STIM1 Knockout Enhances PDGF-Mediated Ca²⁺ Signaling through Upregulation of the PDGFR–PLCγ–STIM2 Cascade

Abstract

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca²⁺ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca²⁺ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca²⁺ release. Store-operated Ca²⁺ entry (SOCE) is the major form of extracellular Ca²⁺ influx following depletion of ER Ca²⁺ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca²⁺ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR–PLCγ–STIM2 signaling, which in turn increases PDGF-BB-induced Ca²⁺ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca²⁺ release from the ER store through PLCγ–IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca²⁺ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca²⁺ signaling.

1. Introduction

Ca²⁺ plays an important and ubiquitous role in intracellular signaling in response to several cellular functions, including cell proliferation, development, differentiation, migration, transcription factor activation, and apoptosis [1,2]. To coordinate these functions, Ca²⁺ signaling is regulated meticulously and is distinguished by several patterns of spatio–temporal parameters with altering amplitude, frequency, or duration [2,3,4]. In cells, Ca²⁺ can be released from the internal store, the endoplasmic reticulum (ER), to increase the intracellular Ca²⁺ concentration. Moreover, there is a common signal transduction pathway to increase cytosolic Ca²⁺ concentration. After the ligand binds to receptor tyrosine kinase (RTK), phospholipase C gamma (PLCγ) is phosphorylated and activated, which in turn hydrolyzes membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 can induce the diffusion of Ca²⁺ from the ER into the cytoplasm through the IP3 receptor (IP3R) on the ER membrane. Subsequent exhaustion of ER Ca²⁺ activates a refill mechanism called the store-operated Ca²⁺ entry (SOCE), which also increases cytosolic Ca²⁺ concentration.

SOCE is the major mechanism that increases and replenishes intracellular Ca²⁺ concentration in most non-excitable and a few excitable cells and is triggered following the depletion of ER Ca²⁺ concentration via the store-operated channels on the plasma membrane [5,6,7]. Stromal interaction molecules (STIMs) are distributed on ER membranes as Ca²⁺ store sensors. Following the depletion of ER Ca²⁺ concentration, the activated STIM proteins aggregate near the plasma membrane and interact with two Ca²⁺-permeable components of store-operated channels (SOCs), viz., Ca²⁺ release-activated calcium modulator 1 (CRAM1, also called Orai1) and transient receptor potential canonical 1 (TRPC1), which contribute to SOCE [8,9,10]. Extracellular Ca²⁺ can be passively transported into the cytoplasm through the STIM–Orai1–TRPC1 complex to regulate Ca²⁺-sensitive enzymes and Ca²⁺-dependent transcription factors, such as cAMP response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT) [11,12,13,14].

The STIM family senses ER Ca²⁺ and activates store-operated channels (SOCs) to regulate cell proliferation [15,16] and migration [17,18]. There are two conserved isoforms of STIM proteins, STIM1 and STIM2, which differ from the ER-plasma membrane junction of the STIM structure [19,20,21]. STIM1 has been reported to play a key role in SOCE and regulates SOCE when ER Ca²⁺ store depletes through interacting with Ca²⁺ release-activated calcium modulator 1 (Orai1) in mouse embryonic fibroblasts (MEF) cells [8,22,23]. Previous studies have shown that STIM2 exhibits more sensitivity but lower affinity to Ca²⁺ in the ER lumen than STIM1. Thus, it has been suggested that STIM2 responds to minor elevation in ER luminal Ca²⁺ concentration [19,20,24]. However, the exact role of STIM2 in Ca²⁺ regulation is still unclear. There are three homologues of Orais, viz., Orai1, Orai2, and Orai3, and all three Orai proteins are expressed in plasma membranes and mediate SOCE [25,26,27,28]. TRPC is a non-selective cation channel that works as an SOCE channel. TRPC is classified into seven subtypes, namely TRPC1–TRPC7, all of which respond to PIP2 hydrolysis [9,29]. Of these, TRPC1 is the major subtype involved in STIM-mediated Ca²⁺ influx via the STIM1–Orai1–TRPC1 complex [30,31]. Previous studies have suggested that STIMs, Orais, and TRPC1 play key roles in SOCE, an important mechanism that regulates intracellular Ca²⁺ hemostasis [32,33].

