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SERCA-mediated endoplasmic reticulum stress facilitates hematopoietic stem cell mobilization

Stem Cell Research & Therapy volume 16, Article number: 208 (2025) Cite this article

Abstract

Background

Hematopoietic stem cell (HSC) transplantation is widely recognized as an effective treatment for various malignant diseases. Enhancing HSC mobilization can improve transplantation outcomes and ultimately increase patient survival rates. Recent studies suggest that mild endoplasmic reticulum (ER) stress promotes HSC self-renewal, anti-apoptotic, and anti-aging capabilities. This led us to investigate whether inducing mild ER stress could facilitate HSC mobilization.

Methods

The phenotype changes in cells treated with ER stress inducers and Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitors were assessed using flow cytometry. The efficacy of these agents on HSC mobilization was evaluated in C57Bl/6 mice, with colony forming unit (CFU) assays used for quantification. Knockdown Jurkat cell lines were constructed to validate the role of SERCA in the mobilization mechanism. Molecular and protein expression levels associated with the pathway were analyzed through quantitative reverse-transcription PCR and western blotting.

Results

Our findings revealed that BHQ, a SERCA inhibitor, efficiently enhanced HSC mobilization in vivo. Mechanistically, BHQ regulated the CaMKII-STAT3-CXCR4 pathway by suppressing SERCA activity. This inhibition led to a reduction in CXCR4 expression on the surface of HSCs, facilitating their migration from the bone marrow into peripheral circulation.

Conclusions

Our study provides novel insights into the role of the SERCA-ER stress pathway in HSC mobilization. By targeting SERCA activity with BHQ, we observed a significant enhancement in the mobilization of HSCs, facilitated by the modulation of the CaMKII-STAT3-CXCR4 signaling pathway. This research highlights the potential of utilizing mild ER stress as a strategy to promote HSC mobilization, with significant implications for improving stem cell-based therapies, including those used in HSC transplantation.

Introduction

Hematopoietic stem cell (HSC) transplantation is an effective treatment for various hematopoietic malignancies and genetic diseases. In clinical practice, peripheral blood (PB) stem cells have gradually replaced bone marrow (BM) as the primary source for stem cell transplantation due to safer and more convenient collection procedures, faster hematological recovery, a lower risk of transplant failure, and improved disease-free survival and overall survival rates [1]. However, in the steady state, HSCs are primarily anchored within the BM microenvironment, with only a small number circulating in PB, rendering it insufficient for collection purposes [2]. HSCs can be mobilized from the BM into the PB through the administration of exogenous cytokines. Transplantation of a sufficient number of mobilized HSCs enhances the graft-versus-tumor effect by promoting hematopoietic reconstitution and reduces the risk of both acute and chronic graft-versus-host disease as well as infection. The clinical outcomes of HSC transplantation largely depend on the quantity and quality of hematopoietic stem/progenitor cells (HSPCs) in the graft.

According to the National Comprehensive Cancer Network (NCCN) guidelines and clinical experience, a minimum of 5 × 106 CD34+ cells per kilogram of recipient body weight is required for successful transplantation and rapid hematopoietic reconstitution [3, 4]. Therefore, effective mobilization is a crucial step for successful HSC transplantation and hematopoietic recovery. Current methods of HSC mobilization primarily involve the continuous administration of granulocyte colony-stimulating factor (G-CSF) to the donor, with apheresis typically starting on the 5th day of G-CSF administration. To achieve the target cell harvest, it is necessary to stimulate the donor for at least 5 days [5]. While G-CSF has benefited many patients, there is still a failure rate ranging from 10–60% [6]. Repeated mobilization attempts also increase the incidence of side effects and the burden on patients or donors. Another newly developed auxiliary mobilization strategy is the use of the C-X-C chemokine receptor type 4 (CXCR4) inhibitor, Plerixafor (AMD3100), which can enhance mobilization when combined with G-CSF. However, even with this combined regimen, approximately 24% of patients still fail to achieve adequate mobilization [7]. A clinical trial involving multiple myeloma patients demonstrated that, despite receiving up to 8 G-CSF injections, 4 plerixafor injections, and 4 apheresis procedures, 15–35% of patients still failed to mobilize an adequate number of CD34+ cells, leading to significant patient distress [5, 8]. These limitations underscore the urgent need to develop more rapid and effective mobilization protocols that minimize side effects and reduce donor discomfort.

Among the currently known regulatory pathways, the chemotactic regulator stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 are recognized as the primary mediators of HSC homing, quiescence, and maintenance [9]. Therefore, the SDF-1/CXCR4 axis remains the most important target for the development of mobilization strategies. CXCR4 belongs to the G-protein-coupled receptor (GPCR) superfamily, and its function is dynamically regulated by the specific phosphorylation of multiple serine residues in the C-terminal domain of the receptor [10,11,12]. Phosphorylation at S339, in particular, is associated with receptor endocytosis [13], and studies have shown that CXCR4 endocytosis reduces its surface expression [14]. The rapid phosphorylation and degradation of CXCR4 may be linked to hematopoietic cell stress.

