TLR3-/4-Priming Differentially Promotes Ca2+ Signaling and Cytokine Expression and Ca2+-Dependently Augments Cytokine Release in hMSCs

In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs. Poly(I:C) exposure rather than LPS incubation not only elevated inositol 1,4,5-triphosphate receptor (IP3R) expression and IP3R-mediated Ca2+ release, but also promoted Orai and STIM expression as well as store-operated Ca2+ entry into hMSCs. In addition, we also observed that 21 Ca2+ signaling genes were significantly up-regulated in response to TLR3 priming of hMSCs by RNA sequencing analysis. Both poly(I:C) and LPS exposure enhanced cytokine release from hMSCs. The enhanced cytokine release vanished upon siRNA knockdown and chelation of intracellular Ca2+. These data demonstrate that TLR3- and TLR4-priming differentially enhance Ca2+ signaling and cytokine expression, and Ca2+ -dependently potentiates cytokine release in hMSCs.


Results
Expression of stem cell markers and differentiation potential in hMSCs. MSC specific surface markers present on hMSCs were analyzed by flow cytometry (Fig. 1a). The cells were positive for an adhesion molecule (CD44), an integrin marker (CD29), and MSC markers (CD90, CD105, CD73), and were negative for a hematopoietic marker (CD34, CD45), an endothelial marker (CD31), and major histocompatibility antigen (HLA-DR). We performed RT-PCR analysis to quantitate the expression of stem cell specific genes. As shown in Fig. 1b, hMSCs expressed markers for pluripotency and self-renewal (OCT4, SOX2), osteogenic state (OPN), mesoderm state (CXCR4), and extracellular matrix molecules (COL10A1). The expression pattern of surface proteins and genes on our hMSC preparations indicated that these cells are primitive to the MSC population. To evaluate the differentiation ability of hMSCs, the cells were cultured in adipogenic and osteogenic medium for 3 weeks. Figure 1c shows the control hMSC morphology (left) and the capacity of hMSCs to differentiate into adipocytes (middle) and osteoblasts (right).

Characterization of Basal [Ca 2+ ] i and Ca 2+ Release and Entry Pathways in hMSCs.
We first characterized basal [Ca 2+ ] i in hMSCs. Real-time single-cell measurements of [Ca 2+ ] i revealed two distinct patterns of basal [Ca 2+ ] i in hMSCs (Fig. 2a). Some hMSCs maintained a stable resting [Ca 2+ ] i , whereas others displayed spontaneous [Ca 2+ ] i oscillations without stimulation.
We next clarified the mechanisms underlying Ca 2+ mobilization from intracellular stores in hMSCs. Stimulation with the muscarinic agonist carbachol (CCH; 50 μM) drastically increased [Ca 2+ ] i in the absence and presence of extracellular Ca 2+ (Fig. 2b). Importantly, the addition of 50 μM CCH evoked similar increases in [Ca 2+ ] i in the absence and presence of extracellular Ca 2+ . Such CCH-induced [Ca 2+ ] i increases suggest that Ca 2+ mobilization from IP 3 -sensitive stores is operational in hMSCs. Furthermore, challenge with the RyR agonist caffeine (10 mM) did not cause a [Ca 2+ ] i increase, but instead slightly decreased [Ca 2+ ] i (Fig. 2e). The inability of caffeine to mobilize Ca 2+ from intracellular stores verifies the presence of few, if any, caffeine/ ryanodine-sensitive Ca 2+ stores in hMSCs. These data reveal that hMSCs employ IP 3 -sensitive stores rather than caffeine/ryanodine-sensitive stores to release Ca 2+ into the cytosolic compartment.