Platelet-derived growth factor (PDGF) is a mitogen that is involved in cellular migration, proliferation, angiogenesis, and extracellular matrix generation [34,35,36,37]. The PDGF family consists of five proteins: PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD, which are structurally and functionally related and contain a disulfide-linked dimer of two polypeptide chains that are conserved similarly to that in the endothelial growth factor (EGF) family [35,38]. The isoforms bind with different affinities to two receptor units, PDGFRα and PDGFRβ. PDGFRs are composed of two related isoforms of RTKs, namely PDGFRα and PDGFRβ, and form three different subtype receptors, viz., PDGFR-αα, PDGFR-ββ, and PDGFR-αβ, which react with different ligands to initiate various reactions [39,40,41]. Each subtype of PDGF activates different combinations of PDGFRs; however, only PDGF-BB has been verified to bind to all kinds of receptors, resulting in auto-phosphorylation of RTK. The active form of PDGFR activates signaling pathways, such as Akt, JNK, ERK, and STAT3, to regulate cell function and hydrolyzes PIP2 to DAG and IP3 to induce ER Ca²⁺ release [42,43]. Previous studies have shown that PDGF-BB activates PDGFRs (PDGFRα and PDGFRβ) and that the phosphorylation of PDGFR activates PLCγ, which hydrolyze PIP2 to DAG and IP3, leading to a depletion in ER Ca²⁺ store and Ca²⁺ re-entering from the extracellular solution through SOCs [44,45]. Recent studies have also shown that PDGF-BB induces SOCE through STIM1 and Orai1 to promote smooth muscle cell migration [46,47].

Mitogens, such as PDGF and EGF, are well recognized as inducing STIM1-mediated Ca²⁺ elevation including ER Ca²⁺ release and SOCE, for which STIM1 and Orai1, but not STIM2, are essential in PDGF-BB-induced Ca²⁺ elevation and cell migration [16,47,48]. However, STIM2 has reportedly responded to minor decreases in basal Ca²⁺ concentration and replenished Ca²⁺ concentration through the STIM–Orai puncta-like complex [20,24]. In most studies, PDGF-BB has been reported as the SOCE activator that leads to ER Ca²⁺ depletion through the PDGFR–PLCγ–IP3 signaling pathway. However, the role between STIM1 and PDGF-BB signaling is still unclear. Thus, in this study, we employed a particular cell line, mouse embryonic fibroblast-STIM1 knockout cells (MEF-STIM1^−/−), to study the effects of STIM1 knockout on PDGF-BB-stimulated Ca²⁺ response.

2. Results

2.1. Stromal Interaction Molecule 1 (STIM1) Knockout Represses Store-Operated Ca²⁺ Entry (SOCE) in Mouse Embryonic Fibroblasts (MEF) Cells