Among the various types of stress, endoplasmic reticulum (ER) stress has been strongly associated with HSC function [15, 16]. Mild ER stress is a characteristic of HSCs [15, 17], as it promotes cell protection and self-renewal capacity [15, 18]. The induction of ER stress is often accompanied by impaired Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) function and the disruption of intracellular Ca2+ homeostasis [19, 20]. SERCA is a key protein responsible for transporting Ca²⁺ from the cytosol into the lumen of the sarco/endoplasmic reticulum (SR/ER), thus maintaining intracellular Ca²⁺ homeostasis. SERCA is encoded by three genes (SERCA1, SERCA2, and SERCA3), which generate several tissue-specific SERCA isoforms (SERCA1-3) through alternative splicing [21]. SERCA2 and SERCA3 are the primarily expressed subtypes in blood cells [22]. Previous studies have reported that ER stress-induced increases in intracellular calcium can activate calmodulin-dependent protein kinase II (CaMKII) [23]. Jisu et al. found that vitamin D deficiency-induced ER stress increased the phosphorylation of CaMKII in human macrophages, without altering the total CaMKII expression. Inhibiting phosphorylated CaMKII (pCaMKII) under conditions of vitamin D deficiency can, in turn, inhibited ER stress [24].

Despite ER stress has been extensively studied in relation to stem cell self-renewal, anti-apoptosis, and anti-aging, its role in HSC mobilization remains poorly understood [15,16,17, 25, 26]. HSC mobilization is a complex process regulated by multiple signaling pathways and microenvironmental cues. Given the well-established link between ER stress, intracellular calcium homeostasis, and cell migration, we hypothesized that modulating SERCA activity, a crucial regulator of ER calcium balance, might serve as a novel approach to enhance HSC mobilization. In this study, we demonstrate that pharmacological inhibition of SERCA using BHQ induces ER stress and leads to a significant downregulation of CXCR4 surface expression on HSCs. This reduction in CXCR4 expression facilitates the migration of HSCs from the BM into the peripheral circulation, effectively enhancing their mobilization. Moreover, our findings suggest that BHQ-mediated SERCA inhibition acts through the CaMKII-STAT3-CXCR4 signaling axis, providing mechanistic insight into how ER stress influences HSC trafficking. These findings offer novel insights into the molecular mechanisms governing HSC mobilization and suggest a potential therapeutic strategy for enhancing HSC mobilization in clinical settings.

Materials and methods

Key small molecular compounds

Table 1 Compounds and abbreviation
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Animals

Adult female C57Bl/6 mice (6–8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Company. Animal anaesthesia was not administered during the experimental procedures in this study. All experimental procedures were carried out in accordance with institutional guidelines, and approved by the Animal Care and Use Committee of Fudan University School of Medicine. The work has been reported in line with the ARRIVE guidelines 2.0.

Cell isolation and culture

Mice were euthanized by cervical dislocation, and bone marrow cells were isolated by flushing the femur with sterile PBS. Lineage-negative (Lin−) cells were enriched using the Lineage Cell Depletion Kit (130-090-858, Miltenyi Biotec) according to the manufacturer’s instructions and previously published studies [27, 28]. Lin− cells were cultured in vitro in SFEM II medium (StemCell, 09655), supplemented with 100 U/mL penicillin/streptomycin, and 50 ng/mL each of recombinant mouse SCF, TPO, and FLT3L for 4 days.

The Jurkat cell line was maintained in RPMI-1640 medium (Gibco, 11875093), and the HEK293T cell line was cultured in DMEM (Gibco, 11965092). Both media were supplemented with 10% FBS (Gibco, 10099158) and 100 U/mL penicillin/streptomycin. All cells were cultured at 37 °C in a humidified incubator with 5% CO₂.

Flow cytometry analysis

Cells were washed at least once with FACS buffer. They were then resuspended in Fc-block (1:100, BD) for 10 min, followed by a 30-minute incubation with an appropriately concentrated antibody staining mixture (if applicable) at 4 °C in the dark. After a final wash, cells were resuspended in 400 µL of FACS buffer. All flow cytometry experiments were performed using a BD Canto II flow cytometer, and data analysis was conducted using FlowJo version 10.4.0.