We also elucidated Ca 2+ entry pathways in hMSCs. As shown in Fig. 2b, depolarization with 150 mM KCl produced no appreciable alteration in [Ca 2+ ] i . This suggests that hMSCs do not use VGCC to mediate Ca 2+ influx. Interestingly, extracellular Ca 2+ efficiently entered hMSCs whose intracellular Ca 2+ stores were depleted by inhibiting the sarcoendoplasmic reticulum Ca 2+ ATPases with the SERCA inhibitor CPA (10 μM) (Fig. 2c,d). Moreover, such Ca 2+ entry was effectively abolished by application of the membrane permeable SOCE antagonist 2APB (50 μM) (Fig. 2d). It is clear that extracellular Ca 2+ enters hMSCs through SOCE. These observations illustrate that SOCE serves as an important mechanism to mediate Ca 2+ entry through the plasma membrane of hMSCs.

TLR3-and TLR4-Priming Up-Regulates the mRNA Expression Levels of TLR3, TLR4 and Cytokines in hMSCs.
To quantify the impact of the TLR3 agonist poly(I:C) and the TLR4 agonist LPS on the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs, we performed RT-PCR and real-time RT-PCR assays. RT-PCR analysis confirmed that control hMSCs expressed both TLR3 and TLR4 mRNAs. This analysis revealed that 4 h exposure to LPS and poly(I:C) elevated TLR4 and TLR3 mRNA expression in hMSCs in a concentration and time-dependent manner (Fig. 3a). Quantification data show the sum of triplicate repeated RT-PCR (Fig. 3a, lower panel). Neither poly(I:C) exposure nor LPS treatment influenced the expression of β -actin. Real-time RT-PCR showed that TLR3 mRNA levels reached the highest level in cells exposed to 5 μg/ml poly(I:C) for 4 h during different exposure times, whereas 1 h treatment with LPS (10 ng/ml) appeared to elevate TLR3 mRNA expression to a plateau level (Fig. 3b). These results suggest that TLR3 expression is more plastic than TLR4 expression following priming of the corresponding receptors.
Interestingly, real-time RT-PCR detection showed that incubation with 5 μg/ml poly(I:C) for 4 h preferably elevated IL4 mRNA levels. In contrast, 4 h treatment with LPS (10 ng/ml) preferentially up-regulated the mRNA expression levels of IL6, IL8 and IP10 (Fig. 3c). These findings reveal that TLR3-and TLR4-priming differentially regulate the mRNA expression of several cytokines including IL4, IL6, IL8 and IP10 in hMSCs.

TLR3-Priming Potently Promotes IP 3 R Expression and IP 3 R-Mediated Ca 2+ Mobilization in hMSCs.
To explore the possible signaling pathways of TLR3 and TLR4 that respond to the highly versatile intracellular signal Ca 2+ , we focused our attention on Ca 2+ mobilization from IP 3 -sensitive stores, which is likely to be the only Ca 2+ release mechanism in hMSCs ( Fig. 2). Therefore, we examined the effects of poly(I:C) and LPS treatments on ITPR (IP 3 R) expression and IP 3 R-mediated Ca 2+ mobilization in hMSCs using RT-PCR analysis, [Ca 2+ ] i measurements, confocal immunofluorescence microscopy and western blot analysis.
[Ca 2+ ] i measurements showed that stimulation with 50 μM CCH evoked [Ca 2+ ] i transients with somewhat different patterns in control cells bathed in extracellular solution without Ca 2+ (Fig. 4a, left panel). Incubation with 5 μg/ml poly(I:C) for 4 h significantly increased CCH-evoked [Ca 2+ ] i responses and the percentage of CCH-responsive cells in the absence of extracellular Ca 2+ (Fig. 4a, right panel and Fig. 4b). However, treatment with 10 ng/ml LPS for 4 h only marginally elevated these two parameters under the same experimental conditions. These results illustrate that TLR3-priming potently promotes IP 3 R-mediated Ca 2+ mobilization in hMSCs, but TLR4-priming is not potent enough to do so.