In order to investigate the effect of STIM1 on SOCE, wild-type (MEF-WT) and STIM1 knockout MEF (MEF-STIM1^−/−) cell lines were used and intracellular concentration of Ca²⁺ was monitored by using a ratiometric fluorescent dye, Fura-2/AM. Thapsigargin (TG), a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor and SOCE activator, is usually used to deplete ER Ca²⁺ store to activate SOCE. The cells were exposed to 2 μM TG in the absence of extracellular Ca²⁺. An initial transient intracellular Ca²⁺ elevation was observed due to Ca²⁺ released from the ER. Next, 2 mM Ca²⁺ was reintroduced into the extracellular solution, leading to Ca²⁺ influx, called SOCE (Figure 1A). Quantification analysis revealed that the ER released Ca²⁺ was similar in both cell types (Figure 1B), but SOCE was significantly reduced in MEF-STIM1^−/− cells compared to that in MEF-WT cells (Figure 1C). In addition, cells were exposed to 2 mM extracellular Ca²⁺ and stimulated with 2 μM TG to mimic normal physiological Ca²⁺ concentration. Representative traces indicate a quick two-fold increase in intracellular Ca²⁺ concentration, which then decreased by 1.4-fold in MEF-WT cells. The resultant Ca²⁺ concentration was higher than the baseline and was sustained for a long period. The initial peak indicated that this Ca²⁺ release from the ER was accompanied by Ca²⁺ influx from the extracellular solution, which sustained the higher Ca²⁺ concentration. In MEF-STIM^−/− cells, the initial peak was 1.4-fold higher, which then quickly reverted to the baseline concentration (Figure 1D). These results suggest that TG-mediated Ca²⁺ elevation after extracellular 2 mM Ca²⁺ exposure showed an initial peak (Figure 1E) and that the total Ca²⁺ elevation (Figure 1F) in MEF-WT cells was more dominant than that in MEF-STIM1^−/− cells. Thus, STIM1 knockout reduced Ca²⁺ elevation in MEF cells, particularly the Ca²⁺ influx.

2.2. PDGF-BB Induced Significant Ca²⁺ Elevation in STIM1 Knockout MEF Cells

PDGF promotes cell migration through regulation of Ca²⁺ signaling. Here, we attempted to investigate the effects of STIM1 knockout on Ca²⁺ elevation in MEF cells following PDGF-BB stimulation. Unexpectedly, PDGF-BB induced Ca²⁺ elevation dose-dependently in MEF-STIM1^−/− cells (Figure 2C,D) but not in MEF-WT cells (Figure 2A,B). Moreover, quantification analysis of the initial Ca²⁺ revealed that PDGF-BB at 100 ng/mL exerted optimal stimulation in MEF-STIM1^−/− cells (Figure 2C) and produced an initial peak that was 1.8-fold higher, which is similar to TG-mediated Ca²⁺ elevation in MEF-WT cells (Figure 1D). In contrast to the TG-mediated Ca²⁺ elevation shown previously, PDGF-BB induced a notable dose-dependent response in MEF-STIM1^−/− cells and had no effect in MEF-WT cells. To clarify how PDGF-BB induces Ca²⁺ elevation, intracellularly or extracellularly, MEF-STIM1^−/− cells were treated with PDGF-BB (0–200 ng/mL) in the absence of extracellular Ca²⁺, after which 2 mM Ca²⁺ was added to the extracellular solution. There was a transient increase in intracellular Ca²⁺ concentration in MEF-STIM1^−/− cells due to the release of ER Ca²⁺, which then reverted to the baseline concentration. Following the addition of 2 mM Ca²⁺, Ca²⁺ influx appeared to replenish the cytosol (Figure 2E). In addition, PDGF-BB at 50 ng/mL triggered substantial Ca²⁺ release from the ER, 100 ng/mL of which was sufficient to induce optimal Ca²⁺ elevation (Figure 2F). More specifically, Ca²⁺ influx analysis with different concentrations of PDGF-BB showed that treatment with 100 ng/mL PDGF-BB induced the highest Ca²⁺ elevation following ER Ca²⁺ depletion (Figure 2G). These data also show that PDGF-BB-mediated Ca²⁺ elevation occurred primarily through intracellular Ca²⁺ release and then through SOCE. Furthermore, knockdown of STIM2 resulted in the downregulation of SOCE upon TG or PDGF-BB treatment in MEF-STIM1^−/− cells (Figure S2).