Mice mobilization assay

C57Bl/6 mice were subcutaneously injected (SC) with 125 µg/kg of G-CSF (Qilu Pharmaceutical) twice daily in 100 µL PBS, or an equivalent volume of PBS alone, for 4 days. BHQ, BD, and KN93 were also administered SC at specific doses in 100 µL of corn oil (See Table 1). At the final time point, the mice were euthanized by cervical dislocation, and peripheral blood was collected via cardiocentesis into anticoagulant tubes containing 18 U/mL heparin. Red blood cells (RBC) were lysed to obtain mononuclear cells (MNCs) for flow cytometry analysis and colony forming unit (CFU) assays [29,30,31,32]. For the hematopoietic progenitor CFU assay, RBC-lysed MNCs were plated in methylcellulose medium (Stem Cell Technologies, M3434) [33] in 24-well culture plates. The plates were then incubated in a humidified incubator for 7 days before the colonies were scored.

RNA extraction and quantitative reverse transcription (qRT)-PCR assay

Total RNA was extracted from cells using Trizol Reagent (TransStart, ET111-01-V2) according to the manufacturer’s instructions. cDNA synthesis was performed using a FastKing-RT Kit (Tiangen, KR118). Real-time PCR amplification was carried out using SYBR-Green I qPCR SuperMix (TransStart, AQ141-01) and detected on a Roche LightCycler® 480 (Roche Diagnostics) following the manufacturer’s recommendations. β-actin was amplified as a housekeeping gene to ensure equal baseline analysis across samples. Primer sequences for β-actin, SERCA1, SERCA2, SERCA3, and XBP1s are listed in Supplemental Table 1. The relative quantification of genes was performed using the 2-ΔΔCt method.

Construction of SERCA2/3-knockdown Jurkat cell line

Lentivirus production and packaging for stable knockdown of SERCA2/3 were performed using pMD2.G and psPAX2 with the hU6-pLKO.1-EGFP vector. The primers used are listed in Supplemental Table 2. To establish a stable SERCA2/3-knockdown Jurkat cell line, GFP-positive cells were sorted for further assays.

Western blot

Equal numbers of cells from each group were washed with ice-cold PBS and lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, 4693132001) for 10 min at 4 °C, with ultrasonic assistance. After centrifugation at 14,000 g for 15 min, the supernatant was collected as the protein extract.

Proteins were separated by polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Biosharp, BS-PVDF-45). The membranes were then blotted with primary antibodies against CXCR4, phospho-CXCR4 (S339), CaMKII, phospho-CaMKII (T286), STAT3, phospho-STAT3 (Y705), and GAPDH, as listed in Supplemental Table 3. Peroxidase-conjugated secondary antibodies (anti-mouse, 1:5000; Immunoway, RS2108; anti-rabbit, 1:5000; Immunoway, RS0002) were applied at room temperature for 1 h. Protein bands were detected using an enhanced chemiluminescence (ECL) system and visualized with a chemiluminescence imaging system (Tanon 4600).

Statistics

Data were analyzed using GraphPad Prism 9 (GraphPad). All data are presented as mean ± SEM. The sample size (n) represents the number of mice per experiment, as specified in the figure legends. Statistical significance was determined using an unpaired, two-tailed Student’s t-test to compare two groups. For all data, differences were considered statistically significant when p < 0.05. Specifically, the following notation was used: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Results

ER stress inducers down-regulate CXCR4 surface expression

Previous studies have reported ER stress leads to the activation of inositol-requiring enzyme 1 (IRE1), which subsequently cleaves X-box-binding protein 1 (XBP1) mRNA, producing the spliced form of XBP1 (XBP1s). Since XBP1s is a widely recognized marker of ER stress [34,35,36], we firstly assessed the mRNA level of XBP1s in Jurkat cells by qPCR method and found that under the action of ER stress inducers BHQ [37] and TG [38], the level of XBP1s significantly increased, with the upregulation becoming more pronounced over time (Fig. 1A, B). These results confirm that BHQ and TG effectively induce ER stress in Jurkat cells.

Fig. 1
figure 1

ER stress inducers suppress CXCR4 surface expression. (A-B) Induction of XBP1s mRNA by ER stress inducers. Jurkat cells were treated with 100 µM BHQ or 100 nM TG for 0.5, 1.5, and 5 h, and XBP1s mRNA levels were quantified by qPCR (n = 3). (C-E) CXCR4 surface expression in Jurkat cells. Cells were treated with 100 µM BHQ for 1.5 h, 100 nM TG for 5 h, or 10 µM BFA for 5 h, and CXCR4 expression was analyzed by flow cytometry (n = 3–6). (F) Schematic diagram of mouse Lin− cells sorting and culture. (G-H) Downregulation of CXCR4 surface expression in mouse LSK cells following treatment with ER stress inducers (100 µM BHQ, 100 nM TG, and 10 µM BFA) (n = 3). (I-J) Downregulation of CXCR4 surface expression in mouse HSCs following treatment with ER stress inducers (100 µM BHQ, 100 nM TG, and 10 µM BFA) (n = 3)