The RT-PCR blot shows that control hMSCs expressed abundant ITPR1 (IP 3 R1), ITPR2 (IP 3 R2) and ITPR3 (IP 3 R3) mRNAs, but very few RYR1 mRNAs and no RYR2 mRNAs (Fig. 4c, upper panel). The real-time RT-PCR  assay showed that priming of TLR3 with poly(I:C) induced significant increases in ITPR1 (IP 3 R1), ITPR2 (IP 3 R2) and ITPR3 (IP 3 R3) mRNAs, whereas priming of TLR4 with LPS did not influence the mRNA expression of all three subtypes of IP 3 Rs (Fig. 4c, lower panel). Furthermore, confocal immunofluorescence microscopy revealed that TLR3-primed hMSCs (Fig. 4d, right panel) displayed more intense IP 3 R3 immunoreactivity than TLR4-primed (Fig. 4d, middle panel) and control hMSCs (Fig. 4d, left panel) under the same experimental conditions. There was no appreciable difference in the intensity of IP 3 R3 immunoreactivity between TLR4-primed  and control hMSCs. Finally, western blot analysis was performed to examine the expression level of the IP 3 R in same condition. Consistent with the immunofluorescence results, treatment with poly(I:C), but not LPS, induced significant increases in the expression of the IP 3 R3 (Fig. 4e). These data verify that TLR3-priming rather than TLR4-priming is potent enough to enhance the expression of IP 3 Rs in hMSCs.

TLR3-Priming Effectively Augments Orai and STIM Expression and SOCE in hMSCs.
To reveal other possibilities that bridge TLR3-and TLR4 priming and [Ca 2+ ] i , we also studied the hMSC-predominant Ca 2+ influx SOCE. We evaluated the influence of TLR3-and TLR4-priming on the expression of three Orai and two STIM proteins as well as SOCE in hMSCs by RT-PCR analysis, [Ca 2+ ] i measurements, confocal immunofluorescence microscopy and western blot analysis.
Single-cell [Ca 2+ ] i analysis revealed that exposure to CPA (10 μM) and the subsequent addition of extracellular Ca 2+ evoked prominent [Ca 2+ ] i transients in control (n = 18), LPS-(n = 17) and poly(I:C)-treated groups (n = 17) in Ca 2+ -free extracellular solution (Fig. 5a). Importantly, the mean net increase of [Ca 2+ ] i reflected by the averaged delta F340/F380 ratios following CPA exposure was significantly higher in the poly(I:C) group than in the control group, whereas this parameter was similar between LPS-treated and control cells (Fig. 5b). More importantly, the mean net increase of [Ca 2+ ] i induced by extracellular application of 4 mM Ca 2+ following Ca 2+ store depletion by CPA was significantly exaggerated in poly(I:C)-treated cells, but just marginally elevated in LPS-treated cells in comparison with that in control cells (Fig. 5c). Moreover, basal [Ca 2+ ] i was mirrored by the averaged F340/F380 ratios prior to application of CPA and was increased significantly in the poly(I:C) group, but was elevated only slightly in LPS the group compared with the control group (Fig. 5g). There is no doubt that TLR3-priming effectively enhances SOCE with a concomitant increase in basal [Ca 2+ ] i in hMSCs.
The RT-PCR assay shows that the mRNA expression levels of three Orai subtypes and two STIM subtypes as well as TRPM4, TRPM7 and TRPC4 occurred clearly in control cells ( Fig. 4d and Figure S1). Real-time RT-PCR analysis illustrates that the mRNA levels of two Orai subtypes and one STIM subtypes significantly elevated in the poly(I:C) group (n = 3), but not in the LPS group (n = 3) in comparison with the control group (n = 3) (Fig. 5d). In addition, the expression of the large-conductance calcium-activated potassium channel gene MaxiK did not change following treatment with either poly(I:C) or LPS in hMSCs ( Figure S2). Furthermore, confocal immunofluorescence microscopy showed that Orai2 (ii) immunofluorescence was significantly brighter in TLR3-primed cells than in control cells under the same experimental conditions. In contrast, this immunofluorescence was only slightly brighter in TLR4-primed cells than in control cells (Fig. 5e). Because poly(I:C) treatment greatly enhanced the mRNA level of Orai2 among the members of SOCE, western blot analysis was employed to examine the protein level of Orai2 under the same condition. Consistent with the previous results, treatment with poly(I:C) but not LPS significantly increased the expression of Orai2 (Fig. 5f). Taken together, these findings suggest that TLR3-priming exaggerates SOCE-mediated Ca 2+ signaling.