2.3. Upregulation and Activation of PDGFRα, PDGFRβ, and Phospholipase C Gamma (PLCγ) in MEF-STIM1^−/− Cells

Previous studies have shown that PDGF-BB activates PDGFRs (PDGFRα and PDGFRβ) and that PDGFR phosphorylation activates PLCγ to hydrolyze PIP2 into DAG and IP3, which leads to a depletion of the ER Ca²⁺ store. Therefore, we examined PDGF-BB-mediated signaling pathways. Immunoblotting showed that expressions of PDGFRα, PDGFRβ, and PLCγ were enhanced in MEF-STIM1^−/− cells compared to those in MEF-WT cells (Figure 3A), indicating that the upregulation was due to PDGF-BB stimulation. Quantification analyses of the ratio of phosphorylated PDGFRβ:PDGFRβ (Figure 3B) and phosphorylated PLCγ:PLCγ (Figure 3C) also confirmed the results, because their activities following PDGF-BB treatment were evidently increased in MEF-STIM1^−/− cells compared to those in MEF-WT cells. CREB activation by phosphorylation can be triggered by both PDGF and Ca²⁺ signal transduction pathways and inhibition of CREB expression or activation decreases PDGF-induced smooth muscle cell migration. Thus, we examined the phosphorylation of CREB in response to PDGF-BB stimulation. The results showed that CREB was phosphorylated in MEF-STIM1^−/− cells and the phosphorylation levels were higher than those in MEF-WT cells (Figure 3D). STIM2 knockdown did not affect the expressions of PDGFRα and PDGFRβ and the PDGF-BB-induced PDGFRβ phosphorylation, whereas STIM1 overexpression downregulated the expressions of PDGFRα and PDGFRβ and the PDGF-BB-induced PDGFRβ phosphorylation (Figure 3E). We then sought to determine other non-Ca²⁺-conducting PDGF-BB-induced downstream signaling molecules, including Akt, JNK, ERK and STAT3 (Figure 4A). Upon PDGF-BB stimulation, Akt phosphorylation increased within 3 min in MEF-STIM1^−/− cells and was sustained for at least 10 min; however, in MEF-WT cells, Akt was activated within 5 min and then decreased quickly (Figure 4B). Although phosphorylation of JNK was triggered by PDGF-BB in both cell types, the levels of phosphorylation were higher in MEF-STIM1^−/− cells than those in the MEF-WT cells (Figure 4C). In addition, PDGF-BB induced higher levels of ERK phosphorylation in MEF-STIM1^−/− cells than that in MEF-WT cells (Figure 4D). Activation of STAT3 upon PDGF-BB stimulation was not significantly different between MEF-WT and MEF-STIM1^−/− cells. Taken together, these findings support the responses of PDGF-BB-induced Ca²⁺ elevation in MEF-STIM1^−/− cells due to the elevated protein levels of PDGFRs, resulting in higher activation of PLCγ, Akt, JNK, and ERK than in MEF-WT cells. PLCγ was the key regulator upstream of IP3-mediated ER Ca²⁺ release and SOCE. The PDGFR kinase inhibitor AG1295 was used to verify the role of PDGFR activation in PDGF-BB-induced PLCγ signaling (Figure S1A). Our results showed that inhibition of PDGFRβ activation using AG1295 (Figure S1B) resulted in cessation of PDGF-BB-mediated PLCγ signaling in MEF-STIM1^−/− cells (Figure S1C). Moreover, PLC inhibitors U73122 and D609 were used to examine PDGF-BB-induced Ca²⁺ responses, such as CREB phosphorylation (Figure S3A). These results indicated that PLC inhibitors did not affect PDGFR phosphorylation upon PDGF-BB stimulation (Figure S3A) but inhibited phosphorylation of PLCγ and CREB (Figure S3B,C).