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To examine the correlation between ER stress and HSPC mobilization, we evaluated the effects of various ER stress inducers (BHQ, TG, and BFA [39] ) on the surface expression of CXCR4 in Jurkat cells. Flow cytometry analysis demonstrated that all three agents significantly reduced CXCR4 surface expression (Fig. 1C-E). Moreover, BHQ exhibited both concentration- and time-dependent effects on CXCR4 expression (Figure. S1D-G). To further verify whether this inhibitory effect extended to primary cells, we isolated Lin− cells from C57Bl/6 mice and assessed CXCR4 surface expression on LSK(Lin−Sca1+c-Kit+) and SLAM LSK cells (phenotypic HSCs). We found that BHQ, TG, and BFA significantly reduced the surface expression of CXCR4 on LSK cells and HSCs (Fig. 1F-J, Fig. S2). In addition, other ER stress inducers have also been shown to significantly inhibit the surface expression of CXCR4 on Jurkat cells (Figure S1A-C), indicating that ER stress inducers may serve as potential mobilizing agents for HSPCs. Given that BHQ exhibited the strongest phenotypic effect with minimal cytotoxicity (Figure S3), it was primarily used as the representative agent in subsequent experiments.

BHQ promotes HSPC mobilization in mice and enhances the mobilization effect of G-CSF

To assess the in vivo mobilization potential of BHQ, C57Bl/6 mice were administered a single subcutaneous injection (SC) of BHQ at a dose of 20 mg/kg. Two hours post-injection, PB samples were collected to enumerate HSPCs (Fig. 2A). Colony forming unit (CFU) assays revealed a significant increase in hematopoietic colony formation in BHQ-treated mice compared to the control group (Fig. 2B). Flow cytometry analysis, which quantified the absolute numbers of LSKs and phenotypic HSCs per milliliter of PB, further demonstrated a greater mobilization of HSPCs in BHQ-treated mice compared to the control group (Fig. 2C-E).

Fig. 2
figure 2

BHQ promotes HSPC mobilization in vivo. (A) Schematic representation of the mouse mobilization model. (B) CFU counts in 1 mL of PB following BHQ treatment. (C) Gating strategy for flow cytometry analysis (n = 7). (D-E) Absolute numbers of mobilized LSK cells and HSCs in 1 mL of PB following BHQ administration (n = 7). (F) Schematic diagram of the combined mobilization experiment using BHQ and G-CSF. (G-I) Synergistic effect of BHQ and G-CSF on mouse HSPC mobilization (n = 6). n indicates the number of mice used per group

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Fig. 3
figure 3

ER Stress triggers calcium signaling pathway to regulate CXCR4 expression. (A-B) Downregulation of CXCR4 surface expression in SERCA2/3 knockdown Jurkat cells (n = 3). (C) The reduced CXCR4 expression could not be restored by the SERCA agonist (CDN1163) in SERCA2/3 knockdown Jurkat cells (n = 3). (D) Flow cytometric analysis of intracellular calcium transients in BHQ-treated Jurkat cells (n = 3). (E) Western blot analysis of p-CaMKII and CaMKII protein levels following BHQ treatment. Full-length blots are presented in Supplementary Fig. 6. (F-G) Suppression of CXCR4 expression in Jurkat cells treated with BD (250 µM for 1.5 h) and KN93 (100 µM for 5 h), similar to the effect of BHQ (n = 3). (H-I) Suppression of CXCR4 expression in mouse LSKs and HSCs by CaMKII inhibitors (n = 3)

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To further investigate the potential of BHQ in augmenting the conventional G-CSF mobilization strategy, mice were initially administered subcutaneous injections of G-CSF (125 µg/kg, twice daily) for four consecutive days. Two hours before PB collection, a single BHQ injection (20 mg/kg) was administered. The mobilization of HSPCs into the peripheral circulation was then assessed by CFU assay and flow cytometry (Fig. 2F). Notably, the combination of BHQ and G-CSF significantly enhanced HSPC mobilization compared to G-CSF alone (Fig. 2G-I). Collectively, these results indicate that BHQ, either alone or in combination with G-CSF, markedly promotes the mobilization of HSPCs in vivo.