TLR3-and TLR4-Priming that is Ca 2+ -Dependent Enhances Cytokine Release from hMSCs.
Cytokine release is considered an important activity in TLR3-and TLR4-primed hMSCs. This led us to study whether the promotion of Ca 2+ signaling by TLR3-and TLR4-priming influences cytokine release from hMSCs. We measured IL6, IL8, IP10 and RANTES from cells exposed to either LPS or poly(I:C) in comparison with control cells.
ELISA assay shows that control cells released undetectable amounts of IL8, IP10 and RANTES, but measurable IL6 from control cells (Fig. 6a). Interestingly, TLR3-and TLR4-priming markedly promoted the release of IL6, IL8, IP10 and RANTES (Fig. 6a). More interestingly, TLR3-and TLR4-priming-induced release of IL6 and RANTES was effectively ablated by chelation of intracellular Ca 2+ with BAPTA/AM (5 μM) (Fig. 6b,c). Type I interferons (IFNs) are mainly involved in the innate immune response against viral infection and have been identified as an important step in the initial inflammatory phase. We analyzed IFN-α and IFN-β cytokine release in TLR3-and TLR4-primed MSCs. Compared to untreated cells, IFN-α was increased in hMSCs following LPS and poly(I:C) treatment. Although the production of IFN-α by untreated cells was also increased. These unusually high constitutive productions of IFN-α are probably due in part to the differences in culture techniques. BAPTA/AM also showed a reducing effect on IFN-α release similar to IL6 and RANTES (Fig. 6d, upper panel). However, these factors did not induce the repression of IFN-β in hMSCs. We assessed the correlation between the BAPTA/AM effect and the mRNA expression of ITPR3, Orai2 and Stim1. Consistent with the cytokine results, real-time RT-PCR analysis illustrates that the mRNA levels of ITPR3, Orai2 and STIM1 were very significantly reduced by BAPTA/AM in both control and TLR3-primed hMSCs with similar patterns (Fig. 6e). These results show that enhanced cytokine release by TLR3-and TLR4-priming critically relies on [Ca 2+ ] i in hMSCs. We also investigated treatment (n = 19), exposure to LPS (n = 19) and incubation with poly(I:C) (n = 19). Herein, n denotes the number of experiments. (c) Representative RT-PCR blots showing the mRNA expression of three IP 3 R subtypes (ITPR1, ITPR2 and ITPR3) and two RyR subtypes (RYR1 and RYR2) in control cells. GAPDH serves as an internal control. NC indicates negative control, i.e., distilled water. Real time RT-PCR quantification illustrating the different mRNA expression profiles of three IP 3 R subtypes (ITPR1, ITPR2 and ITPR3) in the control, LPS and poly(I:C) groups. Experiments were performed three times. (d) Confocal images showing the different intensities of IP 3 R3 immunofluorescence in control cells (left panel) and cells exposed to LPS (middle panel) or poly(I:C) (right panel). (e) Representative western blot of IP 3 R3 in control cells and cells exposed to LPS or poly(I:C) (left panel). Summarized graph showing the normalized level of IP 3 R in indicated conditions (right panel). Pan-Cadherin was used as a loading control. Experiments were performed six times. The significance level was set at *p < 0.05 or **p < 0.005.  2+ ] i reflected by the averaged F340/F380 ratios registered before application of CPA in control cells and cells exposed to LPS or poly(I:C). Experiments were performed nineteen times. The significance level was set at *p < 0.05 or **p < 0.005. whether ITPR3 depletion ( Figure S4) affects cytokine production. Using ELISA assay we found that compared to scrambled siRNA control (NC), IL6 in the supernatants was significantly decreased in ITPR3 siRNA hMSC cells (Fig. 6f).