2.4. PDGF-BB-Mediated ER Store-Depletion Activates STIM2 Translocation and Puncta Formation

STIMs, Orais, and TRPC1 play key roles in the regulation of SOCE. As shown in Figure 2, PDGF-BB induced SOCE in MEF-STIM1^−/− cells but not in MEF-WT cells. To understand how PDGF-BB induced Ca²⁺ elevation in MEF-STIM1^−/− cells, proteins that contribute to SOCE were examined by immunoblotting analysis. Although STIM1 knockout did not affect STIM2; Orai1, Orai2, Orai3, and TRPC1 were decreased in MEF-STIM1^−/− cells (Figure 5A). These results supported the observation that Ca²⁺ influx following TG-mediated ER store depletion in MEF-STIM1^−/− cells is lower than that in MEF-WT cells because of lower SOCE-associated protein levels in MEF-STIM1^−/− cells (Figure 1). To investigate the role of STIM proteins in MEF cells in response to PDGF-BB stimulation, the subcellular localization of endogenous STIM1 and STIM2 was examined by immunostaining. The confocal images showed that STIM1 was not expressed in MEF-STIM1^−/− cells (Figure 5B). Moreover, the STIM2 protein translocated near the plasma membrane and formed distinct intracellular puncta in MEF-STIM1^−/− cells after PDGF-BB treatment from 3 to 10 min. Maximal activity was observed at 5 min, after which it decreased over time. In contrast, PDGF-BB did not induce puncta formation and plasma membrane translocation of STIMs in MEF-WT cells (Figure 5C and Figure 6A,B ). In addition, total internal reflection fluorescence microscopy (TIRFM) and high magnification laser scanning confocal microscopy were applied to verify plasma membrane translocation and intracellular puncta formation of activated STIM2, respectively. TIRFM is a technique used to record the fluorescence signals within 50 to 100 nm range of the basal plasma membrane. We observed a significant plasma membrane translocation of STIM2 after PDGF-BB stimulation for 5 min in MEF-STIM1^−/− cells (Figure 6C). On the other hand, large size of the internal aggregation of the STIM2 puncta was presented in PDGF-BB stimulated MEF-STIM1^−/− cells (Figure 6C). Quantitative results showed that PDGF-BB induced 5–6-fold increases of STIM2 aggregation (Figure 6D). We also demonstrated colocalization between SITM2 and Orai1, Orai2, Orai3, or TRPC1 after PDGF-BB treatment (Figure S4).

2.5. SOCE Inhibitors Decrease PDGF-BB-Induced Ca²⁺ Elevation in MEF-STIM1^−/− Cells

Pharmacological inhibitors of SOCE, such as 2-APB (Figure 7A), SKF96365 (Figure 7B), YM-58483 (Figure 7C), La³⁺ (Figure 7D), and Gd³⁺ (Figure 7E) were used to examine the role of Ca²⁺ mobilization in PDGF-induced Ca²⁺ elevation in MEF-STIM1^−/− cells exposed to 2 mM Ca²⁺ solution. Quantification analysis of the initial Ca²⁺ and total Ca²⁺ elevation following PDGF-BB stimulation showed that Ca²⁺ signaling was inhibited by these inhibitors in a dose-response manner (Figure 7).

2.6. PDGFR–PLCγ–STIM2 Signaling Induces Cell Migration in MEF-STIM1^−/− Cells.

PDGF signaling regulates cell proliferation and migration. To determine the influence of PDGF-BB stimulation in MEF-STIM1^−/− cells, we performed a wound healing assay and cell count with Hoechst 33342 nuclear staining to examine cell migration and proliferation, respectively. To minimize the effect of serum, we reduced the concentration of serum to 0.1%. By comparing MEF-WT and MEF-STIM1^−/− cells, it was observed that the proliferation rate of MEF-WT cells was higher than that of MEF-STIM1^−/− cells and that the number of MEF-STIM1^−/− cells decreased slowly after 24 h (Figure S5A). To avoid cell proliferation interference, cells were treated with 0.1% fecal bovine serum (FBS) as a control and observed over 24 h. We found that 100 ng/mL PDGF-BB with 0.1% FBS did not affect the proliferation of MEF cells (Figure S5B). In addition, MEF-STIM1^−/− cells treated with PDGF-BB led to a substantial increase in wound closure, wherein the wound healed completely after 18 h, unlike MEF-WT cells (Figure 8). These data indicated that STIM1 knockout enhances cell migration when compared with MEF-WT cells.