Fig. 4
figure 4

CaMKII inhibitors promote HSPC mobilization in vivo. (A, D) CFU counts per milliliter of PB following treatment with BD (n = 9) or KN93 (n = 6). (B-C, E-F) Absolute numbers of LSKs and HSCs per milliliter of PB, as determined by flow cytometry, following treatment with BD (n = 9) or KN93 (n = 6). (G-L) Enhanced HSPC mobilization when G-CSF is combined with BD (n = 6) or KN93 (n = 5). n indicates the number of mice used per group

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Fig. 5
figure 5

STAT3 and CXCR4 phosphorylation in Jurkat cells following BHQ or KN93 treatment. (A-E) Western blot analysis of protein levels of STAT3, pSTAT3 (Y705), CXCR4, and pCXCR4 (S339) in Jurkat cells treated with BHQ for 0.5, 1.5, and 5 h. (F-J) Western blot analysis of protein levels of STAT3, pSTAT3 (Y705), CXCR4, and pCXCR4 (S339) in Jurkat cells after KN93 stimulation for 0.5, 1.5, and 5 h. Full-length blots are presented in Supplementary Fig. 6

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BHQ induces ER stress by inhibiting SERCA activity, suppressing camkii and subsequently reducing CXCR4 expression

In line with previous studies [40, 41], BHQ inhibited SERCA activity, leading to calcium release from the ER and triggering ER stress. To examine the role of SERCA in hematopoietic cells, we conducted gene knockdown experiments targeting SERCA isoforms. Given the tissue-specific expression of SERCA isoforms, we first assessed the expression levels of SERCA1, SERCA2, and SERCA3 in Jurkat cells. Our analysis revealed that SERCA2 and SERCA3 were the predominant isoforms expressed (Fig. S4A). Subsequently, we used shRNA to individually knock down these two isoforms (Fig. S4B-C).

Flow cytometry analysis revealed that knockdown of SERCA2 and SERCA3 significantly decreased the surface expression of CXCR4 in Jurkat cells (Fig. 3A-B), despite no significant changes in CXCR4 mRNA levels (Fig. S5). To further verify the role of SERCA in CXCR4 regulation, we treated Jurkat cells, both with and without SERCA knockdown, with the SERCA agonist CDN11633. Interestingly, CXCR4 expression increased significantly in non-knockdown cells, whereas no significant change in CXCR4 expression was observed in the knockdown cells (Fig. 3C). These findings provide evidence that the expression and activity of SERCA play a crucial role in regulating CXCR4 surface expression.

As BHQ inactivated SERCA, intracellular Ca²⁺ failed to enter the ER for storage, resulting in a sudden increase in cytoplasmic Ca²⁺ concentration. This surge (within 30 min) initially upregulated CaMKII activity, but was followed by a sharp decline in intracellular Ca²⁺ levels, which dropped below the baseline. This fluctuation generated a transient calcium wave (Fig. 3D), followed by a decrease in CaMKII levels (Fig. 3E). The level of phosphorylated CaMKII at T286 (CaMKII-T286) continued to decrease, possibly due to the inhibition of phosphorylation triggered by the stress response. These results indicate that SERCA inactivation in hematopoietic cells induces calcium transients, resulting in negative feedback inhibition of downstream CaMKII activity.

To verify whether CaMKII activity plays a key role in the change of CXCR4 surface expression, we employed two classic CaMKII inhibitors, BD [42] and KN93 [43]. Both inhibitors significantly reduced CXCR4 expression in Jurkat cells, mirroring the effect of BHQ (Fig. 3F-G). Further verification in mouse primary cells revealed that BD and KN93 could also significantly inhibit CXCR4 expression in primary LSKs (Fig. 3H) and HSCs (Fig. 3I). Collectively, these findings indicate that the inhibition of CaMKII activity is a critical mechanism driving the reduction of CXCR4 expression.

CaMKII inhibitors promote HSPC mobilization and enhance G-CSF mobilization in mice

To investigate whether CaMKII inhibition also affects HSPC mobilization in vivo, we administered SC injections of BD (30 mg/kg, 3 h) and KN93 (20 mg/kg, 12 h) to C57Bl/6 mice, and then collected PB to analyze HSPC counts. CFU assays revealed that mice injected with BD or KN93 exhibited a significant increase in hematopoietic colony formation compared to the control group (Fig. 4A, D). This effect was also observed in the counts of LSKs and phenotypic HSCs, as detected by flow cytometry (Fig. 4B-C, E-F).

To further determine whether BD or KN93 synergizes with G-CSF, mice were initially administered continuous subcutaneous injections of G-CSF (125 µg/kg, twice daily) for 4 days. Subsequently, BD (30 mg/kg) or KN93 (20 mg/kg) was administered 3–12 h before PB collection. CFU assays and flow cytometry analysis revealed that both BD and KN93 significantly enhanced G-CSF-induced HSPC mobilization (Fig. 4G-L). These findings indicate that CaMKII inhibitors efficiently promote HSPC mobilization in vivo and exhibit a synergistic effect with G-CSF.