Discussion
The present work confirms that two different populations of hMSCs in the same extracellular milieu show two distinct profiles of basal [Ca 2+ ] i , one exhibiting a stable resting [Ca 2+ ] i and the other displaying spontaneous [Ca 2+ ] i oscillations. It is plausible to postulate that one or more [Ca 2+ ] i oscillation-generating devices, e.g., IP 3 R2, somehow become active under basal conditions to create this spontaneous [Ca 2+ ] i oscillation in the latter population of hMSCs. This postulation is supported by the fact that the IP 3 R2 undergoes activation at the lowest concentration of IP 3 among the three IP 3 R subtypes to generate [Ca 2+ ] i oscillations 48 and that all three IP 3 R subtypes are present in hMSCs (see below for details). The distinction in the basal [Ca 2+ ] i profile may serve as a clue for the important aspects, such as cell cycle phase, differentiation fate, self-renewal capacity or immune-modulating features, of hMSCs. These two basal [Ca 2+ ] i profiles deserve further investigation to clarify what exactly they signify.
The results presented herein demonstrate that hMSCs prefer to use IP 3 -sensitive stores rather than engage caffeine/ryanodine-sensitive stores to mobilize stored Ca 2+ into the cytosolic compartment. Indeed, our data show that hMSCs rely on IP 3 Rs rather than RyRs to mediate intracellular Ca 2+ mobilization and are consistently supported by the molecular evidence that hMSCs abundantly express three subtypes of IP 3 Rs with sparse RyR1 and no RyR2 as shown in the present work and previously 42 . IP 3 Rs have been demonstrated to play important roles in embryonic development 49,50 . For example, IP 3 R1 is critical for neuronal development and IP 3 R3 is responsible for a proper pattern of [Ca 2+ ] i oscillations to negatively regulates apoptosis in early differentiating embryonic stem cells 49,50 . These previous findings prompt us to extrapolate that IP 3 Rs are important signaling devices in hMSC development, although their exact roles remain to be explored. In addition, the present work also corroborates that hMSCs acquire Ca 2+ from the extracellular environment through SOCE rather than VGCCs. These functional observations are in accordance with previous studies 42,51 . However, the molecular identity of SOCE in hMSCs has not been convincingly determined, although TRPC4 was detected 42 . Strikingly, the present work not only showed TRPM4 and TRPM7 in addition to TRPC4 but for the first time also visualizes three subtypes of Orais and two subtypes of STIMs in hMSCs. This adds new molecular building blocks for the assembly of SOCE in hMSCs.
hMSCs appropriately sense and efficiently respond to environmental challenges and adequately engraft in inflamed sites or lesion areas to accomplish repair missions 3,[13][14][15][16][17][18]52 . In fact, this is an immune modulating event where TLRs act as key players by recognizing natural ligands, such as the double-stranded RNA of viruses and bacterial cell-surface LPS, and trigger corresponding downstream signaling, e.g., cytokine release 3,13-18 . The present work focused on the most important two TLRs, TLR3 and TLR4 4,10-12 . Their priming can polarize hMSCs into two distinct populations, TLR4-primed hMSCs (MSC1) and TLR3-primed hMSCs (MSC2) 11 . Importantly, we revealed that the TLR3 agonist poly(I:C) boosts TLR3 mRNA expression in a concentration dependent manner, whereas the TLR4 agonist LPS just produces a marginal elevation in TLR4 mRNA but an appreciable increase in TLR3 mRNA in hMSCs. It is well known that TLR3 and TLR4 undergo allosteric alteration, dimerization and genomic up-regulation upon their activation in other cell types 14,15 . Our findings suggest that poly(I:C) and LPS activate TLR3 and TLR4 via different mechanisms in hMSCs; TLR3 activation involves a genomic mechanism in addition to allosteric alteration and dimerization, whereas TLR4 activation relies on only allosteric alteration and dimerization. It is noteworthy that the TLR4 agonist LPS markedly increases TLR3 expression without altering TLR4 expression. This means that LPS transactivates TLR3 because TLR3-and TLR4-primed hMSCs differ in various aspects, including the mRNA expression of IL4, IL6, IL8 and IP10 as revealed in the present work. This strongly supports that TLR3-and TLR4-primed hMSCs execute different immune modulating functions.