3. Discussion

STIM1 plays the major role in controlling Ca²⁺ homeostasis through regulation of SOCE in many non-excitable cells. In this study, we found the unexpected result that PDGF-BB induced higher intracellular Ca²⁺ elevation in MEF-STIM1^−/− cells than that in the parental MEF-WT cells (Figure 2). The observed results were associated with STIM2-dependent SOCE (Figure S2). Immunoblotting revealed that PDGFRα, PDGFRβ, and PLCγ were overexpressed in MEF-STIM1^−/− cells and higher PDGFR and PLCγ phosphorylation was observed after PDGF-BB stimulation (Figure 3). In contrast, the levels of both PDGFRα and PDGFRβ were very low in MEF-WT cells, and hence, PDGF-BB could not induce PDGFR phosphorylation and Ca²⁺ elevation (Figure 2 and Figure 3). Increased PLCγ activation may increase the propensity of IP3 to activate the IP3 receptors and lead to Ca²⁺ leakage from the ER lumen, which is the major source of PDGF-BB-mediated Ca²⁺ elevation in MEF-STIM1^−/− cells (Figure 2). Depletion of the ER Ca²⁺ store through PDGFR–PLCγ axis following extracellular Ca²⁺ influx is termed as SOCE. However, STIM1 was not expressed in MEF-STIM1^−/− cells and Ca²⁺ influx following the re-introduction of 2 mM Ca²⁺ was lower than that of TG-induced SOCE in MEF-WT cells (Figure 1 and Figure 2).

PDGFR (AG1295) and PLC (U73122 and D609) inhibitors were employed to verify whether the PDGFR–PLCγ axis could be activated by PDGF-BB stimulation in MEF-STIM1^−/− cells [49,50,51]. Immunoblotting results supported the essential roles of PDGFRs and PLCγ in PDGF-BB-mediated activation (Figures S1 and S3). In addition, the Ca²⁺-activated transcription factor CREB was also observed in correspondence to activation of PDGFR–PLCγ axis-mediated signaling. These results indicated that PDGF-BB increased Ca²⁺ elevation through the activation of PLCγ.

SOCE inhibitors manifestly lowered intracellular Ca²⁺ elevation in a dose-response manner (Figure 7). Accordingly, SOCE may contribute to PDGF-BB-mediated Ca²⁺ elevation upon PDGF-BB stimulation. Here, we confirmed that STIM2 activation was observed in MEF-STIM1^−/− cells but not in MEF-WT cells (Figure 5 and Figure 6). PDGF-BB induced interaction between STIM2 and SOCE-related channel proteins in MEF-STIM1^−/− cells (Figure S4). However, the level of PDGF-BB-mediated SOCE were lower than ER released Ca²⁺ in MEF-STIM1^−/− cells (Figure 1 and Figure 2), which may be due to the downregulation of SOCs Orais and TRPC1 (Figure 2C and Figure 5A). Our results demonstrated that enhancement of Ca²⁺ signaling in MEF-STIM1^−/− cells was mediated by the PDGFR–PLCγ–STIM2 pathway.