BHQ and KN93 induce CXCR4 phosphorylation and alter its membrane localization by inhibiting STAT3 phosphorylation

To elucidate how ER stress regulates CXCR4 level on the cell surface, we examined STAT3 and its phosphorylated form, pSTAT3 (Y705), a downstream target of CaMKII. Following 1.5 to 5 h of BHQ stimulation and 0.5 to 5 h of KN93 stimulation in Jurkat cells, total STAT3 protein level remained unchanged. However, phosphorylated STAT3 (pSTAT3 Y705) levels were significantly reduced, indicating that CaMKII inhibition affects STAT3 activation. Previous studies have shown that the expression of CXCR4 is regulated by STAT3 [44]. Therefore, we examined the total CXCR4 protein levels and found that in Jurkat cells, the total CXCR4 protein level was significantly downregulated after 0.5 to 5 h of BHQ and KN93 stimulation, which is consistent with literature reports. Additionally, we observed a significant increase in the phosphorylation of CXCR4 at S339, suggesting that CXCR4 phosphorylation plays a crucial role in its membrane localization (Fig. 5).

Fig. 6
figure 6

Phosphorylated protein levels of STAT3 and CXCR4 in mouse LSK cells. (A) Flow cytometry analysis of pSTAT3 (Y705) levels in LSK cells after BHQ stimulation for 5 h (n = 3). (B) Flow cytometry analysis of pCXCR4 (S339) levels in LSK cells after BHQ stimulation for 5 h (n = 3)

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Fig. 7
figure 7

Calcium gluconate rescues CXCR4 expression in SERCA-inhibited cells. (A) Schematic model illustrating ER stress-mediated regulation of CXCR4 expression. (B) Flow cytometric analysis showing that calcium gluconate alleviates BHQ-induced suppression of CXCR4 expression in Jurkat cells (n = 3). (C) Flow cytometric analysis demonstrating that calcium gluconate supplementation significantly increases CXCR4 expression in LSK cells (n = 3). (D) Flow cytometric analysis showing that calcium gluconate supplementation significantly increases CXCR4 expression in SERCA2/3 knockdown cells (n = 3)

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To validate these findings in primary cells, we treated mouse LSK cells with BHQ (100 µM, 5 h). Flow cytometry revealed significant suppression of pSTAT3 (Y705) (Fig. 6A) and elevation of pCXCR4 (S339) (Fig. 6B). These results implicate ER stress in rewiring STAT3-CXCR4 signaling, where CaMKII inhibition dampens STAT3 activity while enhancing CXCR4 phosphorylation—a dual mechanism likely facilitating HSPC mobilization under stress conditions.

Exogenous calcium supplementation restores CXCR4 expression

To investigate whether the expression of CXCR4 can be rescued by replenishing ER calcium reserves with calcium supplementation, we first supplemented Jurkat cells with calcium gluconate (600 µg/mL) prior to BHQ (100–400 µM) treatment. The cells were then incubated together at 37 °C for 30 min. We found that calcium gluconate significantly mitigated the effect of BHQ, thereby restoring CXCR4 surface expression (Fig. 7B). To verify whether calcium supplementation has a similar effect in mouse LSKs, Lin− cells were isolated from C57Bl/6 mice and stimulated with calcium gluconate along with BHQ at 37 °C for 30 min. Flow cytometry analysis demonstrated that calcium gluconate restored CXCR4 expression in LSK cells. Notably, calcium gluconate alone significantly increased the expression of CXCR4 (Fig. 7C). Moreover, in SERCA2/3 knockdown Jurkat cells, additional supplementation with calcium gluconate (600 µg/mL) also significantly restored the expression of CXCR4 (Fig. 7D). These results collectively indicate that calcium supplementation counteracts SERCA inhibition-induced suppression of CXCR4, highlighting the critical role of ER calcium homeostasis in CXCR4 regulation.

Discussion

This study elucidates that inducing ER stress in hematopoietic cells leads to a reduction in intracellular CaMKII activity. Consequently, the downstream STAT3-CXCR4 pathway is inhibited, resulting in a rapid decline in CXCR4 expression on the cell membrane. This process promotes the migration of HSPCs out of the BM, thereby facilitating their mobilization. These findings suggest a potential strategy for enhancing the mobilization of HSPCs through targeted modulation of ER stress.

In the BM microenvironment, stromal cells secrete CXCL12, which binds to CXCR4 on the hematopoietic cell surface, thereby anchoring them within the BM niche [45]. A reduction in CXCR4 surface receptor disrupts the CXCL12-CXCR4 axis, leading to the migration of HSPCs from the BM. Based on this principle, various mobilization strategies for HSPCs have been developed. Our study demonstrates that ER stress inducers significantly reduce CXCR4 expression on hematopoietic cells, suggesting a potential link between ER stress and HSPC mobilization. To verify this hypothesis, we performed relevant in vivo experiments in which mice received an appropriate dose of BHQ via SC injection for short-term stimulation (2 h). PB was then collected for CFU assays and absolute counts of LSKs/HSCs. Under these conditions, we observed a significant increase in the number of mobilized HSPCs in circulation. Furthermore, BHQ enhanced the mobilization effect of G-CSF, a well-established HSPC mobilization agent, leading to a greater yield of HSPC grafts. These results provide preliminary evidence that ER stress plays a regulatory role in HSPC migration, offering potential avenues for optimizing mobilization strategies.

