation of cell proliferation either via calcium influx through calcium channels or by activating CaSR-mediated signaling pathway [13]. Although it has been reported that the voltage-gated calcium channels (VGCCs) antagonist nifedipine exerts antiproliferative effects on rats BMSCs [37], our result In contrast, we found the expression of CaSR in pBM-SCs, which was also expressed in mesangial cells [15], osteoblasts [10], preadipocytes [11], and rat BMSCs [38] and played a vital role in the regulation of cell proliferation.  [42]. Our results also showed that CaSR inhibition by NPS2143 reversed the promotion of pBMSCs proliferation, the change of cell cycle distribution, and the expression levels Numerous studies have shown that the activation of the ERK1/2 signaling pathway is involved in regulating the proliferation of various cells, including kidney epithelial cells [18], smooth muscle cell [19], melanoma cell [20], and preadipocytes [11]. Accordingly, in the present study, the ERK1/2 signaling pathway was activated by [Ca 2+ ] o and the activation of ERK1/2 was abolished by NPS2143. In addition, the inhibition of ERK1/2 signaling pathway with U0126 reversed the stimulation of pBMSCs proliferation, the alteration of cell cycle distributions, and the expression levels of cell cycle marker genes, which were induced by [Ca 2+ ] o . These results provided the evidence that [Ca 2+ ] o stimulated pBMSCs proliferation, at least in part, via activation of CaSR and the linked intracellular ERK1/2 signaling pathway. However, Tfelt-Hansen et al. found that the activation of p38 MAPK and PI3K but not that of ERK1/2 by CaSR promoted the cell proliferation of rat Leydig cancer cells [43]. The reason for the discrepancy between the study of Tfelt-Hansen et al. and our study might be the different cell types.
In conclusion, our findings demonstrated that CaSR was expressed in pBMSCs and that the enhanced proliferation of pBMSCs in response to [Ca 2+ ] o was associated with activation of plasma membrane receptor CaSR, elevation of [Ca 2+ ] i , and the enhancement of the intracellular ERK1/2 signaling pathway. These data may be useful for the application of pBM-SCs in future clinical studies aimed at tissue regeneration and repair.