Proliferation assay of MEF cells also indicated that MEF-STIM1^−/− cells proliferated more slowly than MEF-WT cells [22]. Moreover, we showed that MEF-WT cells proliferated more quickly than MEF-STIM1^−/− cells, even in a low-serum concentration containing 0.1% FBS, whereas PDGF-BB did not affect MEF proliferation in the same condition (Figure S5). Furthermore, our results showed that the ability of cell migration with PDGF-BB treatment in MEF-STIM1^−/− cells was higher than that in MEF-WT cells, regardless of the low cell proliferation rate. It may be that STIM1 knockout evoked migration via PDGFR–PLCγ–STIM2 enhancement, as demonstrated by the wound healing assay (Figure 8). PDGF-BB-induced Ca²⁺ elevation may promote MEF-STIM1^−/− cell migration as well as activation of Akt, ERK, and JNK, which could also participate in cell migration regulation. Both EGF and PDGF share similar signal pathways, regulate many similar functions of the cell, and induce SOCE following ER Ca²⁺ depletion through PLCγ signaling [47,52]. We also observed EGFR downregulation in MEF-STIM1^−/− cells. Moreover, EGF induced higher activity of EGFR, PLCγ, Akt, JNK, and ERK in MEF-WT cells compared to those in MEF-STIM1^−/− cells (Figure S6). PDGF-BB induced higher response in MEF-STIM1^−/− cells, whereas EGF induced higher response in MEF-WT cells. These contradictions resulted from a switch in these different expressions of EGFR and PDGFR.

STIM1 has been vigorously studied and is well-established in response to high Ca²⁺ leakage from the ER lumen, which leads to huge and constitutive Ca²⁺ influx through interacting with Orai1 and TRPC1 in the plasma membrane [8,53]. Without STIM1, STIM2 replaces the role of STIM1 and induces lower Ca²⁺ influx [54]. A recent study shows the essential role of STIM proteins in Ca²⁺ homeostasis and their crucial role in controlling multiple functions of smooth muscle cells [12]. The critical role of STIM1 can be partially rescued by STIM2. STIM1 and Orai1, but not STIM2, are essential for PDGF-BB-induced Ca²⁺ elevation and cell migration in human airway smooth muscle cells, which express both STIM proteins [47]. Our results also showed that STIM1 played a curial role in response to store depletion after TG treatment (Figure 1). Interestingly, we found that PDGF-BB induced Ca²⁺ elevation in MEF-STIM1^−/− cells but not in MEF-WT cells. STIM2 seemed to partially replace the regulatory role of SOCE by STIM1. To our knowledge, this is the first report that shows enhanced Ca²⁺ elevation in MEF-STIM1^−/− cells compared to that in MEF-WT cells because of overexpression of PDGFRs and PLCγ in MEF-STIM1^−/− cells.

4. Materials and Methods

4.1. Cell Culture and Reagents

Wild-type (MEF-WT) and STIM1 knockout (MEF-STIM1^−/−) mouse embryonic fibroblasts were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Caisson, Smithfield, VA, USA) supplemented with 5% fetal bovine serum (FBS; GIBCO, Big Cabin, OK, USA), 100 IU/mL penicillin, 100 μg/mL streptomycin, non-essential amino acid (NEAA; GIBCO), and 1 mM sodium pyruvate (NEAA; GIBCO) under 5% CO₂ at 37 °C. Recombinant mouse PDGF-BB and EGF were purchased from R&D Systems (Minneapolis, MN, USA).

4.2. Single-Cell Ca²⁺ Measurement

Intracellular Ca²⁺ was measured by the ratiometric Fura-2 fluorescence method using a single-cell fluorimeter (Till Photonics, Grafelfing, Germany). Fura-2 were excited alternatively between 340 nm and 380 nm using the Polychrome IV monochromator (Till Photonics), which is highly sensitive and can detect rapid changes in Ca²⁺ concentration with high temporal and spatial resolution and images were captured using an Olympus IX71 inverted microscope equipped with a xenon illumination system and an IMAGO CCD camera (Till Photonics). Emission fluorescence intensity of 510 nm was used to calculate intracellular Ca²⁺ levels using the TILLvisION 4.0 software (Till Photonics).