Recent studies have shown that an appropriate level of ER stress and a moderate unfolded protein response (UPR) can help HSCs stimulate the “hormesis effect”—a phenomenon where low levels of stress enhance the organism’s resistance to subsequent stress, promoting life extension or rejuvenation under various stress conditions [15]. This effect enables HSCs to activate their self-repair, renewal, and maintenance capabilities. Under low-dose ER stress signals, the UPR can induce the activation of IRE1. IRE1 possesses both kinase and endoribonuclease activities, which lead to the splicing of XBP1 mRNA into its active form, XBP1s [37]. XBP1s is then translated into a transcription factor that induces the expression of genes encoding molecular chaperones, folding enzymes, and endoplasmic reticulum-associated degradation (ERAD) components, thereby participating in cell protection programs [15, 46]. In this study, we detected a significant increase in the mRNA levels of XBP1s, the spliced form of XBP1, under the stimulation of low-dose BHQ and TG. This indicates that the ER stress-induced process activated an “hormesis effect” in hematopoietic cells.

BHQ has been widely used as a potent SERCA inhibitor in previous studies [37, 47]. During the ER stress response, inhibition of SERCA prevents intracellular Ca²⁺ from entering the ER for storage through conventional pathways, resulting in an instantaneous decrease in ER calcium reserves while increasing cytoplasmic Ca2+ levels. This increase stimulates the ER stress response, and the excess Ca2+ in the cytoplasm activates the mitochondrial calcium transport channel and enters the mitochondria via the mitochondrial Ca2+ uniporter (MCU) [48,49,50]. When cytoplasmic Ca²⁺ levels increase, CaMKII activity also increases immediately. As Ca²⁺ is taken up by the mitochondria, CaMKII activity decreases in a negative feedback manner. Consequently, the overall effect of this ER stress response is a prolonged inhibition of CaMKII activity. To investigate whether CaMKII inhibition is central to ER stress-induced HSPC mobilization, we employed two CaMKII inhibitors, BD and KN93. Similar to ER stress inducers, these inhibitors suppressed CXCR4 expression and enhanced HSPC mobilization in vivo, strongly supporting our hypothesis that CaMKII acts as a critical mediator in this cascade. This aligns with earlier work identifying CaMKIIγ as a key regulator in hematopoietic cells, where it maintains quiescence in normal HSPCs but drives hyperactivation in leukemic stem cells (LSCs) by modulating NF-κB, Wnt/β-catenin, and STAT3 pathways [42]. Building on these findings, we demonstrate that BHQ- and KN93-mediated CaMKII inhibition reduces phosphorylated STAT3 (pSTAT3) levels, mirroring observations in STAT3-knockout models where IL-30-dependent CXCR4 upregulation was impaired [44]. Our results extend this paradigm by linking ER stress-induced CaMKII suppression to STAT3 inactivation and subsequent CXCR4 downregulation. Furthermore, we uncovered a novel regulatory mechanism: ER stress not only reduced total CXCR4 protein levels but also increased phosphorylation of CXCR4 at S339. This observation aligns with earlier research showing that S339 phosphorylation promotes CXCR4 endocytosis, thereby modulating leukemia cell trafficking in BM niches [13].

Small molecular inhibitors can serve as reversible ER stress triggers, potentially useful in the future development of mobilization strategies to facilitate the regulation of HSPC homing after transplantation. In the current study, to clarify whether the SERCA family is a key component in the ER stress response, we knocked down SERCA2 and SERCA3 using shRNA, which are specifically expressed in hematopoietic cells, to simulate mild ER stress. Notably, simultaneous knockdown of both isoforms resulted in increased apoptosis (data not shown). We found that knocking down either SERCA2 or SERCA3 alone produced a phenotype similar to that induced by BHQ. When treated with SERCA agonists, the surface expression of CXCR4 on non-knockdown Jurkat cells significantly increased. However, in cells expressing shSERCA2 or shSERCA3, the expression of CXCR4 did not increase. This indicates a direct correlation between SERCA activity and CXCR4 expression.