The present work has dissected the mechanisms linking TLR3 and TLR4 to [Ca 2+ ] i . More importantly, we reveal that TLR3-priming produces not only a significant increase in IP 3 R-mediated Ca 2+ mobilization but also a substantial elevation of the molecular expression of IP 3 Rs in hMSCs. In contrast, TLR4-priming has only marginal influences on these two parameters. Likewise, TLR3-priming significantly augments SOCE with a concomitant increase in basal [Ca 2+ ] i and the molecular expression of candidate building blocks of SOCE, including two Orai subtypes and one STIM subtypes as well as TRPM4 and TRPC4 in hMSCs. However, TLR4-priming fails to do so. These findings demonstrate that TLR3-priming but not TLR4-priming exaggerates IP 3 R-and SOCE-mediated Ca 2+ signaling. They also suggest that TLR3-priming does not allosterically modulate IP 3 R and SOCE activity, but instead increases their abundance via genomic mechanisms. In addition to these Ca 2+ channels, K + channels are also present in hMSCs 53,54 . The channel-mediated K + efflux causes a more negative membrane potential and thereby enhances Ca 2+ influx due to the increased electric driving force for Ca 2+ entry 37 . It is possible that TLR3-priming may up-regulate [Ca 2+ ] i through the increased expression of these K + channels. Therefore, we have quantified the mRNA expression of the large-conductance calcium-activated potassium channel gene MaxiK 55 . Neither TLR3-nor TLR-4-priming influences MaxiK expression. Even so, this is particularly interesting because these negative data confirm the relatively selective regulation of TLR-3-priming on IP 3 Rs and SOCE.
Using RNA-sequencing analysis, we observed that 21 Ca 2+ related signaling genes were significantly up-regulated in response to poly(I:C) and strongly correlated with calcium ion transport ( Figure S3). In addition, we found that the putative binding sites for four transcription factors (TFs) were significantly enriched suggesting that these TFs might be involved in the regulation of Ca 2+ signaling genes in TLR3 primed hMSCs. However, we could not observe a significant up-regulation of ITPR3 and STIM1 genes in our RNA-sequencing analysis. Further experiments are required to identify these genes during TLR3 primed hMSCs.
Scientific RepoRts | 6:23103 | DOI: 10.1038/srep23103 Most importantly, the present work demonstrates that TLR3-and TLR4-priming markedly and differentially enhances cytokine releases in a Ca 2+ -dependent fashion in hMSCs. It seems paradoxical that TLR4-priming elevates neither [Ca 2+ ] i nor the molecular expression of IP 3 Rs and SOCE but significantly increases cytokine release, which is diminished by chelation of intracellular Ca 2+ . In fact, this can be explained by the possibility that TLR4-priming acts at other steps in the complex process of cytokine release rather than [Ca 2+ ] i or the molecular expression of IP 3 Rs and SOCE [13][14][15][16][17][18] . Interestingly, in our study, we observed that BAPTA/AM have a much stronger effect on TLR4-primed IL6 and RANTES production than on the TLR3-primed cytokine production. TLR3 primarily activates the TIR-domain-containing adaptor inducing interferon β (TRIF)-dependent pathway, whereas TLR4 activates both myeloid differentiation factor 88 (MyD88) and TRIF dependent pathways 56 . Several studies have reported that the TLR4-MyD88 pathway has been thought to have an important role in TLR4-primed IL6 synthesis 57,58 . It is therefore likely that BAPTA/AM-modulated IL6 and RANTES production depends on both MyD88 and TRIF-dependent pathways rather than only the TRIF-dependent pathway. A better understanding of the consequences of TLR3 or TLR4-primed cytokines/chemokines production modulated by BAPTA/AM in hMSCs warrants a comprehensive investigation.