4.3. Immunoblotting Analyses

Cell lysates were harvested in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris at pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and CompleteTM), and the lysates were analyzed by Western blotting using antibodies against STIM1 (BD, Franklin Lakes, NJ, USA), Orai1, Orai3 (ProSci, Poway, CA, USA), Orai2 (Enzo, Farmingdale, NY, USA), TRPC1 (Proteintech, Rosemont, IL, USA), PDGFRα, JNK, phospho-EGFR, EGFR (Santa Cruz, Santa Cruz, CA, USA), phospho-PDGFRβ, PDGRβ (Abnova, San Francisco, CA, USA), STIM2, phospho-PLCγ, PLCγ, phospho-Akt, Akt, phospho-JNK, phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-CREB, CREB (Cell Signaling, Beverly, MA, USA), and β-actin (Sigma, Saint Louis, MO, USA). The immune complexes were then detected with horseradish peroxidase-conjugated IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and the reaction was developed using an enhanced chemiluminescence (ECL) detection kit under an ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

4.4. Immunofluorescence Staining and Confocal Microscopy

MEF cells (3 × 10⁴ cells) were seeded in a 3-cm glass-bottom dish (Alpha Plus, Taoyuan, Taiwan). After adhesion, cells were starved in a serum-free medium for 12 h and then treated with PDGF-BB (100 ng/mL) for 3, 5, and 10 min. Then, cells were fixed with 4% paraformaldehyde (Alfa Aesar, Haverhill, MA, USA) for 10 min; incubated in PBS washing buffer containing 0.1% Triton X-100 for 10 min; and then in CAS-Block solution (Invitrogen, Carlsbad, CA, USA) for 1 h with gentle shaking. Subsequently, the cells were loaded with primary antibody, anti-STIM1 (Cell Signaling) and anti-STIM2 (Alomone Labs, Jerusalem, Israel) diluted to 1:100 in filtered PBS. Finally, cells were stored overnight at 4°C. Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used to counteract primary antibodies, and Hoechst 33342 was loaded as the nuclear marker. The fluorophores were excited by laser at 405 and 594 nm and detected by a laser scanning confocal microscope (FV-1000, Olympus, Tokyo, Japan). The FV10-ASW software (version 4.2a, Tokyo, Japan) was used to access puncta formation and translocation of the STIM proteins.

4.5. Wound Healing Assay

Culture inserts (ibidi) were applied to assess MEF cell migration. The insert consisted of two wells separated by a 500 μm-thick silicon wall. The culture insert was placed into a 3-cm culture dish and slightly pressed on the top of the insert to ensure adhesion on the dish. MEF-WT and MEF-STIM1^−/− cells were seeded at an equal density (3 × 10⁴ cells in 100 µL) with 0.5% FBS medium and incubated at 37 °C with 5% CO₂ overnight. The insert was removed after cells were well-attached and formed a monolayer. The cells were then incubated in DMEM containing 0.1% FBS with 100 ng/mL PDGF-BB. Cell migrating into the gap (initially ~500 μm) was recorded every 6 h via phase-contrast microscopy. The data were collected from three independent experiments and analyzed as wound closure (%) by the ImageJ software (version 1.47, Bethesda, MD, USA).

4.6. Cell Proliferation Analysis

To assess MEF cell proliferation, cells were stained using Hoechst 33342. MEF cells (WT and STIM1^−/−) were seeded into a 3-cm culture dish with 0.5% FBS medium for cell starvation and incubated at 37 °C and 5% CO₂ overnight. After cells had attached properly to the dish, the medium was changed (0.1% FBS with 100 ng/mL PDGF-BB). The cells were collected and fixed with 4% paraformaldehyde at 0, 24, 48, and 72 h. Next, the cells were stained with Hoechst 33342 for 30 min. Fluorescence images were captured randomly and counted using fluorescence microscopy and ImageJ software, respectively.

4.7. Statistical Analysis

All data were reported as mean ± SEM (standard error of the mean). For statistical analysis, Student’s t-test or one-way ANOVA with Dunnett’s post-hoc test were processed using the Origin software, for which differences were considered significant when p-value was <0.05.