In conditions of mild ER stress, exogenous calcium supplementation can activate stromal interaction molecule 1 (STIM1) on the ER membrane. STIM1 then interacts with calcium release-activated calcium channel protein 1 (Orai1) on the plasma membrane, thereby activating Ca²⁺ influx across the plasma membrane. This process, known as store-operated Ca²⁺ entry (SOCE), replenishes the calcium stores in the ER [18, 48, 51, 52]. In this study, CXCR4 expression was significantly restored by supplementing calcium gluconate in BHQ-treated or shSERCA2/3 knockdown cells. These findings suggest that calcium supplementation may enhance the homing ability of mobilized HSPCs when administered at the time of transplantation.

In this study, we utilized the Jurkat cell line as an in vitro model to investigate ER stress-mediated CXCR4 regulation and HSPC mobilization mechanisms. This choice was based on Jurkat cells’ well-characterized expression of CXCR4, their responsiveness to calcium-dependent signaling pathways, and their historical utility in studying hematopoietic cell behaviors, including chemotaxis and stress responses. These features allowed us to isolate ER stress-specific effects on CXCR4 dynamics in a controlled experimental system. However, while Jurkat cells share phenotypic and functional similarities with primary HSPCs, they lack the heterogeneity and microenvironmental interactions inherent to primary hematopoietic cells in vivo. Additionally, immortalized cell lines like Jurkat may exhibit altered stress response pathways compared to quiescent or mobilized HSPCs. We have verified the changes of pSTAT3 and pCXCR4 levels in mouse primary LSK cells following BHQ treatment. Future studies should further validate these findings through in vivo experiments to provide more comprehensive data support. Overall, our results provide foundational mechanistic insights into how ER stress disrupts CXCR4 signaling, sets the stage for developing novel therapeutic strategies aimed at enhancing HSPC mobilization in clinical settings.

Conclusions

In summary, our study reveals that inducing mild ER stress reversibly inhibits the SERCA-CaMKII-STAT3 pathway, leading to a reduction in CXCR4 surface expression on HSPCs and promoting their mobilization into the peripheral circulation. Moreover, calcium supplementation effectively restores CXCR4 expression, suggesting a potential strategy to enhance the homing capacity of mobilized HSPCs post-transplantation. These findings provide mechanistic insights into ER stress-mediated regulation of HSPC mobilization and highlight its potential therapeutic application as an adjunct to existing mobilization regimens.

Data availability

All data generated during this study are included in this published article and its supplementary information files. The original data supporting the findings of this study are available from the corresponding author on reasonable request.

Abbreviations

HSC:

Hematopoietic stem cell

HSPC:

Hematopoietic stem/progenitor cell

PB:

Peripheral blood

BM:

Bone marrow

G-CSF:

Granulocyte colony stimulating factor

CXCR4:

C-X-C chemokine receptor type 4

SDF-1:

Stromal cell-derived factor 1

GPCR:

G-protein-coupled receptors

ER:

Endoplasmic reticulum

SERCA:

Sarco/endoplasmic reticulum Ca2+-ATPase

CaMKII:

Calmodulin-dependent protein kinase II

MNC:

Mononuclear cells

CFU:

Colony forming unit

IRE1:

Inositol-requiring protein 1

XBP1:

X-box-binding protein 1

SC:

Subcutaneous

UPR:

Unfolded protein response

ERAD:

Endoplasmic reticulum-associated degradation

MCU:

Mitochondrial Ca2+ uniporter

LSC:

Leukemic stem cells

STIM1:

Stromal interaction molecule 1

SOCE:

Store-operated Ca2+ entry

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Acknowledgements

The authors thank the members of the Guoliang Xu, Jinchuan Hu and Xinxin Huang laboratories for their valuable assistance and insightful discussions. The authors declare that no AI-generated content was used in this manuscript.

Funding

This work was funded by Guangdong Major Project of Basic and Applied Basic Research (2023B0303000005), National Excellent Youth Fund of National Natural Science Foundation of China (82222005), General Program of National Natural Science Foundation of China (82070107), and Fudan University Start-up funding to XH.

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  1. Lijun Li and Danhua Xu contributed equally to this work.

Authors and Affiliations

  1. Shanghai Xuhui Central Hospital, Zhongshan-Xuhui Hospital, and The Shanghai Key Laboratory of Medical Epigenetics, The International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China

    Lijun Li, Danhua Xu & Xinxin Huang

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  1. Lijun Li
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All authors participated in the design, interpretation of the studies, analysis of the data, and review of the manuscript; LL and DX conducted the experiments, LL wrote the manuscript, XH conceived the project and revised the manuscript.

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Correspondence to Xinxin Huang.

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The project, entitled “The regulating mechanisms of hematopoietic stem cell expansion and homing” was approved by the Animal Care and Use Committee of Fudan University School of Medicine, with approval number 202311021Z granted on November 24, 2023.

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Li, L., Xu, D. & Huang, X. SERCA-mediated endoplasmic reticulum stress facilitates hematopoietic stem cell mobilization. Stem Cell Res Ther 16, 208 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04345-y

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512