In conclusion, we verified that hMSCs mainly engage Ca 2+ mobilization from IP 3 -sensitive stores and extracellular Ca 2+ entry through SOCE to evoke [Ca 2+ ] i responses. These two Ca 2+ -handling mechanisms undergo differential increases concomitant with the elevation of cytokine production upon TLR3-and TLR4-priming. TLR3-and TLR4-priming-induced cytokine release critically depends on [Ca 2+ ] i . These findings not only clarify the novel signaling cascade from TLR3-and TLR4-priming via [Ca 2+ ] i to cytokine release, but also implicate potential targets for genetic and pharmacological manipulation in hMSC-based therapy. RT-PCR Assays. Total RNA was extracted from hMSCs using RNAiso Plus (Takara, Shiga, Japan) according to the manufacturer's instructions. The obtained RNA was reverse-transcribed with PrimeScript Reverse Transcriptase (Takara, Shiga, Japan). Subsequently, the resultant cDNA was amplified using SYBR Premix Ex Taq TM II (Takara, Shiga, Japan). RT-PCR primer pairs were synthesized by GenoTech (Daejeon, Korea) and their sequences were listed in Table 1. Quantitative real-time PCR was performed on an ABI 7500 real-time PCR system (Applied Biosystems Inc., Carlsbad, CA) using the following parameters: initial denature at 95 °C for 10 min, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for quantitative analysis. The data were analyzed using the critical threshold (ΔCT) and the comparative critical threshold (ΔΔCT) methods in the AB-7500 software. Conventional PCR was carried out with S1000 TM Thermal Cycler (Bio-Rad, Hercules, CA) under the following conditions: initial denature at 95 °C for 5 min, followed by 30-35 cycles of denaturing at 95 °C for 1 min, annealing at 60 °C for 1 min and extending at 72 °C for 1 min. The amplified PCR products were detected by agarose gel electrophoresis and ethidium bromide staining.  (Fig. 1A). The 16-bit grayscale images with a binning of 1 × 1 were captured every 1 s with an exposure time ranging from 100 to 300 ms. ROI signals were calculated by subtracting the background noise signals and the analyzed with MetaFluor software. Western blot analysis. Cell lysates were prepared by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM MgCl 2 , 1% Triton X-100 and a complete protease inhibitor mixture tablet (Roche Applied Science). Equal amounts of protein (30-50 μg) were subjected to 6 or 10% SDS-PAGE and blotted onto a PVDF membrane. The membranes were then incubated with anti-IP 3 R3 (1:1000; Abcam) or anti-Orai2 (1:1000; Abcam) and anti-pancadherin (1:1000; Abcam) or anti-β -actin (1:10000; Sigma) for loading controls and signals were detected with ECL reagent.

Methods
ELISA Detection of Cytokines. hMSCs were seeded at a density of 6 × 10 4 cells/cm 2 into 10 cm dish containing culture medium and cultivated for a day. Thereafter, the normal medium was replaced with the medium supplemented with TLR ligands followed by a 4 h incubation. Cytokine concentrations in the culture media were detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Standard curves were established using mouse recombinant cytokines provided with the ELISA kit (KOMABIOTECH, Seoul, Korea and Elabscience Biotechnology, Beijing, China). The assay detection limit was 16 to 32 pg/ml.